Diamide inhibits pulmonary vasoconstriction induced by hypoxia or prostaglandin F2 alpha

Diamide oxidizes glutathione and other cellular sulfhydryl groups. It decreases calcium ATPase activity and alters mitochondrial calcium flux, probably as a result of the sulfhydryl oxidation. We examined the effect of diamide (5 mg/kg, iv) on pulmonary vascular reactivity in 12 anesthetized dogs. Diamide reversed the pulmonary vasoconstriction caused by hypoxia in seven dogs (control delta PVR + 2.5 +/- 0.6 mm Hg/liter/min; postdiamide delta PVR - 0.1 +/- 0.4 mm Hg/liter/min; P less than 0.01). The pulmonary pressor response to prostaglandin F2 alpha (5 micrograms/kg/min, iv) was also reduced (control delta PVR + 3.8 +/- 0.5 mm Hg/liter/min; postdiamide delta PVR + 1.1 +/- 0.7 mm Hg/liter/min; P less than 0.01). However, in a further five dogs, diamide had only a small effect on the pulmonary vasoconstriction caused by angiotensin II, while the pressor response to hypoxia was again inhibited. The mechanism by which diamide reverses pulmonary vasoconstriction is not certain but the effect is rapid, consistent, and reversible. Because the intravenous infusion of diamide does not produce systemic hypotension, during its period of action on the pulmonary vasculature, unlike the drugs currently available for the clinical treatment of pulmonary hypertension, further studies of its mechanism of action are indicated.     

Treatment with prostaglandin F2alpha increases expression of prostaglandin synthase-2 in the rat corpus luteum

Recent studies indicate that the corpus luteum (CL) may be a source of prostaglandin F2alpha (PGF2alpha) for regression. We investigated expression of mRNA and protein for prostaglandin G/H synthase (PGHS) in the CL of immature superovulated rats following administration of PGF2alpha. We observed an increase in mRNA for PGHS-2, the induced isoform, at 1 h and protein at 8 and 24 h after treatment. One hour after PGF2alpha, there was also a progressive decrease in plasma progesterone concentration. There were no changes, however, in expression of PGHS-1, the constitutive isoform, over the 24 h sampling period. These results indicate that PGHS-2 increases following PGF2alpha treatment and that expression of this enzyme in the rat CL may contribute to the luteolytic mechanism.     

Oxytocin stimulates prostaglandin F2alpha secretion and prostaglandin F synthase protein expression in porcine myometrial tissue

The possibility of PGF(2)alpha production and presence of prostaglandin F synthase (PGFS; PGD(2) 11-ketoreductase) was studied in control and oxytocin (OT)-stimulated myometrial slices isolated from cyclic (Days 14-16) and early pregnant (Days 14-16) sows. Oxytocin (10(-7) M) stimulated (p<0.01) PGF(2)alpha production in both cycling and early pregnant myometrial slices. Prostaglandin F(2)alpha release was higher (p<0.01) in control as well as OT-treated myometrium of early pregnant sows in comparison to cycling myometrium. Prostaglandin F synthase expression at protein level was evident in myometrial slices of cyclic as well as early pregnant sows. The signals of PGFS was stronger (p<0.05) in cycling myometrium exposed to OT compared to that of control. There were no significant differences (p>0.05) in PGFS protein expression between control and OT-stimulated myometrial tissue of early-pregnant sows. The results of this study indicate the local PGF(2)alpha synthesis and the presence of PGFS in porcine cycling and early pregnant myometrial tissue. In addition, OT increased PGD(2) 11-ketoreductase protein expression in myometrium harvested during the porcine estrous cycle. However, the OT-stimulated PGF(2)alpha myometrial secretion was observed in both, cycling and pregnant gilts.     

Peroxisomal chain-shortening of prostaglandin F2 alpha

We have recently reported that prostaglandin F2 alpha can be chain-shortened by isolated rat liver peroxisomes. In the present study it is further established by cell fractionation experiments that the enzymes involved in this reaction are localized to peroxisomes. Under the conditions employed, the highest activity was found in the light mitochondrial fraction. Further fractionation of the light mitochondrial fraction by sucrose density gradient centrifugation showed that the prostaglandin oxidation activity comigrated with peroxisomal marker enzymes. Di(2-ethylhexyl)phthalate treatment resulted in a tenfold increased capacity for the conversion of prostaglandin F2 alpha into tetranorprostaglandin F1 alpha. The reaction was not inhibited by KCN. The reaction was further characterized with respect to cofactor requirements. The prostaglandin oxidation was found to be completely dependent on NAD, CoA, ATP, Mg2+ and was stimulated by FAD. Incubation of prostaglandin E2 with peroxisomes resulted in conversion into several products. After alkaline hydrolysis, one of these was identified as tetranorprostaglandin B1.     

