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In hypertension, increased peripheral vascular resistance results from vascular dysfunction with or without structural changes (vessel wall remodeling and/or microvascular rarefaction). Humans with lower birth weight exhibit evidence of vascular dysfunction. The current studies were undertaken to investigate whether in utero programming of hypertension is associated with in vivo altered response and/or abnormal vascular structure. Offspring of Wistar dams fed a normal (CTRL) or low (LP)-protein diet during gestation were studied. Mean arterial blood pressure response to ANG II was significantly increased, and depressor response to sodium nitroprusside (SNP) infusions significantly decreased in male LP adult offspring relative to CTRL. No arterial remodeling was observed in male LP compared with CTRL offspring. Capillary and arteriolar density was significantly decreased in striated muscles from LP offspring at 7 and 28 days of life but was not different in late fetal life [day 21 of gestation (E21)]. Angiogenic potential of aortic rings from LP newborn (day of birth, PO) was significantly decreased. Striated muscle expressions (Western blots) of ANG II A[T.sub.1] receptor subtype, endothelial nitric oxide synthase, angiopoietin 1 and 2, Tie 2 receptor, vascular endothelial growth factor and receptor, and platelet-derived growth factor C at E21 and P7 were unaltered by antenatal diet exposure. In conclusion, blood pressure responses to ANG II and SNP are altered, and microvascular structural changes prevail in this model of fetal programming of hypertension. The capillary rarefaction is absent in the fetus and appears in the neonatal period, in association with decreased angiogenic potential. The study suggests that intrauterine protein restriction increases susceptibility to postnatal factors resulting in microvascular rarefaction, which could represent a primary event in the genesis of hypertension. microvascular rarefaction; angiotensin; nitric oxide

Developmental programming of hypertension is associated with vascular dysfunction characterized by impaired vasodilatation to nitric oxide, exaggerated vasoconstriction to ANG II, and microvascular rarefaction appearing in the neonatal period. Hypertensive adults have indices of increased oxidative stress, and newborns that were nutrient depleted during fetal life have decreased antioxidant defenses and increased susceptibility to oxidant injury. To test the hypothesis that oxidative stress participates in early life programming of hypertension, vascular dysfunction, and microvascular rarefaction associated with maternal protein deprivation, pregnant rats were fed a normal, low protein (LP), or LP plus lazaroid (lipid peroxidation inhibitor) isocaloric diet from the day of conception until delivery. Lazaroid administered along with the LP diet prevented blood pressure elevation, enhanced vasomotor response to ANG II, impaired vasodilatation to sodium nitroprusside, and microvascular rarefaction in adult offspring. Liver total glutathione was significantly decreased in LP fetuses, and kidney eight-isoprostaglandin F2alpha (8-isoPGF(2alpha)) levels were significantly increased in adult LP offspring; these modifications were prevented by lazaroid. Renal nitrotyrosine abundance and blood levels of 1,4-dihydroxynonene and 4-hydroxynonenal-protein adducts were not modified by antenatal diet exposure. This study shows in adult offspring of LP-fed dams prevention of hypertension, vascular dysfunction, microvascular rarefaction, and of an increase in indices of oxidative stress by the administration of lazaroid during gestation. Lazaroid also prevented the decrease in antioxidant glutathione levels in fetuses, suggesting an antenatal mild oxidative stress in offspring of LP-fed dams. These studies support the concept that perinatal oxidative insult can lead to permanent alterations in the cardiovascular system development.

Opposing effects have been ascribed to nitric oxide (NO) on retinal microvascular survival. We investigated whether changes in the redox state may contribute to explain apparent conflicting actions of NO in a model of oxygen-induced retinal vasoobliteration. Retinal microvascular obliteration was induced by exposing 7-day-old rat pups (P7) for 2 or 5 days to 80% O(2). The redox state of the retina was assessed by measuring reduced glutathione and oxidative and nitrosative products malondialdehyde and nitrotyrosine. The role of NO on vasoobliteration was evaluated by treating animals with nitric oxide synthase (NOS) inhibitors (N-nitro-l-arginine; L-NA) and by determining NOS isoform expression and activity; the contribution of nitrosative stress was also determined in animals treated with the degradation catalyst of peroxynitrite FeTPPS or with the superoxide dismutase mimetic CuDIPS. eNOS, but not nNOS or iNOS, expression and activity were increased throughout the exposure to hyperoxia. These changes were associated with an early (2 days hyperoxia) decrease in reduced glutathione and increases in malondialdehyde and nitrotyrosine. CuDIPS, FeTPPS, and L-NA treatments for these 2 days of hyperoxia nearly abolished the vasoobliteration. In contrast, during 5 days exposure to hyperoxia when the redox state rebalanced, L-NA treatment aggravated the vasoobliteration. Interestingly, VEGFR-2 expression was respectively increased by NOS inhibition after short-term (2 days) exposure to hyperoxia and decreased during the longer hyperoxia exposure. Data disclose that the dual effects of NO on newborn retinal microvascular integrity in response to hyperoxia in vivo depend on the redox state and seem mediated at least in part by VEGFR-2.

Intrauterine programming of hypertension is associated with evidence of increased renin-angiotensin system (RAS) activity. The current study was undertaken to investigate whether arterial baroreflex and blood pressure variability are altered in a model of in utero programming of hypertension secondary to isocaloric protein deprivation and whether activation of the RAS plays a role in this alteration. Pregnant Wistar rats were fed a normal-protein (18%) or low-protein (9%) diet during gestation, which had no effect on litter size, birth weight, or pup survival. Mean arterial blood pressure (MABP; 126 +/- 3 mm Hg 9% versus 108 +/- 4 mm Hg 18%; p < 0.05) and blood pressure variability were significantly greater in the adult offspring of the 9% protein-fed mothers. Arterial baroreflex control of heart rate, generated by graded i.v. infusion of phenylephrine and nitroprusside, was significantly shifted toward higher pressure; i.v. angiotensin-converting enzyme inhibitor normalized MABP and shifted the arterial baroreflex curve of the 9% offspring toward lower pressure without affecting the 18% offspring. For examining whether brain RAS is also involved in programming of hypertension, angiotensin-converting enzyme inhibitor and losartan (specific AT(1) receptor antagonist) were administered intracerebroventricularly; both significantly reduced MABP of the 9% but not the 18% offspring. Autoradiographic receptor binding studies demonstrated an increase in brain AT(1) expression in the subfornical organ and the vascular organ of the lamina terminalis in the 9% offspring. These data demonstrate a major tonic role of brain and peripheral RAS on hypertension associated with antenatal nutrient deprivation.

