The role of renal metabolism of PGE2 in determining its activity as a renal vasodilator in the dog

Since the mammalian renal cortex avidly metabolizes prostaglandin E₂ (PGE₂), we examined the importance of renal metabolism of PGE₂ in determining its renal vascular activity in the dog. We used 13, 14 dihydro PGE₂ (DHPGE₂) as a model compound to study this because DHPGE₂ retains similar activity to the parent prostaglandin, PGE₂, but is a poorer substrate than PGE₂ for both the metabolism and the cellular uptake of the prostaglandins. Using dog renal cortical slices, we found that under similar experimental conditions, PGE₂ was metabolized several-fold faster than DHPGE₂. Both prostaglandins were metabolized to the 15 keto 13, 14 dihydro PGE₂, which was positively identified using GC-MS. In vivo, we infused increasing concentrations of DHPGE₂ into the renal artery of dogs and measured renal hemodynamic changes using radioactive microspheres. DHPGE₂ was a potent renal vasodilator beginning at an infusion rate of 10⁻⁹g/kg/min. When compared to PGE₂, DHPGE₂ was about 10 times more potent in affecting renal vasodilation. The intrarenal redistribution of blood flow towards the inner cortex seen with DHPGE₂ was identical to that seen with PGE₂. We conclude that renal catabolism of PGE₂ is very important in limiting the in vivo biological activity of PGE₂, but regional differences in metabolism of PGE₂ within the cortex are an unlikely determinant of the pattern of redistribution of renal blood flow.     

The excretion of prostacyclin (PGI2) in milk and its possible role as a vasodilator in the mammary gland of goats

1. Prostacyclin production in mammary gland of two lactating goats measured as the excretion in milk of 6-ketoprostaglandin F1 alpha (6-KPGF1 alpha) was followed for 16 days before, during and after exogenous administration of recombinant bovine growth hormone (GH). 2. 6-KPGF1 alpha was detected in all milk samples in concentrations ranging from 32-99 pg/ml milk independently of the time of sampling. 3. GH-treatment significantly increased milk yield, the concentration and excretion of 6-KPGF1 alpha in milk. 4. The concentration of milk 6-KPGF1 alpha was positively correlated with milk yield in the high (R2 = 0.35), but not in the low yielding goat (R2 = 0.003). 5. The possible role of prostacyclin as a local vasodilator in the mammary gland of goats is discussed.     

PGE1 compared to PGE2/PGF2 alpha ratio as a marker for seminal fluid contamination of urine in studies of renal prostaglandin biosynthesis

Occasional and unpredictable seminal plasma leakage (SPL) into the urinary system severely limits the usefulness of primary prostaglandin (PG) excretion rates as markers of renal synthesis in male subjects. Although reports to this effect appeared first some 10 years ago, no specific study has been done to define the magnitude of the problem, while several investigators persisted in the use of primary PG excretion rates, disregarding SPL as a confounding factor. We conducted a systematic study with a group of 5 healthy adult subjects who collected 24-h urine with an average frequency of one collection every other week for a total of 20 weeks. 35% of the urine collected contained seminal plasma. It is imperative to be able to disqualify compromised urine specimens in biological studies. With this objective in mind, we developed a method to identify contaminated samples based on the presence of PGE1. This is a major prostanoid in seminal plasma but is normally absent in 'clear' urine.     

PGE2 is a more potent vasodilator of the lamb ductus arteriosus than is either PGI2 or 6 keto PGF1alpha.

It has been shown in vitro that the lamb ductus arteriosus forms prostaglandins PGE2, PGF2alpha, 6 keto PGF1alpha (and its unstable precursor PGI2). In this study the relative potencies of these endogenous prostaglandins were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO2 and indomethacin. All the prostaglandins (except PGF2alpha) relaxed the vessel. This is consistent with the hypothesis that endogenous prostaglandins inhibit the tendency of the vessel to contract in response to oxygen. Only PGE2, however, relaxed the vessel at concentrations below 10(-8)M. PGI2 and 6 keto PGF1alpha had approximately 0.001 and 0.0001 times the activity of PGE2. Although PGE2 has been observed to be a minor product of prostaglandin production in the lamb ductus arteriosus, the tissue's marked sensitivity to PGE2 might make it the most significant prostaglandin in regulating the patency of the vessel.     

Letter: PGE2 as a vaginal abortifacient - diaphragm effect.

