The role of prostaglandin E2 and its receptors in the regulation of osmotic permeability of the frog bladder epithelium

Arginine-vasotocin-induced enhancement of osmotic water flow was inhibited by PGE2, sulprostone, M&B 28767 and 17-phenyl-omega-trinor PGE2. The inhibitory action of these agents except M&B 28767 was more obvious when the osmotic water flow was activated by dibutyryl-cAMP. The findings suggest that, in the frog urinary bladder, the inhibitory effect of the PGE2 on arginine-vasotocin-induced water flow appears to be mediated via two receptor subtypes coupled with different secondary messengers. The inhibition-sensitive targets are localised both on pre- and post-cAMP steps of the hormonal signal transduction pathway.     

Calciumdependent mechanisms in vasopressin regulation of osmotic water permeability in the mouse kidney collecting duct cells

In the study, the role of PKC and Ca++ in vasopressin regulation of the plasma membrane water permeability was studied in the cells of the mouse kidney collecting duct. Coefficient of osmotic water permeability of total cell surface (Pf) was calculated from the initial rate of cell swelling following the osmotic shock caused by changing the medium osmolarity from isotonic to hypotonic (300 mOsm to 200 mOsm). Desmopressin (dDAVP 1 nM) increased the Pf in hydrated mice from 168.4 +/- 11.8 microm/s up to 231.3 +/- 14.7 microm/s. The Ca++ chelator BAPTA prevented the desmopressin-induced increase in water permeability. Inhibition of PKC (Ro-31-8220 0.1 microM) also abolished the desmopressin-stimulated increase of plasma membrane water permeability, whereas inhibitor of PKC alone did not suppress the stimulation of the water permeability by db-cAMP. The PKC activity and calciumdependent second messengers seem to be important for regulation of water permeability by vasopressin.     

Role of prostaglandin E2 in regulation of urine excretion in saluresis, water and osmotic diuresis in rat

In the saluresis, water and osmotic diuresis were indicating an increase of prostaglandin E2 excretion and a correlation between this index and diuresis. Unselective blockade of cyclooxygenase by diclofenac-natrium leads to a decrease of diuresis in the observed types of urine-production in rats. Inhibition of inducible cyclooxygenase by celebrex didn't change the value of diuresis after water load or administration of osmotic agent, but decreased the diuretic effect of furosemide.     

Molecular mechanisms of sleep-wake regulation: a role of prostaglandin D2

Prostaglandin (PG) D2 is a major prostanoid in the brains of rats and other mammals, including humans. When PGD synthase (PGDS), the enzyme that produces PGD2 in the brain, was inhibited by the intracerebroventricular infusion of its selective inhibitors, i.e. tetravalent selenium compounds, the amount of sleep decreased both time and dose dependently. The amount of sleep of transgenic mice, in which the human PGDS gene had been incorporated, increased several fold under appropriate conditions. These data indicate that PGDS is a key enzyme in sleep regulation. In situ hybridization, immunoperoxidase staining and direct enzyme activity determination of tissue samples revealed that PGDS is hardly detectable in the brain parenchyma but is localized in the membrane systems surrounding the brain, namely, the arachnoid membrane and choroid plexus, from which it is secreted into the cerebrospinal fluid (CSF) to become beta-trace, a major protein component of the CSF. PGD2 exerts its somnogenic activity by binding to PGD2 receptors exclusively localized at the ventrorostral surface of the basal forebrain. When PGD2 was infused into the subarachnoid space below the rostral basal forebrain, striking expression of proto-oncogene Fos immunoreactivity (FosIR) was observed in the ventrolateral preoptic area (VLPO), a putative sleep centre, concurrent with sleep induction. Fos expression in the VLPO was positively correlated with the preceding amount of sleep and negatively correlated with Fos expression in the tuberomammillary nucleus (TMN), a putative wake centre. These observations suggest that PGD2 may induce sleep via leptomeningeal PGD2 receptors with subsequent activation of the VLPO neurons and downregulation of the wake neurons in the TMN area. Adenosine may be involved in the signal transduction associated with PGD2.     