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.

F2-isoprostanes (F2-IsoP's) are biologically active prostanoids formed by free radical-mediated peroxidation of arachidonic acid. Four different F2-IsoP regioisomers (5-, 8-, 12-, and 15-series), each comprising eight racemic diastereomers, total 64 compounds. Information regarding the biological activity of IsoP's is largely limited to 15-F2t-IsoP (8-iso-PGF2alpha). We recently demonstrated that 15-F2t-IsoP and its metabolite, 2,3-dinor-5,6-dihydro-15-F2t-IsoP, evoked vasoconstriction and TXA2 generation in retina and brain microvasculature. We have now examined and compared the biological activities of a series of recently synthesized new 5-, 12-, and 15-series F2-IsoP isomers in pig retinal and brain microvasculature. We hereby show that other 15-series F2-IsoP isomers, 15-epi-15-F2t-IsoP, ent-15-F2t-IsoP, and ent-15-epi-15-F2t-IsoP, are also potent vasoconstrictors. The 12-series isomers tested, 12-F2t-IsoP and 12-epi-12-F2t-IsoP, also caused marked vasoconstriction. Of the 5-series isomers tested, 5-F2t-IsoP and 5-epi-5-F2t-IsoP possessed no vasomotor properties, whereas ent-5-F2t-IsoP caused modest vasoconstriction. The vasoconstriction of ent-5-F2t-IsoP, 12-F2t-IsoP, and 12-epi-12-F2t-IsoP was abolished by removal of the endothelium, by TXA2 synthase and receptor inhibitor (CGS12970, L670,596), and by receptor-mediated Ca2+ channel blockade (SK & F96365); correspondingly, these isomers increased TXB2 formation by activating Ca2+ influx (detected with fura 2-AM) through non-voltage-dependent receptor-mediated Ca2+ entry (SK & F96365 sensitive) in endothelial cells. In conclusion, as seen with 15-F2t-IsoP, ent-5-F2t-IsoP, 12-F2t-IsoP, and 12-epi-12-F2t-IsoP constricted both retinal and brain microvessels by inducing endothelium-dependent TXA2 synthesis. These new findings broaden the scope of our understanding regarding the potential involvement of F2-IsoP's as mediators of oxidant injury.

Oxidant stress contributes to the pathogenesis of hypoxic-ischemic encephalopathies. Platelet-activating factor (PAF) is generated during oxidant stress. We studied the vasomotor mode of actions of PAF on periventricular (PV) microvessels of fetal ( approximately 75% of term), newborn (1-3 days), and adult pigs. PAF constricted PV microvessels from fetal (29.27 +/- 2.6%) and newborn (22.14 +/- 3.2%) pigs but was ineffective in adults (<2.5%). Specific [(3)H]PAF binding was greater in fetus and newborn than in adults; a concordant developmental PAF-induced inositol phosphate formation was observed. PAF-induced vasoconstriction was abrogated by thromboxane A(2) (TXA(2)) synthase and receptor inhibitors, calcium channel blockers, and by removal of endothelium; vasoconstriction to TXA(2) mimetic U-46619 did not differ with age. Immunoreactive TXA(2) synthase expression and PAF-evoked TXA(2) formation revealed a fetus> newborn>adult profile. Thus the greater PAF-induced PV microvascular constriction in younger subjects seems attributable to greater PAF receptor density and mostly secondary to TXA(2) formation from endothelium. The resulting decrease in blood flow may contribute to the increased vulnerability of the PV brain regions to oxidant stress-induced injury in immature subjects.

We determined whether bimatoprost, which has been reported to act via putative prostamide receptors, could be hydrolyzed to its free acid (17-phenyl-PGF(2 alpha)), a potent FP receptor agonist, by human ocular tissue in vitro. We developed a gas chromatography/mass spectrometric method to measure 17-phenyl-PGF(2 alpha) levels at sub-picomolar levels. We then analyzed the amount of 17-phenyl-PGF(2 alpha) present after incubation of 50 microl Lumigan (0.03% bimatoprost) with eye tissue using this assay. We found that cornea, sclera, iris, and ciliary body, all rapidly hydrolyzed bimatoprost to 17-phenyl-PGF(2 alpha) with linear kinetics at a rate of 6.3, 2.0, 2.8, and 1.5 pmol mg tissue(-1) hr(-1), respectively. For cornea, sclera, and ciliary body, this linear rate of hydrolysis continued over a period of at least three hours, while iris-induced hydrolysis did not continue beyond one hour. Our findings suggest that bimatoprost can act as prodrug for FP receptor activation and questions the concept of a "prostamide receptor" agonist.

