Cryobiology and fertility preservation: a perspective on past, current and future studies.

Cryopreservation has been used over many decades for the maintenance of viable biological specimens. Its expansion into the area of fertility preservation has been a natural outcome of the increased risks to human fertility from diseases, such as cancer and its treatment protocols, including radiation and chemo-therapy, and the general lifestyle trend to later marriages. The use of assisted reproductive techniques (ART) in preserving fertility have benefitted significantly from new scientific approaches, such as cryostorage, in which live cells and tissues are stored at low temperatures and revived when necessary. This review focuses on "cryopreservation science monitoring in reproductive biomedicine" to evaluate knowledge, trends, driving forces, impetus, and emerging technologies in order to draw a future roadmap for this field. Our analysis of the field of cryobiology emphasizes the significance of strategic planning of cryobiology research to support more its extensive use in therapeutics in the future. The Royan Institute (Tehran, Iran) recognises this need and has developed a strategic plan to engage in multidisciplinary research on the application of cryobiology, including cryobioengineering, in disease mitigation. We hoped that this study can help improve the quality and quantity of public discourse and expert awareness of the role for cryopreservation in fertility preservation within ART. DOI: 10.54680/fr23410110112.

Atrial and brain natriuretic peptides stimulate the production and secretion of C-type natriuretic peptide from bovine aortic endothelial cells.

C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family which is produced in vascular endothelial cells and may play an important paracrine role in the vasaculature. We sought to determine the regulation of CNP production by other vasoactive peptides from cultured aortic endothelial cells. The vasoconstrictors endothelin-1 and angiotensin II had little effect on the basal secretion of CNP. In contrast, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) strongly stimulated the secretion of CNP. BNP caused as much as a 400-fold enhancement above the basal accumulated secretion of CNP over 24 h at a concentration of 1 microM; this was 20 times greater than the stimulatory effect of ANP, BNP and ANP also significantly enhanced the production of new CNP protein (translation) and mRNA expressed in the BAEC. In contrast, C-ANP-4-23, a truncated form of ANP which selectively binds to the natriuretic peptide clearance receptor, did not stimulate CNP secretion. The enhanced production and secretion of CNP, caused by either ANP or BNP, was significantly prevented by LY 83583, an inhibitor of cGMP generation, and was also attenuated by KT 5823, an inhibitor of cGMP-dependent protein kinase. Our results indicate that ANP and BNP can stimulate CNP production through a guanylate cyclase receptor on endothelial cells. BNP is a much more potent stimulator of CNP secretion, compared to ANP. Our findings suggest that the vasodilatory, and anti-mitogenic effects of ANP and BNP in the vasculature could occur in part through CNP production and subsequent action if these interactions occur in vivo.

High density lipoproteins stimulate the production and secretion of endothelin-1 from cultured bovine aortic endothelial cells.

The concentration of HDL in the blood inversely correlates with the incidence of cardiovascular disease, probably related to the ability of these lipoproteins to efflux cholesterol from vascular cells. it is also possible that HDL affect the production or action of vasoactive peptides implicated in the development of vascular diseases. Therefore, we determined the effects of human HDL on the production and secretion of endothelin-1 (ET-1) from cultured bovine aortic endothelial cells. HDL produced a highly significant stimulation of endothelin secretion (maximum 240% of control), even at very low levels of lipoproteins (1 microgram/ml). HDL also stimulated the translation of ET-1 by twofold in the bovine aortic endothelial cells. In contrast, HDL had no significant effect on steady state mRNA levels, transcript degradation, or transcription. Stimulation of ET-1 secretion by HDL was dependent on protein kinase C activation. Purified apo A-I, the major apoprotein of HDL, increased ET-1 secretion and translation approximately 85% as potently as HDL. Our results indicate that low concentrations of human HDL strongly stimulate the production of ET-1, a powerful vasoconstrictor and mitogen for the vascular smooth muscle cell. We propose that HDL may participate in the regulation of vasomotor tone through this potentially important effect in the vasculature.

