Subcellular localization of coagulation factor II receptor-like 1 in neurons governs angiogenesis.

Neurons have an important role in retinal vascular development. Here we show that the G protein-coupled receptor (GPCR) coagulation factor II receptor-like 1 (F2rl1, previously known as Par2) is abundant in retinal ganglion cells and is associated with new blood vessel formation during retinal development and in ischemic retinopathy. After stimulation, F2rl1 in retinal ganglion cells translocates from the plasma membrane to the cell nucleus using a microtubule-dependent shuttle that requires sorting nexin 11 (Snx11). At the nucleus, F2rl1 facilitates recruitment of the transcription factor Sp1 to trigger Vegfa expression and, in turn, neovascularization. In contrast, classical plasma membrane activation of F2rl1 leads to the expression of distinct genes, including Ang1, that are involved in vessel maturation. Mutant versions of F2rl1 that prevent nuclear relocalization but not plasma membrane activation interfere with Vegfa but not Ang1 expression. Complementary angiogenic factors are therefore regulated by the subcellular localization of a receptor (F2rl1) that governs angiogenesis. These findings may have implications for the selectivity of drug actions based on the subcellular distribution of their targets.

Restoration of renal function by a novel prostaglandin EP4 receptor-derived peptide in models of acute renal failure.

Acute renal failure (ARF) is a serious medical complication characterized by an abrupt and sustained decline in renal function. Despite significant advances in supportive care, there is currently no effective treatment to restore renal function. PGE(2) is a lipid hormone mediator abundantly produced in the kidney, where it acts locally to regulate renal function; several studies suggest that modulating EP(4) receptor activity could improve renal function following kidney injury. An optimized peptidomimetic ligand of EP(4) receptor, THG213.29, was tested for its efficacy to improve renal function (glomerular filtration rate, renal plasma flow, and urine output) and histological changes in a model of ARF induced by either cisplatin or renal artery occlusion in Sprague-Dawley rats. THG213.29 modulated PGE(2)-binding dissociation kinetics, indicative of an allosteric binding mode. Consistently, THG213.29 antagonized EP(4)-mediated relaxation of piglet saphenous vein rings, partially inhibited EP(4)-mediated cAMP production, but did not affect Gα(i) activation or ß-arrestin recruitment. In vivo, THG213.29 significantly improved renal function and histological changes in cisplatin- and renal artery occlusion-induced ARF models. THG213.29 increased mRNA expression of heme-oxygenase 1, Bcl2, and FGF-2 in renal cortex; correspondingly, in EP(4)-transfected HEK293 cells, THG213.29 augmented FGF-2 and abrogated EP(4)-dependent overexpression of inflammatory IL-6 and of apoptotic death domain-associated protein and BCL2-associated agonist of cell death. Our results demonstrate that THG213.29 represents a novel class of diuretic agent with noncompetitive allosteric modulator effects on EP(4) receptor, resulting in improved renal function and integrity following acute renal failure.

Choroidal involution is a key component of oxygen-induced retinopathy.

PURPOSE: Retinopathy of prematurity (ROP) is a major cause of visual handicap in the pediatric population. To date, this disorder is thought to stem from deficient retinal vascularization. Intriguingly, functional electrophysiological studies in patients with mild or moderate ROP and in the oxygen-induced retinopathy (OIR) model in rats reveal central photoreceptor disruption that overlies modest retinal vessel loss; a paucity of retinal vasculature occurs predominantly at the periphery. Given that choroidal circulation is the major source of oxygen and nutrients to the photoreceptors, the authors set out to investigate whether the choroidal vasculature system may be affected in OIR. METHODS: Rat models of OIR treating newborn animals with 80% or 50/10% alternated oxygen level for the first two postnatal weeks were used to mimic ROP in humans. Immunohistology staining and vascular corrosion casts were used to investigate the vessel layout of the eye. To investigate the effect of 15-deoxy-Δ12,14-PGJ(2) (15d-PGJ(2); a nonenzymatic product of prostaglandin D(2)) on endothelial cells, in vitro cell culture and ex vivo choroid explants were employed and intravitreal injections were performed in animals. RESULTS: The authors herein demonstrate that deficient vascularity occurs not only in the retinal plexus but also in the choroid. This sustained, marked choroidal degeneration is specifically confined to central regions of the retina that present persistent photoreceptor loss and corresponding functional deficits. Moreover, the authors show that 15d-PGJ(2) is a prominent contributor to this choroidal decay. CONCLUSIONS: The authors demonstrate for the first time pronounced, sustained choroidal vascular involution during the development of ROP. Findings also suggest that effective therapeutic strategies to counter ROP should consider choroidal preservation.