PGF2alpha is an important smooth muscle contractile agent that exerts significant effects on myometrium and is implicated in labor. THG113 was recently identified as a PGF2alpha receptor (FP) antagonist. We characterized the specificity and selectivity of THG113, tested its effects on PGF2alpha-induced smooth muscle contraction, and assessed its efficacy in a model of endotoxin (LPS)-induced preterm labor. [125I]THG113 bound specifically to FP-expressing but not to native (not expressing FP) HEK293 cells. In FP-expressing HEK293 cells, THG113 markedly reduced PGF2alpha-elicited phosphoinositide hydrolysis (IC50 27 nM). Similarly, PGF2alpha-evoked microvascular (retinal) contraction was noncompetitively blocked (by > 90%) by THG113. In contrast, contraction to agonists of homologous prostanoid receptors EP1 and TP (17-phenyl-trinor PGE2 and U46619) was unaffected (< 1%) by high concentrations of THG113 (100 micromol/L); THG113 (100 micromol/L) also did not affect contraction to numerous other agents including platelet activating factor, endothelin, and angiotensin II. Force and duration of PGF2alpha-evoked contractions of myometrial strips of pig (non-pregnant, luteal phase) and mouse (immediately postpartum) were markedly reduced by THG113. In an endotoxin-induced preterm mouse model, lipopolysaccharide (50 microg intraperitioneal) injection at 16 days' gestation resulted in 100% delivery within 15 h; in contrast, 70% of those treated with THG113 (1 mg/day) delivered > 24 h later (at 18 days' gestation; term: 19 days). In addition, in mice injected with lipopolysaccharide and treated 6 h later with THG113 (0.1 mg bolus followed by 1 mg/day) 40% delivered > 48 h later. Fetuses of pregnant mice treated with THG113 were born alive, had higher birth weights (1.6 +/- 0.1 v 1.4 +/- 0.05 g), and appeared healthy. This study describes an effective and selective noncompetitive FP antagonist, THG113, which significantly delays preterm delivery; this provides the basis for future investigations for its use in tocolysis.

Purpose: To test whether platelet-activating factor (PAF) directly causes retinovascular endothelial cell (EC) death., Methods: Retinovascular density was calculated in rat pups exposed to 80% O(2) from postnatal days (P)6 to P14 (to produce oxygen-induced retinopathy [OIR]), using the adenosine diphosphatase (ADPase) technique, in animals treated with distinct PAF receptor blockers (PCA-4248, BN52021, or THG315). PAF levels were then measured in the retinas. Viability of ECs from piglets and humans in response to C-PAF (a stable PAF analogue) was determined by the reduction of the tetrazolium salt 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) by viable cells, incorporation of propidium iodide (PI), TUNEL assay, and release of lactate dehydrogenase. Release of thromboxane (TX) was measured in the cell media., Results: PAF levels in retina were markedly increased by exposure of isolated rat retinas to H(2)O(2) (1 micro M) and of rat pups placed in 80% O(2). Exposure to 80% O(2) induced retinal vasoobliteration, which was equally significantly inhibited ( approximately 60%) by all PAF receptor blockers tested. C-PAF increased incorporation of PI by isolated rat retinal microvasculature. Also, C-PAF caused time- and concentration-dependent death of cultured retinal ECs, which was prevented by the PAF receptor antagonist CV-3988. This effect of C-PAF was selective on retinal and neurovascular ECs, but not on other ECs. DNA fragmentation (TUNEL) was hardly detected, and inhibition of apoptosis-related processes by nicotinamide, cyclosporin A, and Z-DEVD-FMK and Z-VAD-FMK (caspase inhibitors) barely protected against death in EC, whereas C-PAF increased release of lactate dehydrogenase, implying that necrosis is the nature of EC death. Finally, C-PAF-induced cell death was preceded by an increase in TXB(2) levels and was prevented by TXA(2) synthase inhibition (with CGS12970)., Conclusions: The data suggest PAF plays a major role in vasoobliteration in OIR by triggering death of neuroretinal microvascular ECs. The cell death seems to be mediated at least in part by TXA(2). These effects of PAF may participate in ischemic retinopathies such as diabetes and retinopathy of prematurity.

Purpose: Because prostaglandins (PGs) are implicated in acute hypercapnia-induced hyperemia, this study was conducted to test the hypothesis that prolonged hypercapnia may cause a sustained increase in retinal blood flow (RBF) through a PG-dependent induction of endothelial nitric oxide synthase (eNOS)., Methods: Time-dependent RBF (microsphere technique), PGE(2), nitrite (NO(2)(-)), and NOS protein (reduced nicotinamide adenine dinucleotide phosphate [NADPH]-diaphorase staining) production were measured in hypercapnia (6% CO(2))-treated piglets. From the same species, PGE(2), eNOS mRNA, NOS protein, and vasomotor responses were measured in eyecup preparations, as were Ca(2+) transients in neuroretinovascular endothelial cells., Results: Hypercapnia caused biphasic (at 0.5 hours and 6-8 hours) increases in RBF that were abolished with normalization of the pH. The early phase (0.5 hour) was associated with an increase in PGE(2) levels and the latter phase (6-8 hours) with an increase in NO(2)(-) and NOS protein. Inhibition of cyclooxygenase by diclofenac prevented the early and late increase in RBF. NOS inhibitor L-nitro-arginine prevented only the latter. Hypercapnic acidosis increased retinal PGE(2) levels and eNOS-dependent vasorelaxation ex vivo. The ex vivo time course of eNOS mRNA expression corresponded with the late-phase increase in RBF and was blocked by the transcription inhibitor actinomycin D and the receptor-operated Ca(2+) channel blocker SK&F96365. In neuroretinovascular cells, acidosis increased Ca(2+) transients, which were inhibited by SK&F96365, but not diclofenac., Conclusions: This study discloses a previously unexplored mechanism for late retinal hyperemia during sustained hypercapnia that appears secondary to the induced expression of eNOS mediated by PGE(2).