Schulman et al reported reduction in interval-abortion time and decreased side effects when prostaglandin (PG) E2 vaginal suppositories were administered within a contraceptive diaphragm. The authors conducted a study to confirm Schulman et al' finding. 2 groups of 20 patients with gestational ages ranging from 13 to 20 weeks were matched. 1 group (non-diaphragm) had a 20 mg. suppository inserted every 4 hours high in the vaginal fornix. The other group (diaphragm) had a contraceptive diaphragm containing the suppository inserted at 4-hour intervals. The authors' protocol differed from that of Schulman's in that oxytocin sensitivity was not sought and PGE2 alone was used. Oxytocin, however, was used to promote placental expulsion following fetal delivery in 4 cases. There were no statistically significant differences observed between the groups with respect to total drug dose (84 mg. for diaphragm group and 80 mg. for non-diaphragm), abortion-interval time (15.2 hours vs. 14.3 respectively), or frequency of side effects (vomiting, diarrhea). Schulman's reduced side effects may have followed the decreased absorption rate of PGE2 and avoidance of plasma peaks secondary to reduction in drug-mucosal interface. Schulman also reported onset of uterine activity in the diaphragm group (mean, 41 minutes) versus the control group's mean of 79.4 minutes. There is no known physiologic explanation for such findings.     

Synthesis and biological activity of 2-aminothiazoles as novel inhibitors of PGE2 production in cells

This Letter presents the synthesis and biological evaluation of a collection of 2-aminothiazoles as a novel class of compounds with the capability to reduce the production of PGE(2) in HCA-7 human adenocarcinoma cells. A total of 36 analogs were synthesized and assayed for PGE(2) reduction, and those with potent cellular activity were counter screened for inhibitory activity against COX-2 in a cell free assay. In general, analogs bearing a 4-phenoxyphenyl substituent in the R(2) position were highly active in cells while maintaining negligible COX-2 inhibition. Specifically, compound 5l (R(1)=Me, R(2)=4-OPh-Ph, R(3)=CH(OH)Me) exhibited the most potent cellular PGE(2) reducing activity of the entire series (EC(50)=90 nM) with an IC(50) value for COX-2 inhibition of >5 μM in vitro. Furthermore, the anti-tumor activity of analog 1a was analyzed in xenograft mouse models exhibiting promising anti-cancer activity.     

The role of progesterone metabolism and androgen synthesis in renal blood pressure regulation.

11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a crucial role in converting hormonally active cortisol into inactive cortisone, conferring specificity onto the human mineralocorticoid receptor (MR). Progesterone binds with even higher affinity to the MR, but acts as an MR antagonist. How aldosterone is able to keep its function as predominant MR ligand in clinical situations with high progesterone concentrations, such as pregnancy, is not clear. We have shown in vitro that the human kidney possesses an effective enzyme system that metabolizes progesterone to inactive metabolites in a process similar to the inactivation of cortisol by 11beta-HSD2. In studies on patients with adrenal insufficiency, we have shown that the in vivo anti-mineralocorticoid activity of progesterone is diminished by inactivating metabolism of progesterone, local formation of the deoxycorticosterone mineralocorticoid from progesterone, and inhibition of 11beta-HSD2 by progesterone and its metabolites resulting in decreased inactivation of cortisol and hence increased MR binding by cortisol. The enzymes involved in progesterone metabolism are also responsible for the capability of the human kidney to convert pregnenolone to DHEA and androstenedione leading to the formation of active androgens, testosterone and 5alpha-DH-testosterone. Locally produced androgens might be responsible for the observed difference in blood pressure between men and women and higher susceptibility to hypertension in men.     

Renal cortical intermediary metabolism in the recovery phase of postischemic acute renal failure in the dog.

Renal metabolism has been studied in eight dogs before and 48 hr after a 60-min period of renal ischemia induced by clamping the left renal artery with the simultaneous removal of the right kidney, and in 12 sham-operated animals. The study involved the measurement of renal uptake and production of lactate, glutamine, glutamate, alanine, ammonium, and oxygen, and the measurement of the tissue concentrations of ATP, glutamine, lactate, alpha-ketoglutarate, aspartate, and alanine in the renal cortex. Two days after a temporary renal ischemia, the remaining kidney showed a 22% decrease in glomerular filtration rate (GFR) and a 25% decrease in renal plasma flow. Fractional sodium and potassium excretions were similar to those of control dogs. Renal production or extraction of glutamine, glutamate, alanine, ammonium, and oxygen (all expressed by 100 ml of GFR) was not significantly different in basal conditions or 2 days after ischemia, but lactate extraction was reduced in postischemic kidneys (-101 +/- 29 vs -204 +/- 38 mumol/100 ml GFR in control dogs). The cortical concentrations of glutamine and glutamate were lower in postischemic than in control kidneys. No differences were found in cortical concentration of alpha-ketoglutarate, aspartate, lactate, pyruvate, or ATP, but total nucleotides and inorganic phosphate were decreased in postischemic kidneys. It is concluded that in the recovery phase of the ischemia, a decreased lactate uptake is the main metabolic change, and total ATP production is adapted to the decrease of GFR and sodium reabsorption.     