Ischemic neurons prevent vascular regeneration of neural tissue by secreting semaphorin 3A.

The failure of blood vessels to revascularize ischemic neural tissue represents a significant challenge for vascular biology. Examples include proliferative retinopathies (PRs) such as retinopathy of prematurity and proliferative diabetic retinopathy, which are the leading causes of blindness in children and working-age adults. PRs are characterized by initial microvascular degeneration, followed by a compensatory albeit pathologic hypervascularization mounted by the hypoxic retina attempting to reinstate metabolic equilibrium. Paradoxically, this secondary revascularization fails to grow into the most ischemic regions of the retina. Instead, the new vessels are misdirected toward the vitreous, suggesting that vasorepulsive forces operate in the avascular hypoxic retina. In the present study, we demonstrate that the neuronal guidance cue semaphorin 3A (Sema3A) is secreted by hypoxic neurons in the avascular retina in response to the proinflammatory cytokine IL-1ß. Sema3A contributes to vascular decay and later forms a chemical barrier that repels neo-vessels toward the vitreous. Conversely, silencing Sema3A expression enhances normal vascular regeneration within the ischemic retina, thereby diminishing aberrant neovascularization and preserving neuroretinal function. Overcoming the chemical barrier (Sema3A) released by ischemic neurons accelerates the vascular regeneration of neural tissues, which restores metabolic supply and improves retinal function. Our findings may be applicable to other neurovascular ischemic conditions such as stroke.

Sustained hypercapnia induces cerebral microvascular degeneration in the immature brain through induction of nitrative stress.

Hypercapnia is regularly observed in chronic lung disease, such as bronchopulmonary dysplasia in preterm infants. Hypercapnia results in increased nitric oxide synthase activity and in vitro formation of nitrates. Neural vasculature of the immature subject is particularly sensitive to nitrative stress. We investigated whether exposure to clinically relevant sustained high CO(2) causes microvascular degeneration in the newborn brain by inducing nitrative stress, and whether this microvascular degeneration has an impact on brain growth. Newborn rat pups were exposed to 10% CO(2) as inspired gas (Pa(CO(2)) = 60-70 mmHg) starting within 24 h of birth until postnatal day 7 (P7). Brains were notably collected at different time points to measure vascular density, determine brain cortical nitrite/nitrate, and trans-arachidonic acids (TAAs; products of nitration) levels as effectors of vessel damage. Chronic exposure of rat pups to high CO(2) (Pa(CO(2)) approximately 65 mmHg) induced a 20% loss in cerebrovascular density at P3 and a 15% decrease in brain mass at P7; at P30, brain mass remained lower in CO(2)-exposed animals. Within 24 h of exposure to CO(2), brain eNOS expression and production of nitrite/nitrate doubled, lipid nitration products (TAAs) increased, and protein nitration (3-nitrotyrosine immunoreactivity) was also coincidently augmented on brain microvessels (lectin positive). Intracerebroventricular injection of TAAs (10 microM) replicated cerebrovascular degeneration. Treatment of rat pups with NOS inhibitor (L-N(omega)-nitroarginine methyl ester) or a peroxynitrite decomposition catalyst (FeTPPS) prevented hypercapnia-induced microvascular degeneration and preserved brain mass. Cytotoxic effects of high CO(2) were reproduced in vitro/ex vivo on cultured endothelial cells and sprouting microvessels. In summary, hypercapnia at values frequently observed in preterm infants with chronic lung disease results in increased nitrative stress, which leads to cerebral cortical microvascular degeneration and curtails brain growth.