We reported upregulation of endothelial nitric oxide synthase (eNOS) by PGE(2) in tissues and presence of perinuclear PGE(2) receptors (EP). We presently studied mechanisms by which PGE(2) induces eNOS expression in cerebral microvessel endothelial cells (ECs). 16,16-Dimethyl PGE(2) and selective EP(3) receptor agonist M&B28767 increased eNOS expression in ECs and the NO-dependent vasorelaxant responses induced by substance P on cerebral microvessels. These effects could be prevented by prostaglandin transporter blocker bromcresol green and actinomycin D. EP(3) immunoreactivity was confirmed on plasma and perinuclear membrane of ECs. M&B28767 increased eNOS RNA expression in EC nuclei, and this effect was augmented by overexpression of EP(3) receptors. M&B28767 also induced increased phosphorylation of Erk-1/2 and Akt, as well as changes in membrane potential revealed by the potentiometric fluorescent dye RH421, which were prevented by iberiotoxin; perinuclear K(Ca) channels were detected, and their functionality corroborated by NS1619-induced Ca(2+) signals and nuclear membrane potential changes. Moreover, pertussis toxin, Ca(2+) chelator, and channel blockers EGTA, BAPTA, and SK&F96365, as well as K(Ca) channel blocker iberiotoxin, protein-kinase inhibitors wortmannin and PD 98059, and NF-kappaB inhibitor pyrrolidine dithiocarbamate prevented M&B28767-induced increase in Ca(2+) transients and/or eNOS expression in EC nuclei. We describe for the first time that PGE(2) through its access into cell by prostaglandin transporters induces eNOS expression by activating perinuclear EP(3) receptors coupled to pertussis toxin-sensitive G proteins, a process that depends on nuclear envelope K(Ca) channels, protein kinases, and NF-kappaB; the roles for nuclear EP(3) receptors seem different from those on plasma membrane.

Prostanoids exert significant effects on circulatory beds. They play a role in the response of the vasculature to adjustments in perfusion pressure and oxygen and carbon dioxide tension, and they mediate the actions of numerous factors. The role of prostanoids in governing circulation of the perinate is suggested to surpass that in the adult. Prostanoids are abundantly generated in the perinate. They have been implicated in autoregulation of blood flow as studied in brain and eyes. Prostaglandins are also dominant regulators of ductus arteriosus tone. The effects of these autacoids are mediated through specific G protein-coupled receptors. In addition to the pharmacological characterization of the prostanoid receptors, important advances in understanding the biology of these receptors have been made in the last decade. Their cloning and the development of animals with disrupted genes of these receptors have been very informative. The involvement of prostanoid receptors in the developing subject, especially on brain and ocular vasculature and on ductus arteriosus, has also begun to be investigated; the expression of these receptors changes with development. Some but not all of the ontogenic changes in these receptors are attributed to homologous regulation. Interestingly, in the process of elucidating their effects, functional perinuclear prostaglandin E2 receptors have been uncovered. This article reviews prostanoid receptors and addresses implications on the developing subject with attention to vascular physiology.

Microvascular degeneration is an important event in oxygen-induced retinopathy (OIR), a model of retinopathy of prematurity. Because oxidant stress abundantly generates thromboxane A2 (TxA2), we tested whether TxA2 plays a role in retinal vasoobliteration of OIR and contributes to such vascular degeneration by direct endothelial cytotoxicity. Hyperoxia-induced retinal vasoobliteration in rat pups (80% O2 exposure from postnatal days 5-14) was associated with increased TxB2 generation and was significantly prevented by TxA2 synthase inhibitor CGS-12970 (10 mg x kg(-1) x day(-1)) or TxA2-receptor antagonist CGS-22652 (10 mg x kg(-1) x day(-1)). TxA2 mimetics U-46619 (EC50 50 nM) and I-BOP (EC50 5 nM) caused a time- and concentration-dependent cell death of neuroretinovascular endothelial cells from rats as well as newborn pigs but not of smooth muscle and astroglial cells; other prostanoids did not cause cell death. The peroxidation product 8-iso-PGF2, which is generated in OIR, stimulated TxA2 formation by endothelial cells and triggered cell death; these effects were markedly diminished by CGS-12970. TxA2-dependent neuroretinovascular endothelial cell death was mostly by necrosis and to a lesser extent by apoptosis. The data identify an important role for TxA2 in vasoobliteration of OIR and unveil a so far unknown function for TxA2 in directly triggering neuroretinal microvascular endothelial cell death. These effects of TxA2 might participate in other ischemic neurovascular injuries.

We studied the mechanisms of retinal and choroidal vasorelaxation elicited by nitric oxide (NO) using piglet eyes. The NO donors sodium nitroprusside (SNP) and diethylamine-NONOate caused comparable concentration-dependent relaxation that was partially (approximately 40%) attenuated by the guanylate cyclase inhibitors methylene blue and LY83583 and reduced to a lesser extent (approximately 25%) by the inhibitor of cGMP-dependent kinase, KT 5823. In contrast, NO-induced dilatation (by NO donors and endogenous NO after stimulation with bradykinin) was substantially (approximately 70%) diminished by the KCa channel blockers tetraethylammonium (TEA), charybdotoxin, and iberiotoxin; by the cyclooxygenase inhibitors indomethacin and ibuprofen; by the prostaglandin I (PGI2) synthase inhibitor trans-2-phenyl cyclopropylamine (TPC); and by the removal of endothelium; whereas relaxation of endothelium-denuded vasculature to SNP was unaltered by indomethacin, TPC, and charybdotoxin but was nearly nullified by methylene blue and the Kv channel blocker 4-aminopyridine. NO donors significantly increased PGI2 synthesis and the putative PGI2 receptor-coupled second messenger cAMP, from ocular vasculature (retinal microvessels and choroidal perfusate), and this increase in PGI2 formation was markedly reduced by TPC, tetraethylammonium, charybdotoxin, and/or the removal of endothelium, but it was only slightly reduced by methylene blue and LY83583. Also, SNP and KCa channel openers NS1619 and NS004 caused an increase in PGI2 synthesis in cultured endothelial cells, which was virtually abolished by KCa blockers. Finally, vasorelaxation to a cGMP analogue, 8-bromo cGMP, and protein kinase G stimulant beta-phenyl-1,N2-etheno-8-bromoguanosine 3':5'-cyclic monophosphate was mostly Kv dependent and, in contrast to NO, largely unrelated to PGI2 formation. In conclusion, data indicate that NO-induced ocular vasorelaxation is partly mediated by cGMP through its action on smooth muscle, and more importantly, by stimulating PGI2 formation of endothelial origin via a mechanism mostly independent of guanylate cyclase, which involves the opening of a KCa channel.