The vasodilator effect of thiamylal in dog mesenteric artery: contribution of intracellular action.

The effects of thiamylal on contractions induced by various mechanisms were investigated in mesenteric arteries isolated from dogs. Thiamylal (10(-4) to 10(-3) M) significantly inhibited contractions induced by KCl (20 mM) in normal media, and those induced by norepinephrine (10(-5) M) in normal and Ca(2+)-free media. Caffeine-induced contraction was significantly inhibited by thiamylal in the concentrations greater than 3 x 10(-5) M in intact fibers and 10(-5) M in chemically skinned fibers. Chemically skinned fibers that were precontracted with Ca2+ were relaxed by thiamylal in concentrations lower than those required to relax intact fibers that were precontracted with KCl (20 mM); the ED50 was 1.52 x 10(-5) M in skinned fibers and 5.50 x 10(-4) M in intact fibers. These results suggest that intracellular mechanisms are involved in thiamylal-induced vasodilatation of dog mesenteric artery.     

Intramitochondrial substrate concentration as a factor controlling metabolism. The role of interanion competition

1. The concentration-dependence of the intramitochondrial accumulation of l-malate and succinate was measured and expressed in the form of adsorption isotherms. The accumulation, however, may arise because of an internal positive potential. 2. The competition for accumulation offered by some other anions, including phosphate, was measured and is expressed conventionally by additional terms in the adsorption equation. 3. The interactions between anions were also studied when one was acting as oxidized substrate. 4. In some examples there is a parallel between the effects of an added anion on both accumulation and oxidation; in other cases chemical participation of the added substance in metabolism is presumed to remove the correlation. 5. It is suggested that by combining kinetic data on penetration with stoicheiometric data on accumulation and specific reaction rates it may be possible to account for the rates of respiration obtained with intact mitochondria. 6. It is possible to show that there is a certain phosphate/substrate ratio for maximum phosphorylation rate with some substrates. This is to be expected when phosphate and substrate compete for accumulation.     

Production of corticosterone in vitro: role of PGE2

The Authors have studied corticosteron output and tissue levels of cAMP during superfusion with ACTH and/or PGE2 before and after preincubation with indometacin (2 microgram/ml) in beef adrenal glands isolated and superfused with Ringer solution. The Authors on the basis of Mieir studies, conclude that PGE2 can be considered as fixed intermediares of the corticosteroidogenetic action of ACTH.     

Dissection of antibacterial and toxic activity of melittin: a leucine zipper motif plays a crucial role in determining its hemolytic activity but not antibacterial activity

Melittin, a naturally occurring antimicrobial peptide, exhibits strong lytic activity against both eukaryotic and prokaryotic cells. Despite a tremendous amount of work done, very little is known about the amino acid sequence, which regulates its toxic activity. With the goal of understanding the basis of toxic activity and poor cell selectivity in melittin, a leucine zipper motif has been identified. To evaluate the possible structural and functional roles of this motif, melittin and its two analogs, after substituting the heptadic leucine by alanine, were synthesized and characterized. Functional studies indicated that alanine substitution in the leucine zipper motif resulted in a drastic reduction of the hemolytic activity of melittin. However, interestingly, both the designed analogs exhibited antibacterial activity comparable to melittin. Mutations caused a significant decrease in the membrane permeability of melittin in zwitterionic but not in negatively charged lipid vesicles. Although both the analogs exhibited similar secondary structures in the presence of negatively charged lipid vesicles as melittin, they failed to adopt a significant helical structure in the presence of zwitterionic lipid vesicles. Results suggest that the substitution of heptadic leucine by alanine impaired the assembly of melittin in an aqueous environment and its localization only in zwitterionic but not in negatively charged membrane. Altogether, the results suggest the identification of a structural element in melittin, which probably plays a prominent role in regulating its toxicity but not antibacterial activity. The results indicate that cell selectivity in some antimicrobial peptides can probably be introduced by modulating their assembly in an aqueous environment.     