Canine prostaglandin F2alpha receptor (FP) and prostaglandin F2alpha synthase (PGFS): molecular cloning and expression in the corpus luteum

In the dog luteolysis is not affected by hysterectomy. This observation led to the hypothesis that paracrine/autocrine rather than endocrine mechanisms of PGF2alpha are responsible for luteal regression in the dioestric bitch. The present experiments tested for the capacity of canine CL to produce and respond to PGF2alpha by qualitatively and quantitatively determining the expressions of PGFS, the enzyme converting PGH2 into PGF2alpha, and the PGF2alpha-receptor (FP) in CL of non-pregnant dogs during dioestrus. Canine PGFS and FP were isolated and cloned; both genes show a high homology (82-94%) when compared to those of other species. Relatively weak FP mRNA expression was detected on day 5 of dioestrus. It had increased by day 25 and remained constant thereafter. In situ hybridization (ISH) localized FP solely to the cytoplasm of the luteal cells, suggesting that these cells are the only luteal targets of PGF2alpha in this species. Only negative results were obtained for the expression of PGFS in canine CL by routine qualitative RT-PCR. When Real Time (TaqMan) PCR was applied, repetitively more negative than positive results were obtained at all timepoints. Any positive measurements observed at any point were neither repeatable nor related to the stage of dioestrus. This led us to conclude that expression of PGFS is either absent or present at very low level only. These data suggest that luteal regression in non-pregnant bitches is not modulated by PGF2alpha. However, the FP seems to be constitutionally expressed, explaining the receptivity of canine CL to exogenous PGF2alpha.     

Influence of prostaglandin F2 alpha on sperm production and seminal characteristics of the stallion

Six mature stallions were used to test the effect of prostaglandin F2 alpha (PGF2 alpha ) on sperm production and seminal characteristics. Semen was collected from each stallion twice weekly 1 hr following a 10 mg intramuscular injection of PGF2 alpha or a sham injection. A switchback design was used so that three stallions received PGF2 alpha and three served as controls during the first 9 weeks (period 1). Treatment regimens were reversed during the second 9 weeks (period 2). Treatment of stallions with PGF2 alpha resulted in an increase (P less than .05) in gel free seminal volume and a decrease in sperm cell concentration. Total spermatozoa, sperm cell motility, and percentage of primary and secondary sperm abnormalities of ejaculates were not significantly affected by treatment of stallions with PGF2 alpha before semen collection. All treated stallions exhibited a pronounced sweating response to the drug. During the experiment, two of the six stallions masturbated within 20 to 30 minutes after PGF2 alpha treatment without achieving an erection.     

Histophysiological studies of prostaglandin F2alpha on isolated organs. I. Effect of prostaglandin F2alpha on the heart.

The physiological and histochemical effects of PGF2alpha on isolated rabbit hearts were examined. The results showed a positive inotropic effect. The coronary flow increased. From the histochemical studies, adenosine triphosphatase (ATP-ase) and succinic dehydrogenase activities were increased while that of alkaline phosphatase was decreased. Glycogen granules were depleted. These findings were discussed on a histophysiological basis.     

Prostaglandin E1 inhibits the pulmonary vascular pressor response to hypoxia and prostaglandin F2alpha.

In the anesthetised dog an infusion of exogenous prostaglandin E1 (100muG/min) inhibits the pulmonary vascular pressor response to hypoxia. Both 25 and 100muG/min PGE1 can reduce the transient pulmonary hypertension caused by a bolus of prostaglandin F2alpha. This suggests that hypoxia and PGF2alpha may share a final common pathway in producing pulmonary vasoconstriction. These results may help to explain the mechanism by which endotoxin inhibits the pulmonary vascular response to hypoxia. This effect is probably achieved by stimulating the production of an endogenous dilator prostaglandin. Exogenous PGE1 can mimic this effect.     