Role of vascular endothelial cell growth factor in Ovarian Hyperstimulation Syndrome.

Controlled ovarian hyperstimulation with gonadotropins is followed by Ovarian Hyperstimulation Syndrome (OHSS) in some women. An unidentified capillary permeability factor from the ovary has been implicated, and vascular endothelial cell growth/permeability factor (VEGF) is a candidate protein. Follicular fluids (FF) from 80 women who received hormonal induction for infertility were studied. FFs were grouped according to oocyte production, from group I (0-7 oocytes) through group IV (23-31 oocytes). Group IV was comprised of four women with the most severe symptoms of OHSS. Endothelial cell (EC) permeability induced by the individual FF was highly correlated to oocytes produced (r2 = 0.73, P < 0.001). Group IV FF stimulated a 63+/-4% greater permeability than FF from group I patients (P < 0. 01), reversed 98% by anti-VEGF antibody. Group IV fluids contained the VEGF165 isoform and significantly greater concentrations of VEGF as compared with group I (1,105+/-87 pg/ml vs. 353+/-28 pg/ml, P < 0. 05). Significant cytoskeletal rearrangement of F-actin into stress fibers and a destruction of ZO-1 tight junction protein alignment was caused by group IV FF, mediated in part by nitric oxide. These mechanisms, which lead to increased EC permeability, were reversed by the VEGF antibody. Our results indicate that VEGF is the FF factor responsible for increased vascular permeability, thereby contributing to the pathogenesis of OHSS.

Differential effect of neuraminidase-treatment on the surface charge-associated properties of rat reticulocytes and erythrocytes. Studies by partitioning in two-polymer aqueous phases.

Rat reticulocytes undergo charge-associated surface changes, detectable by cell partitioning in charged dextran-poly(ethylene glycol) aqueous phase systems, as they become mature erythrocytes. Young reticulocytes have a lower partition coefficient, i.e., quantity of cells in the top phase as a percentage of total cells added, than do mature erythrocytes. Sialic acid is the main charge-bearing group on red blood cells and, in the case of the rat, most of the sialic acid can be removed by treatment of the cells with neuraminidase (Vibrio cholerae). By combining isotopic 59Fe-labeling of reticulocytes with countercurrent distribution of the entire red blood cell population in charged dextran-poly(ethylene glycol) aqueous phases we have now studied the relative effect of neuraminidase-treatment on rat reticulocytes and mature erythrocytes. It was found that neuraminidase-treatment (a) does not eliminate surface differences, detectable by partitioning, between rat reticulocytes and erythrocytes and (b) reduces the partition coefficient of mature erythrocytes to a greater extent than the partition coefficient of reticulocytes indicating a differential effect of this enzyme on the two cell populations.

Effect of surface modification of rat erythrocytes of different ages on their partitioning behavior in charge-sensitive two-polymer aqueous phases.

Partitioning differences between cells in two-polymer aqueous phase systems originate from subtle differences between the surface properties of cells. Because of the exponential relation between the parameters affecting the partition ratio (P) and the P itself, differences in membrane components suspected of effecting the differential partitioning of closely related cell populations cannot be directly established by conventional chemical assay techniques. In order to study the chemical nature of the components responsible for the age-related changes in surface properties of rat red cells we have devised an approach which uses a combination of isotopic labeling of erythrocyte subpopulations of distinct cell age with different enzyme and/or chemical treatments followed by countercurrent distribution in charge-sensitive two-polymer aqueous phase systems. These studies show that: neuraminidase-susceptible sialic acid is not responsible for the cell age-related surface differences detected by partitioning; the component(s) responsible for the cell age-related surface differences can be extracted (from aldehyde-fixed red cells) with ethanol or cleaved with dilute sulfuric acid. Our data are consistent with the hypothesis that ganglioside-linked sialic acid is the chemical moiety responsible for the cell charge-associated surface differences among rat red blood cells of different ages.