Purpose: To determine the relative contribution of cyclooxygenase (COX)-1 and COX-2 in regulating prostaglandin (PG) E2 and PGF2alpha receptors (EP and FP, respectively) densities and their functions in retinal vasculature of neonatal pigs., Methods: Newborn pigs were treated intravenously every 8 hours for 48 hours with saline, 40 mg/kg nonselective COX inhibitor ibuprofen, 80 mg/kg COX-1 inhibitor valeryl salicylate, or 5 mg/kg DuP697 and 5 mg/kg NS398, COX-2 inhibitors. Retinal microvessel EP and FP receptor densities were measured by radioligand binding and receptor-coupled effects by determining second-messenger inositol 1,4,5-trisphosphate (IP3) and vasomotor responses. Retinal blood flow (RBF) response to incremental increases in blood pressure (BP) was measured by a microsphere technique., Results: Valeryl salicylate, DuP697, and NS398 reduced retinal PGE2 and PGF2alpha concentrations in the newborn by approximately half, whereas ibuprofen caused further reduction to levels observed in adults. Retinal vessel EP1, EP3, and FP receptor densities increased approximately threefold after treatments with COX-1 or COX-2 inhibitors, and five- to sixfold after ibuprofen treatment. EP and FP receptor upregulation was associated with corresponding increases in IP3 production and retinal vasoconstriction in response to PGF2alpha, fenprostalene (an FP agonist), PGE2, 17-phenyl trinor PGE2 (an EP1 agonist), and M&B28,767 (an EP3 agonist) and with enhanced RBF autoregulation of high BP (> or =125 mm Hg). Conversely, EP2 receptor density and coupled functions were minimally affected by COX inhibition., Conclusions: Data suggest that increased COX-1- and COX-2-catalyzed prostaglandin synthesis contribute equivalently to the downregulation of retinovascular EP1, EP3, and FP receptors and their vasoconstrictor functions in newborn pigs; the EP2 receptor was not significantly influenced by ontogenic alterations in prostaglandin levels.

Ibuprofen, a cyclooxygenase (COX) inhibitor nonselective for either COX-1 or COX-2 isoform, upregulates cerebrovascular prostaglandin E2 (PGE2) and PGF2alpha receptors in newborn pigs. COX-2 was shown to be the predominant form of COX and the main catalyst of prostaglandin synthesis in the newborn brain. We proceeded to establish direct evidence that COX-2-generated prostaglandins govern PGE2 and PGF2alpha receptor density and function in the cerebral vasculature of the newborn. Hence, we determined PGE2 and PGF2alpha receptor density and functions in brain vasculature by using newborn pigs treated with saline, ibuprofen, COX-1 inhibitor (valerylsalicylate), or COX-2 inhibitors (DUP-697 and NS-398). Newborn brain PGE2 and PGF2alpha concentrations were significantly reduced by ibuprofen, DUP-697, and NS-398 but not by valerylsalicylate. In newborn pigs treated with DUP-697, NS-398, and ibuprofen, PGE2 and PGF2alpha receptor densities in brain microvessels were increased to adult levels; there was also a significant increase in inositol 1,4,5-trisphosphate (IP3) production and cerebral vasoconstrictor effects of 17-phenyl trinor PGE2 (EP1 receptor agonist), M&B-28767 (EP3 receptor agonist), PGF2alpha, and fenprostalene (PGF2alpha analog). Treatment with ibuprofen or DUP-697 also increased the upper blood pressure limit of cerebral cortex and periventricular blood flow autoregulation from 85 to > or = 125 mmHg (uppermost blood pressure studied). However, valerylsalicylate treatment did not affect cerebrovascular PGE2 and PGF2alpha receptors, IP3 production, or vasoconstrictor effects in newborn animals. These in vivo and in vitro observations indicate that COX-2 is mainly responsible for the regulation of PGE2 and PGF2alpha receptors and their functions in the newborn cerebral vasculature.

Ontogenic changes in choroidal vascular prostaglandin E2 (PGE2) receptors (EP1, EP2, EP3, and EP4), changes in receptor-coupled functions, and the possible role of high perinatal prostaglandin levels in regulating expression and function of these receptors were studied. PGE2 receptors and their functions on choroidal tissues were characterized by radioligand binding; by measurements of second messengers to receptor stimulation; and by vasomotor response to EP1, EP2, EP3, and EP4 ligands on perfused choroidal vascular beds from saline- and ibuprofen-treated (40 mg/kg every 4 every 4 hours for 48 hours) newborn pigs and from adult animals. PGE2 as well as EP2- and EP4-attributed choroidal stimulation elicited greater vasorelaxation in the saline-treated newborn and was associated with higher nitrite (oxidation product of NO, N omega-nitro-L-arginine inhibitable) production than in adult tissues. In contrast, EP1 and EP3 stimulation caused significantly more constriction in the adult than in the newborn, and this was associated with increased production of inositol 1,4,5-trisphosphate (IP3) and greater reduction of cAMP synthesis in the adult. Maximum [3H]PGE2 binding was also higher (3-fold) in adult than in newborn tissues. Competition binding studies revealed that of the PGE2 receptors in the adult choroid, approximately 55% were of the EP1 subtype, 8% were EP2, 22% were EP3, and 15% were EP4. Newborn choroid contained approximately 33% each of EP1 and EP2 receptors, 20% of EP3, and 15% of EP4. Inhibition of endogenous prostaglandin synthesis for 48 hours with ibuprofen in newborns to attain levels found in the adult resulted in an upregulation of [3H]PGE2 binding, EP1- and EP3-mediated vasoconstriction, and increases and decreases in IP3 and cAMP production, respectively, in newborn tissues compared with adult tissues. On the other hand, ibuprofen treatment of newborns led to a decrease in PGE2- and EP4-mediated vasorelaxation and nitrite synthesis (associated with decreased expression of endothelial NO synthase) to levels observed in adults: EP2-elicited responses in newborns were not affected by ibuprofen. In conclusion, fewer EP1 receptors (associated with vasoconstriction), more EP2 receptors, and greater EP4-coupled NO production (coupled to vasorelaxation) seem to be responsible for the increased vasodilation to PGE2 in the newborn. The decrease in prostaglandin levels with age appears to cause, on one hand, upregulation of EP1 and EP3 receptors and receptor-coupled vasoconstriction and, on the other hand, decreased EP4-coupled NO synthesis and choroidal vasodilation. Altogether, these factors result in increased vasorelaxation to PGE2 in the newborn compared with the adult. These findings may help to explain the inability of the newborn to autoregulate choroidal blood flow.