Thromboxane B2, 6-keto-prostaglandin F1 alpha, and prostaglandin F2 alpha production by contracting pregnant rate uteri in vitro

While prostaglandin production by uterine tissue has been shown to be involved in the contractile mechanism of this tissue, less attention has focused upon the involvement of other prostanoids. We have simultaneously measured in vitro isometric contractility of pregnant rat uteri with the release of prostaglandin F2 alpha (PGF), 6-keto-prostaglandin F1 alpha (6-k-PGF1 alpha) and thromboxane B2 (TXB2) into the bathing medium under various conditions. Frequency of uterine contractions and integrated contractile force (ICF) increased from 15 days of gestation and peaked at the time of parturition. Activity was generally greatest during the first 15 min of incubation except during parturition and on Day 1 postpartum when the uterine segment remained active for 1 h experimental period. Indomethacin (INDO) significantly reduced contractile activity regardless of gestational stage. PGF, TXB2, and 6-k-PGF1 alpha increased with gestational age, peaking at the time of parturition. Production was greatest during the first 15 min of incubation and INDO inhibited production of each prostanoid regardless of gestational stage. Imidazole (100 micrograms/ml) inhibited TXB2 production without affecting PGF or 6-k-PGF1 alpha levels. Frequency of contraction and ICF were not affected by imidazole treatment despite TXB2 reduction. These data demonstrate that the in vitro uterus from pregnant rats is capable of producing prostanoids other than prostaglandins and their production generally parallels uterine contractile activity. Thus, the possibility that these prostanoids are involved in physiologic changes during parturition warrants further investigation.     

Role of prostaglandin F2alpha in ovulation.

Using radioimmunoassay procedures, the levels of plasma, uterine and ovarian prostaglandin (PG) F2alpha, and those of plasma estradiol and progesterone were measured in intact, hysterectomized or ovariectomized immature female rats pretreated with PMS and subsequent HCG. Occurrence of ovulation was confirmed at 8 hours after the HCG administration not only in the intact rats but also in the hysterectomzied rats. The levels of plasma estradiol and progesterone, and of uterine and ovarian PGF2alpha rose with the PMS injection alone, but they did not reach the peaks before the HCG administration. Both plasma estradiol and uterine PGF2alpha showed a peak at 2 hours after the HCG injection. These peaks were antecedent 2 or 6 hours before the peaks of ovarian and plasma PGF2alpha, respectively. However, such increase of uterine PGF2alpha does not seem to be indispensable for ovulation, because ovulation could occur in the hysterectomized rats. The levels of ovarian PGF2alpha showed a high plateau from 4 to 8 hours after the HCG injection, and then rapidly decreased after ovulation. The levels of plasma PGF2alpha peaked not only in the intact rats but also in the hysterectomized rats at 8 hours after the HCG treatment. But in the ovariectomized rats, this plasma PGF2alpha peak at 8 hours disappeared and there was no statistical change of plasma PGF2alpha throughout the PMS-HCG treatment. Plasma progesterone gradually increased and reached the maximum at 10 hours after the HCG injection. These results conclude that the main source of increased plasma PGF2alpha during the ovulatory process induced with the PMS-HCG treatment is the ovary, and it is strongly suggested that a rapid increase of PGF2alpha in the ovary may play some important role(s) in the ovulatory process.     

Autocrine production of prostaglandin F2alpha enhances phenotypic transformation of normal rat kidney fibroblasts

We have used normal rat kidney (NRK) fibroblasts as an in vitro model system to study cell transformation. These cells obtain a transformed phenotype upon stimulation with growth-modulating factors such as retinoic acid (RA) or transforming growth factor- (TGF-). Patch-clamp experiments showed that transformation is paralleled by a profound membrane depolarization from around –70 to –20 mV. This depolarization is caused by a compound in the medium conditioned by transformed NRK cells, which enhances intracellular Ca²⁺ levels and thereby activates Ca²⁺-dependent Cl^– channels. This compound was identified as prostaglandin F₂ (PGF₂) using electrospray ionization mass spectrometry. The active concentration in the medium conditioned by transformed NRK cells as determined using an enzyme immunoassay was 19.7 ± 2.5 nM (n = 6), compared with 1.5 ± 0.1 nM (n = 3) conditioned by nontransformed NRK cells. Externally added PGF₂ was able to trigger NRK cells that had grown to density arrest to restart their proliferation. This proliferation was inhibited when the FP receptor (i.e., natural receptor for PGF₂) was blocked by AL-8810. RA-induced phenotypic transformation of NRK cells was partially (25%) suppressed by AL-8810. Our results demonstrate that PGF₂ acts as an autocrine enhancer and paracrine inducer of cell transformation and suggest that it may play a crucial role in carcinogenesis in general. membrane potential; intracellular calcium; mass spectrometry; FP receptor     

Effects of prostaglandin F2 alpha on agonist-induced progesterone production in cultured bovine luteal cells