The succinate receptor GPR91 in neurons has a major role in retinal angiogenesis.

Vascularization is essential for tissue development and in restoration of tissue integrity after an ischemic injury. In studies of vascularization, the focus has largely been placed on vascular endothelial growth factor (VEGF), yet other factors may also orchestrate this process. Here we show that succinate accumulates in the hypoxic retina of rodents and, via its cognate receptor G protein-coupled receptor-91 (GPR91), is a potent mediator of vessel growth in the settings of both normal retinal development and proliferative ischemic retinopathy. The effects of GPR91 are mediated by retinal ganglion neurons (RGCs), which, in response to increased succinate levels, regulate the production of numerous angiogenic factors including VEGF. Accordingly, succinate did not have proangiogenic effects in RGC-deficient rats. Our observations show a pathway of metabolite signaling where succinate, acting through GPR91, governs retinal angiogenesis and show the propensity of RGCs to act as sensors of ischemic stress. These findings provide a new therapeutic target for modulating revascularization.

Intracrine signaling through lipid mediators and their cognate nuclear G-protein-coupled receptors: a paradigm based on PGE2, PAF, and LPA1 receptors.

Prostaglandins (PGs), platelet-activating factor (PAF), and lysophosphatidic acid (LPA) are ubiquitous lipid mediators that play important roles in inflammation, cardiovascular homeostasis, and immunity and are also known to modulate gene expression of specific pro-inflammatory genes. The mechanism of action of these lipids is thought to be primarily dependent on their specific plasma membrane receptors belonging to the superfamily of G-protein-coupled receptors (GPCR). Increasing evidence suggests the existence of a functional intracellular GPCR population. It has been proposed that immediate effects are mediated via cell surface receptors whereas long-term responses are dependent upon intracellular receptor effects. Indeed, receptors for PAF, LPA, and PGE(2) (specifically EP(1), EP(3), and EP(4)) localize at the cell nucleus of cerebral microvascular endothelial cells of newborn pigs, rat hepatocytes, and cells overexpressing each receptor. Stimulation of isolated nuclei with these lipids reveals biological functions including transcriptional regulation of major genes, namely c-fos, cylooxygenase-2, and endothelial as well as inducible nitric oxide synthase. In the present review, we shall focus on the nuclear localization and signaling of GPCRs recognizing PGE(2), PAF, and LPA phospholipids as ligands. Mechanisms on how nuclear PGE2, PAF, and LPA receptors activate gene transcription and nuclear localization pathways are presented. Intracrine signaling for lipid mediators uncover novel pathways to elicit their effects; accordingly, intracellular GPCRs constitute a distinctive mode of action for gene regulation.

Field stimulation-induced tetrodotoxin-resistant vasorelaxation is mediated by sodium hypochlorite.

This study was done to determine the mechanism of field stimulation-induced tetrodotoxin (TTX)- and NG- nitro-l-arginine (LNA)-resistant vasorelaxation. Field stimulation with platinum and carbon, but not with silver, electrodes (30 V, 30 HZ, 2-5 ms pulse width) as well as electrically stimulated salt (0.9% NaCl) solution (ESSS) or Krebs solution caused 100% relaxation of phenylephrine-contracted rat aortic strips, which was TTX and LNA resistant and endothelium independent. ESSS also relaxed other vascular preparations (rabbit aorta and renal artery, dog coronary artery, pig ductus arteriosus, and rat portal vein). The electric current generated hypochlorite (OCl-) and H2O2 from the salt solution; however, vasorelaxation was caused by NaOCl and not by H2O2. ESSS and NaOCl caused contraction failure of spontaneously beating right atria of rats and did not affect uterine contractions, vascular cAMP, cGMP, or the pH of the tissue bath. Field stimulation, ESSS, and NaOCl did not relax aortic preparations contracted by 32 mmol/L potassium and their vasorelaxant effects on phenylephrine-contracted rat aortic strips and rings were completely reversed by tetraethylammonium and partially by glibenclamide and iberiotoxin. We conclude that electric pulses generate the oxidant OCl- from the salt solution, which causes vasorelaxation by increasing K+ conductance.