Insulin stimulates production and secretion of endothelin from bovine endothelial cells.

Endothelin, a vasoconstrictor peptide secreted from endothelial cells, has been thought to play a role in various forms of vascular disease. Diabetes mellitus is well known for its association with accelerated atherosclerosis and microvascular damage. Although the basis for the vessel insult is multifactorial, hyperinsulinemia is thought to contribute by an unknown mechanism. In this study, we sought to determine whether insulin stimulates the production and secretion of ET-1 as a possible basis for the association of hyperinsulinemia and vascular disease. We demonstrated that insulin significantly stimulates the gene expression and secretion of ET-1 from cultured BAEC, and that insulin increases ET-1 mRNA expressed in BBCEC. Insulin caused a maximal twofold inducement above control ET-1 mRNA expression in a dose-related fashion in BAEC. The increased mRNA resulted from increased transcription, as determined by nuclear run-off studies. Increased ET-1 mRNA was seen after 4 h of incubation with insulin: the peak occurred at 6-8 h and persisted for 24 h. Insulin caused as much as a fourfold stimulation of ET-1 secretion from BAEC in a dose-related fashion, including a twofold increase at a physiological concentration (10(-9) M): The increase began at 1 h of incubation and continued for the entire 24-h incubation period. The insulin-induced increases in both ET-1 mRNA and ET-1 protein secretion were significantly attenuated by genistein, a tyrosine kinase inhibitor. This stimulation probably occurred through the insulin receptor, because IGF-1 had no effect on ET-1 gene expression or secretion from these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma membrane estrogen receptors signal to antiapoptosis in breast cancer.

Chemotherapy or irradiation treatment induces breast cancer cell apoptosis, but this can be limited by estradiol (E2) through unknown mechanisms. To investigate this, we subjected estrogen receptor-expressing human breast cancer cells (MCF-7 and ZR-75-1) to paclitaxel (taxol) or to UV irradiation. Marked increases in cell apoptosis were induced, but these were significantly reversed by incubation with E2. Taxol or UV stimulated c-Jun N-terminal kinase (JNK) activity, which was inhibited by E2. Expression of a dominant-negative Jnk-1 protein strongly prevented taxol- or UV-induced apoptosis, whereas E2 inhibition of apoptosis was reversed by expression of constituitively active Jnk-1. As targets for participation in apoptosis, Bcl-2 and Bcl-xl were phosphorylated in response to JNK activation by taxol or UV; this was prevented by E2. Taxol or UV activated caspase activity in a JNK-dependent fashion and caused the cleavage of procaspase-9 to caspase-9, each inhibited by E2. Independently, the steroid also activated extracellular signal-regulated protein kinase activity, which contributed to the antiapoptotic effects. We report novel and rapid mechanisms by which E2 prevents chemotherapy or radiation-induced apoptosis of breast cancer, probably mediated through the plasma membrane estrogen receptor.

Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ERalpha and ERbeta expressed in Chinese hamster ovary cells.