Molecular basis of pH and Ca2+ regulation of aquaporin water permeability

Aquaporins facilitate the diffusion of water across cell membranes. We previously showed that acid pH or low Ca(2+) increase the water permeability of bovine AQP0 expressed in Xenopus oocytes. We now show that external histidines in loops A and C mediate the pH dependence. Furthermore, the position of histidines in different members of the aquaporin family can "tune" the pH sensitivity toward alkaline or acid pH ranges. In bovine AQP0, replacement of His40 in loop A by Cys, while keeping His122 in loop C, shifted the pH sensitivity from acid to alkaline. In the killifish AQP0 homologue, MIPfun, with His at position 39 in loop A, alkaline rather than acid pH increased water permeability. Moving His39 to His40 in MIPfun, to mimic bovine AQP0 loop A, shifted the pH sensitivity back to the acid range. pH regulation was also found in two other members of the aquaporin family. Alkaline pH increased the water permeability of AQP4 that contains His at position 129 in loop C. Acid and alkaline pH sensitivity was induced in AQP1 by adding histidines 48 (in loop A) and 130 (in loop C). We conclude that external histidines in loops A and C that span the outer vestibule contribute to pH sensitivity. In addition, we show that when AQP0 (bovine or killifish) and a crippled calmodulin mutant were coexpressed, Ca(2+) sensitivity was lost but pH sensitivity was maintained. These results demonstrate that Ca(2+) and pH modulation are separable and arise from processes on opposite sides of the membrane.     

Molecular mechanisms of sleep-wake regulation: roles of prostaglandins D2 and E2

Although sleep-wake cycles are repeated every day and night and almost one-third of our lifetime is spent sleeping, the molecular mechanisms of sleep-wake regulation have remained little understood. Recent experimental evidence indicates that prostaglandins (PG) D2 and E2 are probably two of the major endogenous sleep-regulating substances, one promoting sleep and the other wakefulness, in rats, dogs, rabbits, monkeys, and probably in humans as well. Preliminary evidence indicates that the sites of action of PGD2 and E2 are located in the sleep and wake centers in or near the preoptic area and posterior hypothalamus, respectively.     

A role for the kallikrein-kinin system in the regulation of osmotic water permeability in the toad bladder

Captopril (CA), a specific inhibitor of kininase II, did not alter osmotic water permeability (Posm) when present in the mucosal bath of the urinary bladder isolated from the toad Bufo arenarum at a concentration of 2.3 X 10(-3) M. This treatment, however, caused a 65% enhancement in the increase in Posm following serosal exposure to vasopressin, oxytocin or theophylline, agents that increase intracellular cyclic AMP levels. The effect of captopril was prevented by procedures that reduce the kallikrein (KK)-like alkaline esterase activity present in the bladder (such as simultaneous exposure to 2.3 X 10(-5) M aprotinin, or pretreatment of the toads with 0.1 N NaCl for several days before the experiment) or by replacing the mucosal bath with fresh solution of identical composition after exposure to captopril. In contrast, changing the serosal bath did not alter the effect of the drug. These results are consistent with an effect of CA at a step beyond cAMP generation, and suggest it is mediated by release of a soluble factor, probably a kinin, into the mucosal bath. These observations, together with data previously published, suggest that the KK-kinin system may participate in the control of epithelial water and electrolyte permeability in the toad bladder. In particular, under environmental stress, it may become important in the regulation of the animal's extracellular fluid volume, thus exhibiting an adaptive value.     