Redox-dependent effects of nitric oxide on microvascular integrity in oxygen-induced retinopathy.

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.

Intracellular-specific colocalization of prostaglandin E2 synthases and cyclooxygenases in the brain.

Prostaglandin E2 (PGE2) is the major primary prostaglandin generated by brain cells. However, the coordination and intracellular localization of the cyclooxygenases (COXs) and prostaglandin E synthases (PGESs) that convert arachidonic acid to PGE2 in brain tissue are not known. We aimed to determine whether microsomal and cytosolic PGES (mPGES-1 and cPGES) colocalize and coordinate activity with either COX-1 or COX-2 in brain tissue, particularly during development. Importantly, we found that cytosolic PGES also associates with microsomes (cPGES-m) from the cerebrum and cerebral vasculature of the pig and rat as well as microsomes from various cell lines; this seemed dependent on the carboxyl terminal 35-amino acid domain and a cysteine residue (C58) of cPGES. In microsomal membranes from the postnatal brain and cerebral microvessels of mature animals, cPGES-m colocalized with both COX-1 and COX-2, whereas mPGES-1 was undetectable in these microsomes. Accordingly, in this cell compartment, cPGES could coordinate its activity with COX-2 and COX-1 (partly inhibited by NS398); albeit in microsomes of the brain microvasculature from newborns, mPGES-1 was also present. In contrast, in nuclei of brain parenchymal and endothelial cells, mPGES-1 and cPGES colocalized exclusively with COX-2 (determined by immunoblotting and immunohistochemistry); these PGESs contributed to conversion of PGH2 into PGE2. Hence, contrary to a previously proposed model of exclusive COX-2/mPGES-1 coordination, COX-2 can coordinate with mPGES-1 and/or cPGES in the brain, depending on the cell compartment and the age group.

Cytotoxicity of the E(2)-isoprostane 15-E(2t)-IsoP on oligodendrocyte progenitors.

Oxidant stress plays a significant role in the pathogenesis of periventricular leukomalacia (PVL). Isoprostanes (IsoPs) are bioactive products of lipid peroxidation abundantly generated during hypoxic-ischemic injuries. Because loss of oligodendrocytes (OLs) occurs early in PVL, we hypothesized that IsoPs could induce progenitor OL death. 15-E(2t)-IsoP but not 15-F(2t)-IsoP elicited a concentration-dependent death of progenitor OLs by oncosis and not by apoptosis, but exerted minimal effects on mature OLs. 15-E(2t)-IsoP-induced cytotoxicity could not be explained by its conversion into cyclopentenones, because PGA(2) was hardly cytotoxic. On the other hand, thromboxane A(2) (TxA(2)) synthase inhibitor CGS12970 and cyclooxygenase inhibitor ibuprofen attenuated 15-E(2t)-IsoP-induced cytotoxicity. Susceptibility of progenitor OLs was independent of TxA(2) receptor (TP) expression, which was far less in progenitor than in mature OLs. However, TxA(2) synthase was detected in precursor but not in mature OLs, and TxA(2) mimetic U46619 induced hydroperoxides generation and progenitor OL death. The glutathione synthesis enhancer N-acetylcysteine prevented 15-E(2t)-IsoP-induced progenitor cell death. Depletion of glutathione in mature OLs with buthionine sulfoximine rendered them susceptible to cytotoxicity of 15-E(2t)-IsoP. These novel data implicate 15-E(2t)-IsoP as a product of oxidative stress that may contribute in the genesis of PVL.