The existence of a putative membrane estrogen receptor (ER) has been supported by studies accomplished over the past 20 yr. However, the origin and functions of this receptor are not well defined. To study the membrane receptor, we transiently transfected cDNAs for ERalpha or ERbeta into Chinese hamster ovary (CHO) cells. Transfection of ERalpha resulted in a single transcript by Northern blot, specific binding of labeled 17beta-estradiol (E2), and expression of ER in both nuclear and membrane cell fractions. Competitive binding studies in both compartments revealed near identical dissociation constants (K(d)S) of 0.283 and 0.287 nM, respectively, but the membrane receptor number was only 3% as great as the nuclear receptor density. Transfection of ERbeta3 also yielded a single transcript and nuclear and membrane receptors with respective Kd values of 1.23 and 1.14 nM; the membrane receptor number was only 2% compared with expressed nuclear receptors. Estradiol binding to CHO-ERalpha or CHO-ERbeta activated Galphaq and G(alpha)s proteins in the membrane and rapidly stimulated corresponding inositol phosphate production and adenylate cyclase activity. Binding by 17-beta-E2 to either expressed receptor comparably enhanced the nuclear incorporation of thymidine, critically dependent upon the activation of the mitogen-activated protein kinase, ERK (extracellular regulated kinase). In contrast, c-Jun N-terminal kinase activity was stimulated by 17-beta-E2 in ERbeta-expressing CHO, but was inhibited in CHO-ERalpha cells. In summary, membrane and nuclear ER can be derived from a single transcript and have near-identical affinities for 17-beta-E2, but there are considerably more nuclear than membrane receptors. This is also the first report that cells can express a membrane ERbeta. Both membrane ERs activate G proteins, ERK, and cell proliferation, but there is novel differential regulation of c-Jun kinase activity by ERbeta and ERalpha.

Lower Oncogenic Potential of Human Mesenchymal Stem Cells Derived from Cord Blood Compared to Induced Pluripotent Stem Cells.

BACKGROUND: In regenerative medicine, use of each of the mesenchymal stem cells derived from bone marrow, cord blood, and adipose tissue, has several cons and pros. Mesenchymal stem cells derived from cord blood have been considered the best source for precursor transplantation. Direct reprogramming of a somatic cell into induced pluripotent stem cells by over-expression of 6 transcription factors Oct4, Sox2, Klf4, lin28, Nanog, and c-Myc has great potential for regenerative medicine, eliminating the ethical issues of embryonic stem cells and the rejection problems of using non-autologous cells. OBJECTIVE: To compare reprogramming and pluripotent markers OCT4, Sox-2, c-Myc, Klf4, Nanog, and lin28 in mesenchymal stem cells derived from cord blood and induced pluripotent stem cells. METHODS: We analyzed the expression level of OCT4, Sox-2, c-Myc, Klf4, Nanog and lin28 genes in human mesenchymal stem cells derived from cord blood and induced pluripotent stem cells by cell culture and RT-PCR. RESULTS: The expression level of pluripotent genes OCT4 and Sox-2, Nanog and lin28 in mesenchymal stem cells derived from cord blood were significantly higher than those in induced pluripotent stem cells. In contrast to OCT-4A and Sox-2, Nanog and lin28, the expression level of oncogenic factors c-Myc and Klf4 were significantly higher in induced pluripotent stem cells than in mesenchymal stem cells derived from cord blood. CONCLUSION: It could be concluded that mesenchymal stem cells derived from human cord blood have lower oncogenic potential compared to induced pluripotent stem cells.

Natriuretic peptides suppress vascular endothelial cell growth factor signaling to angiogenesis.

Vascular endothelial cell growth factor (VEGF) is essential for angiogenesis. Atrial natriuretic peptide (ANP) inhibits the production of VEGF, but whether this important vascular peptide also inter- rupts VEGF signaling to angiogenesis is unknown. In cultured bovine aortic endothelial cells, VEGF significantly stimulated extracellular signal-regulated protein kinase activity and phosphorylation, which was inhibited 60% by coincubation with ANP or a natriuretic peptide clearance receptor specific ligand (NPRC), C-type NAP-(4-23) [C-ANP-(4-23)]. VEGF also stimulated c-Jun N-terminal kinase (JNK) and p38 activities/phosphorylation that were prevented by the two natriuretic peptides (NP). A specific NP guanylate cyclase (GC) receptor antagonist, HS-142-1, blocked the actions of ANP [but not those of C-ANP-(4-23)], supporting the involvement of both GC and NPRC receptors. VEGF and expression of constituitively active JNK each stimulated the synthesis of cyclin D1 and increased the activity of the cyclin-dependent kinase-4, which was inhibited 55% by ANP. VEGF induced endothelial cell proliferation and migration, which was significantly blocked by NP or by expressing a dominant negative JNK-1. VEGF stimulated human microvascular endothelial cells to form capillary tubes, which was significantly inhibited by expressing dominant negative JNK-1 and by NP. Therefore, VEGF induction of critical steps in angiogenesis is enhanced through JNK activation. The actions are significantly prevented by NP, which act through both the NPRC and GC receptors to block growth factor signaling. Thus, NP are candidate antiangiogenesis factors that inhibit both the synthesis and function of VEGF.