We performed open-circuit perfusions of newborn and adult pig eyes to study the age-dependent metabolism of 4,7,10,13,16,19-docosahexaenoic acid (DHA) in this organ. DHA taken up by the perfused eyes was partitioned into glycerolipids, beta-oxidation, and the intracellular nonesterified fatty acid pool. In newborn eyes, DHA was incorporated into structural lipids to a greater extent than in adult eyes. Competition experiments suggest that the adult eye is more selective for DHA than the newborn eye. Finally, pulse-chase data indicate that DHA transport from the circulation across the retinal pigment epithelium and into the retina is more rapid in adult than in newborn eyes. The results are discussed with respect to the rapid accumulation of retinal DHA in early life and the avid retention of this fatty acid by the adult retina.

The density of PGF2 alpha (FP) and PGE2 (EP) receptors on the cerebral microvasculature of the newborn is less than on that of the adult animal. High levels of prostaglandins in the newborn brain could be responsible for down-regulation of FP and EP receptors and their functions in the cerebral microvasculature. Cerebrovascular FP and EP receptor density, receptor-coupled second messenger production and cerebral vasoconstrictor responses to PGF2 alpha and PGE2 were studied in newborn pigs (1 to 2 days old) treated intravenously with ibuprofen (40 mg/kg every 6 hours for 48 hours) or saline, and compared with adults. Ibuprofen treatment in the newborn increased brain microvascular FP and EP receptor densities to levels found in that of adult pigs. Likewise brain microvessel inositol 1,4,5-triphosphate production in response to PGF2 alpha, fenprostalene (PGF2 alpha analog), PGE2, 17-phenyl trinor PGE2 (EP1 receptor subtype agonist) and M&B 28,767 (EP3 agonist) was greater in ibuprofen-treated newborn pigs; the latter was associated with increased vasoconstriction to these agents to levels comparable with those of adults. Steady-state levels of FP receptor mRNA in cerebral microvas-culature did not differ between saline-treated newborn, and ibuprofen-treated newborn and adult pigs. It is concluded that the low FP and EP receptor densities and receptor-coupled functions on newborn brain microvessels seem to be secondary to high levels of brain prostaglandins; the changes in receptor levels do not seem to be related to steady-state levels of receptor mRNA in brain microvessels.

The role of nitric oxide (NO) as well as its interaction with prostaglandins (PG) in setting the limits of autoregulation of retinal blood flow (RBF) and choroidal blood flow (ChBF) were studied in newborn pigs (1-5 d old). Blood flows were measured by the microsphere technique. Low and high ocular perfusion pressures (OPP) were induced by inflating balloon-tipped catheters placed at the aortic root and isthmus, respectively. Animals were treated with the NO synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME, 1 mg/kg followed by 50 mu g/kg/min; n = 12) or NG-monomethyl-L-arginine (L-NMMA, same dose as L-NAME; n = 3), or with saline (n = 12). In separate animals (n = 42), guanosine 3',5'-cyclic monophosphate (cGMP), the second messenger for NO, and PG were measured at an average OPP of 90 mm Hg and 125 +/- 6 mm Hg; cGMP levels served as an index of NO release. The effect of the NO donor sodium nitroprusside on choroidal vessel diameter was determined using video imaging of isolated eyecup preparations. In control animals RBF was constant only within a range of 30 to 80 mm Hg OPP (r = 0.03, p > 0.9). There was no autoregulation of ChBF which increased as a function of OPP (tau = 0.58-0.72, p < 0.01). L-NAME and L-NMMA prevented a change in RBF and ChBF from 30 to 146 mm Hg [the highest OPP studied (r < 0.3, p > 0.15)] and caused an increase in retinal as well as choroidal vascular resistance as OPP was raised; these agents did not affect ocular blood flow at OPP < 30 mm Hg. Elevated OPP caused increases in cGMP, 6-keto-PGF1alpha, and PGE2 in the choroid (a vascular tissue), which were prevented by L-NAME and L-NMMA. Sodium nitroprusside caused a dilatation of choroidal vessels in isolated eyecup preparations, which was significantly attenuated by indomethacin. Data suggest a role for NO in the autoregulation of RBF and ChBF in the newborn such that a release of NO during a rise in OPP prevents adequate constriction necessary for maintaining RBF and ChBF constant; data also suggest that the vasodilator effect of NO might in part be mediated through a release of PG.