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.

Mechanism of the negative inotropic effects of alpha 1-adrenoceptor agonists on mouse myocardium.

This study was done to identify the mechanism of the alpha1-adrenoceptor (AR) mediated negative inotropic effects of phenylephrine (PE) on adult mouse myocardium. As reported by others, we also found that the nonselective alpha1AR agonist PE produced a negative inotropic effect on ventricular strips from adult mice that was inhibited by the alpha1AAR antagonist 5-methylurapidil (5MU) but not by the alpha1BAR antagonist chloroethylclonidine (CEC) or the alpha1DAR antagonist BMY 7378. The selective alpha1AAR agonist A61603 also produced a negative inotropic effect, which was antagonized by 5MU. Phorbol 12,13-dibutyrate (activator of all PKC isoforms) mimicked the negative inotropic responses to PE and A61603. The negative inotropic effects of PE were inhibited by bisindolylmaleimide (inhibitor of all PKC isoforms) but not by Gö 6976 (inhibitor of Ca2+-dependent PKC). Rottlerin, an inhibitor of Ca2+-independent PKCdelta, antagonized the negative inotropic effects of PE and A61603. PE and A61603 increased the translocation of PKCdelta, which was prevented by rottlerin. These data suggest that the alpha1AR-mediated negative inotropy on adult mouse myocardium is signaled by Ca2+-independent PKCdelta.

Modulation of L-type Ca2+ channels in neonatal rat heart by a novel Ca2+ channel agonist.

L-type Ca2+ channels are essential in triggering the intracellular Ca2+ release and contraction in heart cells. In this study, we used patch clamp technique to compare the effect of two pure enantiomers of L-type Ca2+ channel agonists: (+)-CGP 48506 and the dihydropyridine (+)-SDZ-202 791 in cardiomyocytes from rats 2-5 days old. The predominant Ca2+ current activated by standard step pulses in these myocytes was L-type Ca2+ current. The dihydropyridine antagonist (+)-PN200-110 (5 microM) blocked over 90% of Ca2+ currents in most cells tested. CGP 48506 lead to a maximum of 200% increase in currents. The threshold concentration for the CGP effect was at 1 microM and the maximum was reached at 20 microM. SDZ-202 791 had effects in nanomolar concentrations and a maximum effect at about 2 microM. The maximal effect of (+)-SDZ-202 791 was a 400% increase in the amplitude of Ca2+ currents and was accompanied by a 10-15 mV leftward shift in the voltage dependence of activation. CGP 48506 increased the currents equally at all voltages tested. Both compounds slowed the deactivation of tail currents and lead to the appearance of slowly activating and slowly deactivating current components. However, SDZ-202 791 had larger effects on deactivation and CGP 48506 had larger effect on the rate of Ca2+ current activation. The effect of SDZ-202 791 was fully additive to that of CGP 48506 even after maximum concentrations of CGP. This observation suggests that the two Ca2+ channel agonists may act at two different sites on the L-type Ca2+ channel. We suggest that CGP 48506 would be a potential cardiotonic agent without the deleterious proarrhythmic effects attributable to the dihydropyridine agonists.

Selective neuromicrovascular endothelial cell death by 8-Iso-prostaglandin F2alpha: possible role in ischemic brain injury.