The present study examines the effects of prostaglandin F2 alpha (PGF2 alpha) on basal and agonist-stimulated progesterone (P4) production utilizing long-term, serum-free cultures of bovine luteal cells. During the first 24 h of culture, PGF2 alpha had no significant effect on P4 production, and was unable to inhibit either luteinizing hormone (LH)- or dibutyryl cAMP (dbcAMP)-stimulated increases in P4. Treatment with PGF2 alpha on Day 1 produced a moderate, nonsignificant (P greater than 0.05) inhibition of cholera toxin (CT)- and forskolin (FKN)-stimulated P4 synthesis. Beyond Day 1 of culture (Days 3-11), PGF2 alpha continued to have no significant effect on basal P4 production, but suppressed all stimulatory effects of LH, dbcAMP, CT and FKN. Treatment with indomethacin inhibited prostaglandin synthesis by the cultured cells and also elevated levels of P4 from Days 3 to 11 of culture. Concurrent treatment with PGF2 alpha suppressed the steroidogenic effect of indomethacin. From these studies it was concluded that in cultured bovine luteal cells, PGF2 alpha does not affect basal P4 production, but is able to inhibit agonist-stimulated P4 production at a site beyond the accumulation of cAMP. This inhibitory effect is not apparent during the first 24 h of culture, but appears after Day 1 and persists throughout the remaining 10 days of the culture period.     

Increased prostaglandin F2 alpha and E2 production in late passage WI38 diploid fibroblasts

The prostaglandin (PG) F2alpha and E2 concentrations in the culture medium of WI38 cells were determined at various passage levels. It was found that late passage cells produce significantly higher amounts of both PG F2alpha and PG E2.     

Urinary excretion of prostaglandin E2 and prostaglandin F2alpha in potassium-deficient rats

Potassium-deficiency was induced in rats by dietary deprivation of potassium. The animals became polyuric and urine osmolality decreased more then three-fold compared to controls. Urinary excretion of prostaglandin E2 (PGE2) and prostaglandin F2alpha (PGF2alpha) did not increase during 2 weeks of potassium depletion. Partial inhibition of renal prostaglandin synthesis by meclofenamate did not increase the urine osmolality after water deprivation. These results make unlikely the hypothesis that the polyuria of potassium-deficiency, is the result of enhanced renal synthesis of prostaglandins with subsequent antagonism of the hydro-osmotic effect of vasopressin. Male animals consistently excreted less PGE2 than female animals.     

Matrix metalloproteinase expression and activity following prostaglandin F(2 alpha)-induced luteolysis

Luteal tissue contains matrix metalloproteinases (MMPs) that cleave specific components of the extracellular matrix (ECM) and are inhibited by tissue inhibitors of metalloproteinases (TIMPs). We previously reported a decrease in luteal TIMP-1 within 15 min of prostaglandin F(2 alpha) (PGF(2 alpha))-induced luteolysis. An increase in the MMP:TIMP ratio may promote ECM degradation and apoptosis, as observed in other tissues that undergo involution. The objectives of these experiments were to determine whether 1) PGF(2 alpha) affects expression of mRNA encoding fibrillar collagenases (MMP-1 and -13), gelatinases A and B (MMP-2 and -9), membrane type (mt)-1 MMP (MMP-14), stromelysin (MMP-3), and matrilysin (MMP-7), and 2) PGF(2 alpha) increases MMP activity during PGF(2 alpha)-induced luteolysis in sheep. Corpora lutea (n = 3-10/time point) were collected at 0, 15, and 30 min and 1, 2, 4, 6, 12, 24, and 48 h after PGF(2 alpha) administration. Northern blot analysis confirmed the presence of all MMPs except MMP-9. Expression of mRNA for the above MMPs (except MMP-2) increased significantly (P < 0.05) by 30 min, and all MMPs increased significantly (P < 0.05) by 6 h after PGF(2 alpha) administration. Expression of MMP-14 mRNA increased significantly (P < 0.05) by 15 min post-PGF(2 alpha) and remained elevated through 48 h. MMP activity in luteal homogenates (following proenzyme activation and inactivation of inhibitors) was increased significantly (P < 0.05) by 15 min and remained elevated through 48 h post-PGF(2 alpha). MMP activity was localized (in situ zymography) to the pericellular area of various cell types in the 0-h group and was markedly increased by 30 min post-PGF(2 alpha). MMP mRNA expression and activity were significantly increased following PGF(2 alpha) treatment. Increased MMP activity may promote ECM degradation during luteolysis.