Insulin stimulates endothelin binding and action on cultured vascular smooth muscle cells.

Hyperinsulinemia has been implicated as a separate risk factor for the development of accelerated cardiovascular disease, but the mechanism is unknown. Recently, we and several other groups have shown that insulin stimulates the production and secretion of the vasoconstrictor peptide endothelin-1 (ET-1) from vascular endothelial cells, and hyperinsulinemia results in increased plasma ET levels in vivo. However, the interactive effects of diabetes, insulin, and glucose on ET target tissues, like those on vascular smooth muscle cells (VSMC), are not well defined. In these studies, we examined the effects of the diabetic factors on ET receptors and [3H]thymidine incorporation into cultured cells prepared from control, streptozocin-diabetic, insulin-treated diabetic, and hyperinsulinemic rats. Scatchard analysis of saturation binding studies revealed a 2-fold increase in ET receptor number in normal VSMC treated in vitro with insulin, whereas glucose had no significant effect. Neither treatment affected receptor affinity. Similarly, aortic smooth muscle cells, brain capillary pericytes, and kidney afferent arteriolar smooth muscle cells from rats made hyperinsulinemic in vivo each showed approximately a 2-fold increase in receptor number. This increase in receptor density probably resulted from the stimulation of receptor protein production, because insulin caused a maximal 2.3 +/- 0.3 (+/- SEM) fold increase in the ETA receptor mRNA expressed in cultured VSMC by 4 h. Both insulin and ET significantly increased thymidine incorporation in aortic VSMC, but ET-1 was much more potent in this regard. However, the combined effects of insulin plus ET-1 resulted in a 10-fold increase in this index of cell proliferation, significantly different from the effects of either peptide alone. We postulate that hyperinsulinemia in vivo may potentiate ET release and receptor-mediated action, thereby contributing to vascular disease in the setting of diabetes.

Estrogen and progesterone inhibit vascular smooth muscle proliferation.

Estrogen (E) has been identified in epidemiologic and prospective studies to protect against the development of cardiovascular disease in women. It is unclear whether progesterone (P) is similarly beneficial. The mechanisms by which E or P might act are incompletely defined. One possibility is that sex steroids inhibit the proliferation of vascular smooth muscle, an early/important event in vascular pathology. We examined the ability of E and P to inhibit the growth of human umbilical vein smooth muscle cells (hUVSMC) in culture, when stimulated by serum or the mitogen, endothelin-1 (ET-1). Serum and ET-1 stimulated hVSMC cell numbers by approximately 110% and 43% respectively, compared with control, after 3 days in culture. This stimulation was maximally reversed 75% by E and 64% by P. No synergistic or additive effects of the two steroids were found. ET-1 and serum stimulated mitogen-activated protein kinase (MAP-K) and MAP-kinase kinase activities, and these were critical for mitogenesis. Mitogen-stimulated MAP-kinase kinase and MAP-K activities were significantly inhibited by either E or P. The steroids also inhibited mitogen-stimulated c-fos and c-myc, downstream targets for MAP-K action. Critical signaling and molecular events through which mitogens stimulate VSMC proliferation can be significantly inhibited by E or P, providing a potential cellular mechanism for their vascular protective actions.

Insulin stimulates thyroid hormone receptor alpha gene expression in cultured bovine aortic endothelial cells.