Epigenetic mechanisms regulate the prostaglandin E receptor 2 in breast cancer

The increase in local oestrogen production seen in oestrogen receptor positive (ER+) breast cancers is driven by increased activity of the aromatase enzyme. CYP19A1, the encoding gene for aromatase, is often overexpressed in the oestrogen-producing cells of the breast adipose fibroblasts (BAFs) surrounding an ER+ tumour, and the molecular processes underlying this upregulation is important in the development of breast-specific aromatase inhibitors for breast cancer therapy. Prostaglandin E2 (PGE2), a factor secreted by tumours, is known to stimulate CYP19A1 expression in human BAFs. The hormonal regulation of this process has been examined; however, what is less well understood is the emerging role of epigenetic mechanisms and how they modulate PGE2 signalling. This present study characterises the epigenetic processes underlying expression of the prostanoid receptor EP2 in the context of ER+ breast cancer. Sodium bisulphite sequencing of CpG methylation within the promoter region of EP2 revealed that an inverse correlation existed between methylation levels and relative EP2 expression in breast cancer cell lines MDA-MB-231, MCF7 and MCF10A but not in HS578t and T47D. Inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5aza) and histone deacetylation with Trichostatin A (TSA) resulted in upregulation of EP2 mRNA in all cell lines with varying influences of each epigenetic process observed. Expression of EP2 was detected in human BAFs despite a natively methylated promoter, and this expression was further increased upon 5aza treatment. An examination of 3 triple negative, 3 ductal carcinoma in situ and 3 invasive ductal carcinoma samples revealed that there was no change in EP2 promoter methylation status between normal and cancer associated stroma, despite observed differences in relative mRNA levels. Although EP2 methylation status is inversely correlated to expression levels in established breast cancer cell lines, we could not identify that such a correlation existed in tumour-associated stroma cells.     

Hormonal regulation of cell junction permeability: Upregulation by catecholamine and prostaglandin E1

Summary By cellular activation with hormones, we test the proposition (Loewenstein, W.R.,Physiol. Rev. 61:829, 1981) that the permeability of cell junction is upregulated through elevation of the level of cyclic AMP. Cultured rat glioma C-6 cells, with -adrenergic receptors, and human lung WI-38 cells, with prostaglandin receptors, were exposed to catecholamine (isoproterenol) and prostaglandin E₁, respectively, while their junctions were probed with microinjected fluorescent-labelled mono-, di-, and triglutamate. Junctional permeability, as indexed by the proportion of cell interfaces transferring the probes, rose after the hormone treatments. The increase in permeability took several hours to develop and was associated with an increase in the number of gap-junctional membrane particles (freeze-fracture electron microscopy). Such interaction between hormonal and junctional intercellular communication may provide a mechanism for physiological regulation of junctional communication and (perhaps as part of that) for physiological coordination of responses of cells in organs and tissues to hormones.     

Developmental regulation of prostaglandin E2 synthase in porcine ductus arteriosus

The synthesis of PGE(2), the major vasodilator prostanoid of the ductus arteriosus (DA), is catalyzed by PGE(2) synthases (PGES). The factors implicated in increased PGE(2) synthesis in the perinatal DA are not known. We studied the developmental changes of PGES along with that of cyclooxygenase (COX)-2 and cytosolic phospholipase A(2) (cPLA(2)) in the DA of fetal (75-90% gestation) and immediately postnatal newborn (NB) piglets. Levels of microsomal PGES (mPGES), COX-2, and PGE(2) in the DA of NB were approximately 7-fold higher than in fetus; activities of cytosolic PGES (cPGES) and cPLA(2) in DA of the fetus and NB did not differ. Because platelet-activating factor (PAF) could regulate COX-2 expression, the former was measured and found to be more abundant in the DA of the NB than of fetus. PAF elicited an increase in mPGES, COX-2, and PGE(2) in fetal DA to levels approaching those of the NB; cPGES, cPLA(2), and COX-1 were unaffected. In perinatal NB DA, PAF receptor antagonists BN-52021 and THG-315 reduced mPGES, COX-2, and PGE(2) levels and were associated with increased DA tone. It is concluded that PAF contributes in regulating DA tone by governing mPGES, COX-2, and ensuing PGE(2) levels in the perinate.     

Homologous regulation of prostaglandin E2 receptors in rat renal medulla

Prostaglandin (PG) receptors are present on enzymatically dissociated cells from the rat renal medulla and are subject to homologous regulation both in vivo and in vitro. One hour after injection of 100 micrograms of 16,16'-dimethyl-PGE2, the number of PGE2 binding sites on renal cells declines to 40% of controls. In vitro exposure of renal cells to PGE2 or dimethyl-PGE2 also results in a time- and concentration-dependent "down" regulation of prostaglandin receptors. In the absence of indomethacin in the incubation medium, endogenously synthesized prostaglandins mediate a similar time-dependent loss of cell-associated receptors. This loss is reversible since, after agonist removal and reincubation of the cells at 37 degrees C, there is a rapid (within 15 min) reappearance of PGE2 receptors (to 60-93% of controls). Reappearance occurs whether down regulation is induced in vitro by endogenously synthesized prostaglandins, added PGE2 or dimethyl-PGE2, or in vivo after injection of dimethyl-PGE2. Cycloheximide does not affect down regulation but significantly prevents subsequent recovery of the receptors. In contrast, neither colchicine nor chloroquine influences homologous regulation of renal prostaglandin receptors. These results document an agonist-induced reversible cycling of renal prostaglandin receptors which may determine the effectiveness of prostaglandin action in normal and pathologic states.     