On the role of phylogeny in determining activity patterns of rodents

Evolutionary plasticity is limited, to a certain extent, by phylogenetic constraints. We asked whether the diel activity patterns of animals reflect their phylogenies by analyzing daily activity patterns in the order Rodentia. We carried out a literature survey of activity patterns of 700 species, placing each in an activity time category: diurnal, nocturnal, or active at both periods (a-rhythmic). The proportion of rodents active at these categories in the entire order, was compared to the activity patterns of species of different families for which we had data for over ten species each: Dipodidae, Echimyidae, Geomyidae, Heteromyidae, Muridae, and Sciuridae. Activity times of rodents from different habitat types were also compared to the ordinal activity time pattern. We also calculated the probability that two random species (from a particular subgroup: family, habitat, etc.) will be active in the same period of the day and compared it to this probability with species drawn from the entire order. Activity patterns at the family level were significantly different from the ordinal pattern, emphasizing the strong relationship between intra-family taxonomic affiliation and daily activity patterns. Large families (Muridae and Sciuridae) analyzed by subfamilies and tribes showed a similar but stronger pattern than that of the family level. Thus it is clear that phylogeny constrains the evolution of activity patterns in rodents, and may limit their ability to use the time niche axis for ecological separation. Rodents living in cold habitats differed significantly from the ordinal pattern, showing more diurnal and a-rhythmic activity patterns, possibly due to physiological constraints. Ground-dwelling rodents differed significantly, showing a high tendency towards a-rhythmic activity, perhaps reflecting their specialized habitat. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Androgen deprivation increases neuronal nitric oxide metabolism and its vasodilator effect in rat mesenteric arteries.

This study examines the effects of male sex hormones on the vasoconstrictor response to electrical field stimulation (EFS), as well as neuronal NO modulation of this response. For this purpose, denuded superior mesenteric artery from orchidectomized and control male Sprague-Dawley rats was used. EFS induced similar frequency-dependent contractions in segments from both groups. The NO synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester strengthened EFS-elicited contractions more in arteries from orchidectomized than from control male rats. The expression of nNOS was more pronounced in segments from control than from orchidectomized animals. Basal and EFS-induced NO release was similar in segments from both groups. In noradrenaline (NA)-precontracted segments, sodium nitroprusside (SNP) induced a concentration-dependent relaxation, that was greater in segments from orchidectomized than control male rats. 8-Bromo-cGMP induced a similar concentration-dependent relaxation in NA-precontracted segments from either group, and the cGMP levels induced by SNP were also similar in the two groups. Superoxide dismutase (SOD), a superoxide anion scavenger, did not modify the relaxation in segments from control male rats. In contrast, SOD enhanced the relaxation induced by SNP in segments from orchidectomized rats, and the effect was reversed by preincubation with SOD plus catalase. The generation of superoxide anion and of peroxynitrite was greater in segments from orchidectomized than control rats. In NA-precontracted segments from control or orchidectomized rats, exogenous peroxynitrite and H(2)O(2) induced a concentration-dependent relaxation. These results suggest that EFS induces a similar nNOS-derived NO release in segments from orchidectomized and control male rats, despite the decrease in nNOS expression in orchidectomized rats. The NO metabolism is higher in segments from orchidectomized male rats due to the increases in anion superoxide generation and peroxynitrite formation. The vasodilator effects of the peroxynitrite and H(2)O(2)0 generated from the NO metabolism are what enhance the functional role of the nNOS-derived NO release in the orchidectomized rats.     

Renal sympathetic nerve activity modulates afferent renal nerve activity by PGE2-dependent activation of alpha1- and alpha2-adrenoceptors on renal sensory nerve fibers

Increasing efferent renal sympathetic nerve activity (ERSNA) increases afferent renal nerve activity (ARNA). To test whether the ERSNA-induced increases in ARNA involved norepinephrine activating alpha-adrenoceptors on the renal sensory nerves, we examined the effects of renal pelvic administration of the alpha(1)- and alpha(2)-adrenoceptor antagonists prazosin and rauwolscine on the ARNA responses to reflex increases in ERSNA (placing the rat's tail in 49 degrees C water) and renal pelvic perfusion with norepinephrine in anesthetized rats. Hot tail increased ERSNA and ARNA, 6,930 +/- 900 and 4,870 +/- 670%.s (area under the curve ARNA vs. time). Renal pelvic perfusion with norepinephrine increased ARNA 1,870 +/- 210%.s. Immunohistochemical studies showed that the sympathetic and sensory nerves were closely related in the pelvic wall. Renal pelvic perfusion with prazosin blocked and rauwolscine enhanced the ARNA responses to reflex increases in ERSNA and norepinephrine. Studies in a denervated renal pelvic wall preparation showed that norepinephrine increased substance P release, from 8 +/- 1 to 16 +/- 1 pg/min, and PGE(2) release, from 77 +/- 11 to 161 +/- 23 pg/min, suggesting a role for PGE(2) in the norepinephrine-induced activation of renal sensory nerves. Prazosin and indomethacin reduced and rauwolscine enhanced the norepinephrine-induced increases in substance P and PGE(2). PGE(2) enhanced the norepinephrine-induced activation of renal sensory nerves by stimulation of EP4 receptors. Interaction between ERSNA and ARNA is modulated by norepinephrine, which increases and decreases the activation of the renal sensory nerves by stimulating alpha(1)- and alpha(2)-adrenoceptors, respectively, on the renal pelvic sensory nerve fibers. Norepinephrine-induced activation of the sensory nerves is dependent on renal pelvic synthesis/release of PGE(2).