Thyroid hormones and insulin regulate numerous cell processes and potentially interact through the transcriptional regulation of key genes. For instance, thyroid hormones stimulate the transcription of the fatty acid synthase and malic enzyme genes in chick embryonic hepatocytes, while insulin amplifies these effects. It is possible that insulin augments these actions of thyroid hormone by stimulating production of the thyroid hormone nuclear receptor (TR). In these studies, we examined the regulation of TR production/gene expression by insulin in bovine aortic endothelial cells (BAEC). We demonstrate that insulin significantly stimulates the gene expression of the TR alpha receptor, from BAEC. Insulin causes a maximal threefold induction above control TR alpha steady state mRNA levels in time and dose-related fashion in these cells. The increased mRNA mainly resulted from a twofold increase in transcription, as determined by nuclear run on. Insulin also increases thyroid receptor number and thyroid hormone binding, determined by Scatchard analysis of competitive inhibition binding studies. An established observation is that insulin can synergistically augment thyroid hormone-induced transcriptional activation of several important genes. It has also been previously determined that thyroid hormone action correlates closely to TR nuclear receptor number. Therefore, our studies, which show that insulin stimulates TR alpha production, suggests a potential mechanism whereby insulin can augment thyroid hormone transcriptional action.

Increased capillary permeability induced by human follicular fluid: a hypothesis for an ovarian origin of the hyperstimulation syndrome.

OBJECTIVE: To examine the effect of follicular fluid (FF) and peritoneal fluid (PF) from patients undergoing assisted reproductive technology procedures on endothelial barrier function. This was determined in vitro by measuring the permeability of filter-grown bovine aortic endothelial cell monolayers to a permeability marker. DESIGN: Endothelial cells obtained from bovine thoracic arotas were treated with collagenase solution and plated on millicell filters, on which they formed confluent monolayers. Flux rate was determined at 60 minutes by measuring the radioactive tracer (3H mannitol) permeating from the apical to the basolateral part of the filter. Fifty-eight samples of FF and PF, both from stimulated and natural cycles were analyzed and grouped according to the number of eggs retrieved. Follicular fluid and PF samples from natural cycles were used as controls. RESULTS: There was an augmentation in the permeability rate of both FF and PF from patients undergoing controlled ovarian hyperstimulation (COH) who responded with an increasing number of eggs compared with controls (51% and 39%, respectively). When analyzing samples from patients who responded with a low number of oocytes, no significant increase was observed. CONCLUSIONS: It is known that in OHSS, the increase in capillary permeability is related to the administration of gonadotropins, and is believed to be mediated by a vasoactive substance of ovarian origin. In this study, FF and PF from patients undergoing COH showed a significant increase in the permeability rate through endothelial cells in vitro. Based on these findings, it could be hypothesized that if the same events took place in vivo, the isolation of this factor from ovarian source could be of significant importance to elucidate the pathogenesis of OHSS.

Tamoxifen regulates cell fate through mitochondrial estrogen receptor beta in breast cancer.

Tamoxifen (TAM) has both cytostatic and cytotoxic properties for breast cancer. TAM engaged mitochondrial estrogen receptor beta (ERß) as an antagonist in MCF7-BK cells, increasing reactive oxygen species (ROS) concentrations from the mitochondria that were required for cytotoxicity. In part, this derived from TAM downregulating manganese superoxide dismutase (MnSOD) activity by causing the nitrosylation of tyrosine 34, thereby increasing ROS. ROS-activated protein kinase C delta and c-jun N-terminal kinases, resulting in the mitochondrial translocation of Bax and cytochrome C release. Interestingly, TAM failed to cause high ROS levels or induce cell death in MCF7-BK-TR cells due to stimulation of MnSOD activity through agonistic effects at mitochondrial ERß. In several mouse xenograft models, lentiviral shRNA-induced knockdown of MnSOD caused tumors that grew in the presence of TAM to undergo substantial apoptosis. Tumor MnSOD and mitochondrial ERß are therefore targets for therapeutic intervention to reverse TAM resistance and enhance a cell death response.

Esophageal cancer in northwestern Iran.