Ontogeny and regulation of ovine placental prostaglandin E2 synthase

Recent evidence suggests that ovine placental output of prostaglandin (PG) E2 rises through late gestation partly because of a direct effect of cortisol on PGH2 synthase 2 (PGHS-2) expression and activity within trophoblast tissue. Synthesis of PGE2 is also dependent, however, on PGE2 synthase (PGES), which converts PGH2 to PGE2. We hypothesized that PGES is expressed in the ovine placenta, and that, similar to PGHS-2, expression increases through gestation and is regulated positively by cortisol. Placental tissues from pregnant ewes in mid and late gestation, at term, and during early and active labor were analyzed to determine the gestational profile of PGES. The regulation of PGES expression was assessed in placental tissues from pregnant ewes in which intrafetal cortisol infusion was administered in late gestation, in the presence or absence of an aromatase inhibitor, to block the cortisol-stimulated rise in estradiol. Expression of PGES was analyzed by in situ hybridization, Western blot analysis, and immunohistochemistry. In the placentome, PGES localized to fetal trophoblast cells and endothelial cells in maternal blood vessels, consistent with its contribution to the rise in placental PGE2 output toward the onset of labor and with a role of PGE2 in the local regulation of uteroplacental blood flow, respectively. Expression of PGES mRNA and protein increased with gestation. However, there was no significant further change with labor or during cortisol infusion in the presence or absence of a rise in fetal plasma estradiol, in contrast to reported changes in PGHS-2. These results suggest that PGES is not coregulated with PGHS-2 in the sheep placenta at term. The progressive increase in PGES, however, likely contributes to the rise in circulating PGE2 in the fetus in late pregnancy.     

An osmotic hypothesis for the regulation of oxygen permeability in soybean nodules

Nodule permeability (P) controls the amount of O₂ entering the nodule, and thereby the rates of both nodule respiration and N₂ fixation. P may be regulated by changes in the effective thickness of a water-filled diffusion barrier in the nodule cortex. Regulation of diffusion barrier thickness was hypothesized to result from changes in the water content of intercellular spaces. Modulation of intercellular water would be a response to osmotic potential gradients in the tissue. To test this hypothesis, preliminary experiments examined three classes of solutes (soluble sugars, free amino acids, and ureides) in nodules of intact plants exposed to 10 or 21 kPa O₂ for 24 h. Neither soluble sugars nor free amino acids in nodules were responsive to O₂ treatments. However, nodule ureides accumulated after exposure to 10kPa O₂ for 24 h. A symplastic increase in nodule ureides under the 10kPa O₂ treatment compared to the 21 kPa O₂ treatment may have removed water from intercellular spaces in the nodule cortex and increased P. In addition, the nodule cortex of intact plants was infiltrated with water, polyethylene glycol (PEG), KC1, or Na-succinate solutions to determine the effect of intercellular water and osmoticants on dinitrogenase activity and P. Results from infiltrating the apoplast of the nodule cortex with osmotic solutions indicated that both increases in intercellular water and decreases in the apoplastic water potential decrease dinitrogenase activity and P. Furthermore, the inability to recover dinitrogenase activity and P following the infiltration of the cortex with PEG compared to either KCl or Na-succinate treatments may indicate that recovery was dependent upon removal of the solute from the apoplast.     