The vasoconstrictor effects of PGE2 and PGF2 alpha are less pronounced on retinal vessels of the newborn than of the adult pig. We tested the hypothesis that the decreased vasomotor response to these prostaglandins might be due to relatively fewer receptors and/or different receptor subtypes (in the case of PGE2) on retinal vessels of the newborn animal. Binding studies using [3H]PGE2 and [3H]PGF2 alpha revealed that PGE2 (EP) and PGF2 alpha (FP) receptor densities in retinal microvessel membrane preparations from newborn animals were approximately 25% of those found in vessels from the adult. The Kd for PGF2 alpha did not differ; however, the Kd for PGE2 was less in newborn than in adult vessels. Competition binding studies using AH 6809 (EP1 antagonist), butaprost (EP2 agonist), M/+B 28,767 (EP3 agonist), and AH 23848B (EP4 antagonist) suggested that the retinal vessels of the newborn contained approximately equal number of EP1 and EP2 receptor subtypes whereas the main receptor subtype in the adult vessels was EP1. In addition, PGE2 and butaprost produced comparable increases in adenosine 3',5'-cyclic monophosphate synthesis in newborn and adult vessels. PGE2, 17-phenyl trinor PGE2 (EP1 agonist) and PGF2 alpha caused a 2.5 to 3-fold greater increase in inositol 1,4,5-triphosphate (IP3) formation in adult than in newborn preparations. It is concluded that fewer PGF2 alpha receptors and an associated decrease in receptor-coupled IP3 formation in the retinal vessels of the newborn could lead to weaker vasoconstrictor effects of PGF2 alpha on retinal vessels of the newborn than of adult pigs; fewer EP1 receptors (associated with vasoconstriction) and a relatively greater proportion of EP2 receptors (associated with vasodilation) might be responsible for the reduced retinal vasoconstrictor effects of PGE2 in the newborn.

We tested whether the ontogenic differences in the constrictor effects of peroxides on the retinal vasculature were modulated by dilator cyclo-oxygenase products. Retinal arteriole (100-200 microns) vasomotor response to H2O2, t-butyl hydroperoxide, and cumene hydroperoxide were studied in isolated eyecup preparations using video camera monitoring of vessel diameter. A time- and dose-dependent biphasic retinal vasomotor response to all peroxides was observed on tissues of newborn and adult pigs. A rapid vasoconstriction (first 2 min) was followed by a relaxation which was greater in the adult than in the newborn tissues. The constrictor as well as the dilator response to peroxides and the observed increase in prostanoids were blocked by the cyclo-oxygenase inhibitor indomethacin. The peroxide-induced relaxation was inhibited or markedly attenuated by the prostaglandin I2 synthase blockers, trans-2-phenyl cyclopropylamine and minoxidil on tissues of newborn and adult animals. These agents also prevented the increase of the prostaglandin I2 receptor-coupled second messenger, cyclic 3',5'-adenosine monophosphate. Our data indicate that prostaglandin I2 plays a major role in counteracting the initial constrictor effects of peroxides in the retinal vasculature, and that the reversal of this constriction is greater in the adult than the newborn. These findings suggest that reduced reversal of vasoconstriction by the dilator prostaglandin I2 during an oxidative stress in the newborn may facilitate vasoconstriction by the dilator prostaglandin I2 during an oxidative stress in the newborn may facilitate neovascularization in retinopathy of prematurity.

In this study, we used a sensitive and specific radioimmunoassay to detect a substance which appears to be melatonin, an acetylation and methylation product of serotonin, in the eyes and central nervous system of the opisthobranch mollusc Aplysia californica. This identification was confirmed in the eyes by HPLC with fluorimetric detection. Melatonin activity was high in the eyes during the day and in the cerebral ganglia during the night. Only small amounts of melatonin were present at midday or midnight in the pedal ganglia. A single 1-hr exposure to light in the middle of the dark phase resulted in a sharp increase of melatonin in the eyes, whereas no such activity was detectable in cerebral and pedal ganglia. Eyes maintained in culture exhibited a diurnal rhythm of released melatonin activity over a 3-day period. These results suggest that melatonin is produced in A. californica in a rhythmic pattern different from that associated with pineal melatonin production in vertebrates.

The upper blood pressure limit of retinal blood flow autoregulation is lower in the newborn than in the adult; this suggests an insufficient vasoconstrictor response in the newborn when perfusion pressure is increased. Because prostaglandins (PGs) have an important role in autoregulation of retinal blood flow, we compared the effects of PGE2, PGF2 alpha, carbacyclin (PGI2 analogue), and U46619 (thromboxane analogue), as well as that of agonists for the three different PGE2 receptor subtypes, 17-phenyl trinor PGE2 (EP1), butaprost (EP2), and M&B 28,767 (EP3), on the retinal vasculature of newborn and adult pigs, using isolated eyecup preparations. PGF2 alpha and PGE2 caused a markedly greater constriction of retinal arteries and veins of the adult than of the newborn animals. Further analysis of the response to PGE2, using receptor subtype agonists, revealed that the EP1 receptor agonist, 17-phenyl trinor PGE2, and the EP3 receptor agonist, M&B 28,767, caused a significant constriction of adult arteries and veins but produced minimal effects on newborn vessels; the EP2 receptor agonist, butaprost, caused a small and comparable dilation of newborn and adult arteries and veins. The PGI2 analogue, carbacyclin, caused a greater dilation of the adult than of the newborn arteries, but produced comparable dilation of veins from both newborn and adult animals. In contrast to the effects of PGF2 alpha and PGE2, the thromboxane analogue, U46619, as well as the alpha 1-adrenoceptor agonist, phenylephrine, significantly constricted newborn arteries and veins, and this effect was comparable with that observed on retinal vessels of the adult.(ABSTRACT TRUNCATED AT 250 WORDS)