BACKGROUND AND PURPOSE: Free radical-induced peroxidation is an important factor in the genesis of hypoxic-ischemic encephalopathy, including that of the preterm infant. Isoprostanes are major peroxidation products. Since microvascular dysfunction seems to contribute to ischemic encephalopathies, we studied the cytotoxicity of 8-iso-prostaglandin F2alpha (PGF2alpha) on cerebral microvascular cells. METHODS: Microvascular endothelial, astroglial, and smooth muscle cells from newborn brain were cultured. The cytotoxicity of 8-iso-PGF2alpha on these cells was determined by MTT assays and lactate dehydrogenase (LDH) release, propidium iodide incorporation, and DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling [TUNEL]). In addition, effects of intraventricular injections of 8-iso-PGF2alpha and possible involvement of thromboxane in 8-iso-PGF2alpha-induced cytotoxicity were determined. RESULTS: 8-Iso-PGF2alpha induced time- and concentration-dependent endothelial cell death (EC50=0.1 nmol/L) but exerted little effect on smooth muscle and astroglial cells; endothelial cell death seemed mostly of oncotic nature (propidium iodide incorporation and LDH release). Cell death was associated with increased endothelial thromboxane A2 (TXA2) formation and was prevented by TXA2 synthase inhibitors (CGS12970 and U63557A); TXA2 mimetics U46619 and I-BOP also caused endothelial cell death. Intraventricular injection of 8-iso-PGF2alpha induced periventricular damage, which was attenuated by CGS12970 pretreatment. CONCLUSIONS: These data disclose a novel action of 8-iso-PGF2alpha involving TXA2 in oxidant stress-induced cerebral microvascular injury and brain damage.

Increased platelet-activating factor-induced periventricular brain microvascular constriction associated with immaturity.

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.

Altered vascular function in fetal programming of hypertension.

BACKGROUND AND PURPOSE: Reduced endothelium-dependent vasorelaxation partly due to loss of nitric oxide (NO) bioavailability occurs in most cases of chronic hypertension. Intrauterine nutritional deprivation has been associated with increased risk for hypertension and stroke, associated with relaxant dysfunction and decreased vascular compliance, but the underlying mechanisms are not known. The present studies were undertaken to investigate whether endothelial dysfunction associated with altered NO-dependent vasodilatation pathways is also observed in a model of in utero programming of hypertension. METHODS: Pregnant Wistar rats were fed a normal (18%), low (9%), or very low (6%) protein isocaloric diet during gestation. Vasomotor response of resistance cerebral microvessels (<50 micro m) was studied in adult offspring of dams fed the 18% and 9% protein diets by a video imaging technique. Endothelial NOS (eNOS), soluble guanylate cyclase (sGC), and K(Ca) channel expression were measured by Western blot. NO synthase (NOS) activity was measured enzymatically as well as in situ by NADPH diaphorase staining. RESULTS: Litter size and survival to adulthood were not affected by the diets. Birth weights of offspring of dams fed the 6% diet were markedly lower than those of dams fed the 9% diet, which were marginally lower than those of controls. Systolic blood pressures of adult offspring of mothers in the 6% and 9% groups were comparably greater (156+/-2 and 155+/-1 mm Hg, respectively) than that of control offspring (137+/-1 mm Hg); we therefore focused on the 9% and 18% groups. Cerebral microvessel constriction to thromboxane A(2) mimetic and dilation to carba-prostaglandin I(2) did not differ between diet groups. In contrast, vasorelaxation to the NO-dependent agents substance P and acetylcholine was diminished by 50% in low protein-exposed offspring, but eNOS expression and activity were similar between the 2 diet groups. Vasorelaxant response to the NO donor sodium nitroprusside was also decreased and was associated with reduced (by 50% to 65%) cGMP levels and sGC expression. cGMP analogues caused comparable vasorelaxation in the 2 groups. Expression of K(Ca) (another important mediator of NO action) and relaxation to the K(Ca) opener NS1619 were unchanged by antenatal diet. CONCLUSIONS: Maternal protein deprivation, which leads to hypertension in the offspring, is associated with diminished NO-dependent relaxation of major organ (cerebral) microvasculature, which seems to be largely attributed to decreased sGC expression and cGMP levels. The study provides an additional explanation for abnormal vasorelaxation in nutrient-deprived subjects in utero.