Dynamic changes in the osmotic water permeability of protoplast plasma membrane

The osmotic water permeability coefficient (P(f)) of plasma membrane of maize (Zea mays) Black Mexican Sweet protoplasts changed dynamically during a hypoosmotic challenge, as revealed using a model-based computational approach. The best-fitting model had three free parameters: initial P(f), P(f) rate-of-change (slope(P(f))), and a delay, which were hypothesized to reflect changes in the number and/or activity of aquaporins in the plasma membrane. Remarkably, the swelling response was delayed 2 to 11 s after start of the noninstantaneous (but accounted for) bath flush. The P(f) during the delay was < or =1 microm s(-1). During the swelling period following the delay, P(f) changed dynamically: within the first 15 s P(f) either (1) increased gradually to approximately 8 microm s(-1) (in the majority population of low-initial-P(f) cells) or (2) increased abruptly to 10 to 20 microm s(-1) and then decreased gradually to 3 to 6 microm s(-1) (in the minority population of high-initial-P(f) cells). We affirmed the validity of our computational approach by the ability to reproduce previously reported initial P(f) values (including the absence of delay) in control experiments on Xenopus oocytes expressing the maize aquaporin ZmPIP2;5. Although mercury did not affect the P(f) in swelling Black Mexican Sweet cells, phloretin, another aquaporin inhibitor, inhibited swelling in a predicted manner, prolonging the delay and slowing P(f) increase, thereby confirming the hypothesis that P(f) dynamics, delay included, reflected the varying activity of aquaporins.     

Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis

Here we report the molecular identification of cytosolic glutathione (GSH)-dependent prostaglandin (PG) E(2) synthase (cPGES), a terminal enzyme of the cyclooxygenase (COX)-1-mediated PGE(2) biosynthetic pathway. GSH-dependent PGES activity in the cytosol of rat brains, but not of other tissues, increased 3-fold after lipopolysaccharide (LPS) challenge. Peptide microsequencing of purified enzyme revealed that it was identical to p23, which is reportedly the weakly bound component of the steroid hormone receptor/hsp90 complex. Recombinant p23 expressed in Escherichia coli and 293 cells exhibited all the features of PGES activity detected in rat brain cytosol. A tyrosine residue near the N terminus (Tyr(9)), which is known to be critical for the activity of cytosolic GSH S-transferases, was essential for PGES activity. The expression of cPGES/p23 was constitutive and was unaltered by proinflammatory stimuli in various cells and tissues, except that it was increased significantly in rat brain after LPS treatment. cPGES/p23 was functionally linked with COX-1 in marked preference to COX-2 to produce PGE(2) from exogenous and endogenous arachidonic acid, the latter being supplied by cytosolic phospholipase A(2) in the immediate response. Thus, functional coupling between COX-1 and cPGES/p23 may contribute to production of the PGE(2) that plays a role in maintenance of tissue homeostasis.     

Inhibition of prostaglandin E2 formation and histamine action in cancer immunotherapy

The activity of cell-mediated defense systems is stimulated by consecutive formation of interleukin-1 (IL-1), interleukin-2 (IL-2) and interferon (IFN). The system is inhibited by interleukin-4 (IL-4) and also by prostaglandin E₂ (PGE₂) and histamine, which are released when the immune system is activated. The inhibition is strong in cancer patients, because PGE₂ is formed in many cancer cells and its formation is stimulated by IL-1. The release of histamine is also stimulated by IL-1. Tus PGE₂ and histamine are feedback inhibitors of cell-mediated immunity. This inhibition can be abolished by inhibitors of the cyclo-oxygenase (e. g. indomethacin) and H-2 receptor antagonists (e. g. cimetidine). This may offer a new option to stimulate the immune system to kill cancer cells.     

Molecular mechanisms of glutamine action

Glutamine is the most abundant free amino acid in the body and is known to play a regulatory role in several cell specific processes including metabolism (e.g., oxidative fuel, gluconeogenic precursor, and lipogenic precursor), cell integrity (apoptosis, cell proliferation), protein synthesis, and degradation, contractile protein mass, redox potential, respiratory burst, insulin resistance, insulin secretion, and extracellular matrix (ECM) synthesis. Glutamine has been shown to regulate the expression of many genes related to metabolism, signal transduction, cell defense and repair, and to activate intracellular signaling pathways. Thus, the function of glutamine goes beyond that of a simple metabolic fuel or protein precursor as previously assumed. In this review, we have attempted to identify some of the common mechanisms underlying the regulation of glutamine dependent cellular functions.