BACKGROUND: Esophageal cancer (EC) is one of the most frequent and serious cancers worldwide, but its geographic distribution is disparate. Northern Iran is known as one of the hot spots for EC, but there is inadequate evidence available regarding its characteristics in northwestern region of Iran. Therefore, this study aimed to describe some demographic and histopathologic features of EC in this region of Iran. MATERIALS AND METHODS: 166 hospital referral patients from a hospital in the northwestern region of Iran who underwent endoscopic biopsy for the chief complaint of dysphagia or odynophagia, and were admitted with the pathologic diagnosis of esophageal cancer during 3 years were enrolled in this study. RESULTS: The mean age of the patient was 61.8 ± 12.0 years old. Male/female ratio was 0.84. With respect to the site of tumor, tumor was located in cervical esophagus in 7 cases (4.2%), upper thoracic in 5 patients (3%), middle thoracic in 64 patients (38.6%), lowers thoracic in 68 cases (41%), and cardia in 22 cases (13.2%). There was a significant difference among the site of tumor in different age groups (P = 0.021) and different sex groups (P = 0.001). In men, EC usually involves the lower parts, whereas in women it usually involves the upper parts of esophagus. Squamous cell carcinoma was the most common type of EC in all age groups, but the prevalence rate of adenocarcinoma seems to increase with age (P = 0.045). CONCLUSIONS: Demographic and histopathologic pattern of esophageal cancer in northwestern region of Iran is different from its histopathologic pattern in western countries in accordance with other reports from Golestan province in north-eastern region of Iran.

16,16-Dimethyl Prostaglandin E2 modulation of endothelial monolayer paracellular barrier function.

Prostaglandins prevent gastrointestinal mucosal injury and promote healing following mucosal injury by various noxious agents. Preservation or repair of microvascular function appears to be crucial in these processes. The processes involved in prostaglandin-mediated repair and preservation of endothelial function are unclear. In the present study, we investigated the role of prostaglandins on endothelial paracellular barrier function using the filter-grown bovine aortic endothelial cell monolayers. Endothelial paracellular barrier function as assessed using a paracellular marker, mannitol. Prostaglandin analogs 16,16-dimethyl prostaglandin E2 (DMPGE2) and prostaglandin I2 (PGI2) caused an enhancement of endothelial monolayer paracellular barrier function as evidenced by a dose-dependent decrease in endothelial paracellular permeability. DMPGE2 induced enhancement of endothelial paracellular barrier function correlated directly with increasing intracellular cAMP levels. Agents which increase intracellular cAMP levels at different stages of cAMP amplification cascade including phosphodiesterase inhibitor (3-isobutyl-1 methylxanthine [IBMX]), membrane permeable cAMP (8-bromo cAMP), and adenylate cyclase activators (isoproterenol and forskolin) also produced enhancement in endothelial paracellular barrier function. DMPGE2 enhancement of paracellular barrier function correlated with dense accumulation of actin microfilaments near the intercellular junctions. IBMX, isoproterenol, forskolin, and 8-bromo cAMP also produced similar changes in endothelial actin microfilaments. Cytochalasin B prevented the DMPGE2 enhancement of paracellular barrier function. Indomethacin (INDO), a cyclooxygenase inhibitor, caused a dose-dependent increase in endothelial paracellular permeability. Pharmacologic doses of INDO resulted in condensation and disruption of actin microfilaments with formation of large paracellular openings or gaps between the adjacent cells. Pretreatment of endothelial monolayers with DMPGE2 prevented INDO-induced disturbance of actin microfilaments and paracellular barrier function. IBMX, isoproterenol, forskolin, and 8-bromo cAMP also prevented INDO-induced changes in actin microfilaments and paracellular barrier function. These findings indicate that DMPGE2 has a paracellular barrier enhancing effect on filter-grown endothelial monolayers. This effect appears to be mediated through intracellular cAMP and actin microfilaments.