Purpose: To study the role of free radicals in autoregulation of retinal blood flow (RBF) and choroidal blood flow (ChBF) and the contribution of the cyclooxygenase pathway in free radical formation during blood pressure changes in 1- to 3-day-old pigs., Methods: Blood pressure was adjusted by inflating balloon-tipped catheters placed at the aortic isthmus and the aortic root to induce hypertension and hypotension, respectively. Blood flow was measured using the microsphere technique. Also, the effects of peroxides on retinal artery diameter were studied on eyecup preparations using time-frame photography processed by digital imaging., Results: Blood gases and intraocular pressure (13 +/- 1 mm Hg) remained stable throughout the experiments. In control animals, RBF was constant only between 30 and 75 mm Hg of ocular perfusion pressure and ChBF increased as a function of ocular perfusion pressure (tau = 0.58, P < 0.01). Inhibition of peroxidation with the free radical scavenger 21-aminosteroid U74389F (2.5 mg/kg) widened the range of RBF and ChBF autoregulation (ocular perfusion pressure from 30 to 131 mm Hg). Hypertension caused an increase in the products of peroxidation, malondialdehyde, and hydroperoxides, as well as in prostaglandin E2, prostaglandin F2 alpha, and 6-keto-prostaglandin F1 alpha in the retina and choroid of control animals; these changes were inhibited by the free radical scavengers U74389F (2.5 mg/kg) and high-dose allopurinol (140 mg/kg) as well as by the cyclooxygenase inhibitors ibuprofen (40 mg/kg) and indomethacin (5 mg/kg). In isolated eyecup preparations, H2O2 and cumene hydroperoxide dilated retinal vessels, and this effect was completely blocked by indomethacin., Conclusions: These findings indicate that free radicals play a major role in setting the upper limit of RBF and ChBF autoregulation of the newborn animal. In addition, there exists a positive feedback interaction between free radicals and cyclooxygenase activity in ocular tissues, such that during hypertension the cyclooxygenase pathway is an important producer of free radicals and in turn is also activated by them.

Free radicals have been implicated in the development of injury to the immature retina. Asphyxia increases free radicals as well as prostaglandins (PG) in neural tissues. We assessed whether in the retina the cyclooxygenase pathway contributes to free radical formation after oxidative insults such as asphyxia, which in turn disrupts retinal function. Newborn pigs were treated with either saline, ibuprofen (194 mumol/kg i.v.), or allopurinol (1 mmol/kg i.v.), and retinal malondialdehyde (MDA), hydroperoxides, PGE2 and PGF2 alpha levels, and the amplitudes and implicit times of the a- and b-waves of the full-field electroretinogram were measured before and 1 h after a 5-min period of asphyxia. In saline-treated animals, asphyxia caused a marked increase (p < 0.01) in MDA, hydroperoxides, PGE2, and PGF2 alpha concentrations in the retina. This was associated with a significant decrease (p < 0.01) in the b-wave amplitude measured under scotopic and photopic conditions and an increase in the b-wave implicit times. Ibuprofen and another cyclooxygenase inhibitor, indomethacin (28 mumol/kg i.v.), decreased PGE2 and PGF2 alpha levels and prevented the increase in MDA and hydroperoxides after asphyxia. Allopurinol maintained low concentrations of MDA and hydroperoxides after asphyxia. Both ibuprofen and allopurinol prevented the postasphyxial changes in the b-wave amplitude and diminished the delay in implicit time observed after asphyxia in saline-treated pigs. Our findings suggest that in the retina after asphyxia free radicals appear to originate primarily from the cyclooxygenase pathway and contribute to the deterioration in retinal electrophysiologic function of the newborn animal. Cyclooxygenase inhibitors, like free radical scavengers, may protect retinal function from deteriorating after oxidative stresses.

Myeloid bodies are believed to be differentiated areas of smooth endoplasmic reticulum membranes, and they are found within the retinal pigment epithelium in a number of lower vertebrates. Previous studies demonstrated a correlation between phagocytosis of outer segment disc membranes and myeloid body numbers in the retinal pigment epithelium of the newt. To test the hypothesis that myeloid bodies are directly involved in outer segment lipid metabolism and to further characterize the origin and functional significance of these organelles, we examined the effects on myeloid bodies of eliminating the source of outer segment membrane lipids (neural retina removal) and of the subsequent return of outer segments (retinal regeneration) in the newt Notophthalmus viridescens. Light- and electron-microscopic analysis demonstrated that myeloid bodies disappeared from the pigment epithelium within six days of neural retina removal. By week 6 of regeneration, rudimentary photoreceptor outer segments were present but myeloid bodies were still absent. However, at this time, the smooth endoplasmic reticulum in some areas of the retinal pigment epithelial cells had become flattened, giving rise to small (0.5 micron long), two-to-four layer-thick lamellar units, which are myeloid body precursors. Small myeloid bodies were first observed one week later at week 7 of retinal regeneration. This study revealed that newt myeloid bodies are specialized areas of smooth endoplasmic reticulum. It also showed that a contact between functional photoreceptors and the retinal pigment epithelium is essential to the presence of myeloid bodies in the epithelial cells.

The phospholipid composition of a myeloid body (MB) enriched subcellular fraction of chick retinal pigment epithelium (RPE) was determined in order to further characterize the origin and functional significance of these lamellar membrane organelles. The major MB phospholipids found were phosphatidylcholine and phosphatidylethanolamine which represented 43% and 34% of the total MB lipids respectively. Sphingomyelin and phosphatidylinositol comprised the remaining detectable phospholipids. The fatty acyl chain composition of all detected phospholipids showed that the long-chain polyunsaturated fatty acids [arachidonic (20:4 n-6), docosapentaenoic (22:5 n-3) and docosahexaenoic (22:6 n-3)] account for greater than 45% of the fatty acids in MB membranes. This high proportion of long-chain polyunsaturated fatty acids in MBs is particularly striking when compared to the long-chain fatty acid composition of the photoreceptor outer segments from this predominantly cone retina which contains less than 25% long-chain polyunsaturated fatty acids. The results from this study clearly demonstrate that MB lipids represent a significantly enriched pool of long-chain polyunsaturated fatty acids.

The absorption and regenerability characteristics are compared for rhodopsin contained in rod outer segment membranes and purified in a series of alkyl sucrose esters. It is found that membrane-bound rhodopsin has maximum absorbance from 504 to 500 nm between 1.5 and 40 degrees C. After purification, rhodopsin absorbance can be blue-shifted by up to 6 nm, depending on the detergent species used. Only the longest chain sucrose esters give purified rhodopsin with maximum absorbance comparable to that of the native pigment. In the same manner, detergent-purified rhodopsin will be easily regenerated as long as its native spectral characteristics are maintained. Sucrose esters thus prove to be mild enough to maintain rhodopsin functionality with respect to these two properties and could probably be used successfully to maintain other membrane proteins' integrity.