NO/cGMP signaling and HSP90 activity represses metamorphosis in the sea urchin Lytechinus pictus.

Nitric oxide (NO) signaling repressively regulates metamorphosis in two solitary ascidians and a gastropod. We present evidence for a similar role in the sea urchin Lytechinus pictus. NO commonly signals via soluble guanylyl cyclase (sGC). Nitric oxide synthase (NOS) activity in some mammalian cells, including neurons, depends on the molecular chaperone heat shock protein 90 (HSP90); this may be so in echinoid larvae as well. Pluteus larvae containing juvenile rudiments were treated with either radicicol L- or D-nitroarginine-methyl-ester (L-NAME and D-NAME), or IH-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), inhibitors of HSP90, NOS, and sGC, respectively. In all instances, drug treatment significantly increased the frequency of metamorphosis. SNAP, a NO donor, suppressed the inductive properties of L-NAME and biofilm, a natural inducer of metamorphosis. NADPH diaphorase histochemistry indicated NOS activity in cells in the lower lip of the larval mouth, the preoral hood, the gut, and in the tube feet of the echinus rudiment. Histochemical staining coincided with NOS immunostaining. Microsurgical removal of the oral hood or the pre-oral hood did not induce metamorphosis, but larvae lacking these structures retained the capacity to metamorphose in response to ODQ. We propose that the production of NO repressively regulates the initiation of metamorphosis and that a sensory response to environmental cues reduces the production of NO, and consequently cGMP, to initiate metamorphosis.     

Regulatory roles of nitric oxide during larval development and metamorphosis in Ciona intestinalis

Metamorphosis in the ascidian Ciona intestinalis is a very complex process which converts a swimming tadpole to an adult. The process involves reorganisation of the body plan and a remarkable regression of the tail, which is controlled by caspase-dependent apoptosis. However, the endogenous signals triggering apoptosis and metamorphosis are little explored. Herein, we report evidence that nitric oxide (NO) regulates tail regression in a dose-dependent manner, acting on caspase-dependent apoptosis. An increase or decrease of NO levels resulted in a delay or acceleration of tail resorption, without affecting subsequent juvenile development. A similar hastening effect was induced by suppression of cGMP-dependent NO signalling. Inhibition of NO production resulted in an increase in caspase-3-like activity with respect to untreated larvae. Detection of endogenously activated caspase-3 and NO revealed the existence of a spatial correlation between the diminution of the NO signal and caspase-3 activation during the last phases of tail regression. Real-time PCR during development, from early larva to early juveniles, showed that during all stages examined, NO synthase (NOS) is always more expressed than arginase and it reaches the maximum value at late larva, the stage immediately preceding tail resorption. The spatial expression pattern of NOS is very dynamic, moving rapidly along the body in very few hours, from the anterior part of the trunk to central nervous system (CNS), tail and new forming juvenile digestive organs. NO detection revealed free diffusion from the production sites to other cellular districts. Overall, the results of this study provide a new important link between NO signalling and apoptosis during metamorphosis in C. intestinalis and hint at novel roles for the NO signalling system in other developmental and metamorphosis-related events preceding and following tail resorption.     

Impaired NO signaling in small pulmonary arteries of chronically hypoxic newborn piglets

We performed studies to determine whether chronic hypoxia impairs nitric oxide (NO) signaling in resistance level pulmonary arteries (PAs) of newborn piglets. Piglets were maintained in room air (control) or hypoxia (11% O(2)) for either 3 (shorter exposure) or 10 (longer exposure) days. Responses of PAs to a nonselective NO synthase (NOS) antagonist, N(omega)-nitro-L-arginine methylester (L-NAME), a NOS-2-selective antagonist, aminoguanidine, and 7-nitroindazole, a NOS-1-selective antagonist, were measured. Levels of NOS isoforms and of two proteins involved in NOS signaling, heat shock protein (HSP) 90 and caveolin-1, were assessed in PA homogenates. PAs from all groups constricted to L-NAME but not to aminoguanidine or 7-nitroindazole. The magnitude of constriction to L-NAME was similar for PAs from control and hypoxic piglets of the shorter exposure period but was diminished for PAs from hypoxic compared with control piglets of the longer exposure period. NOS-3, HSP90, and caveolin-1 levels were similar in hypoxic and control PAs. These findings indicate that NOS-3, but not-NOS 2 or NOS-1, is involved with basal NO production in PAs from both control and hypoxic piglets. After 10 days of hypoxia, NO function is impaired in PAs despite preserved levels of NOS-3, HSP90, and caveolin-1. The development of NOS-3 dysfunction in resistance level PAs may contribute to the progression of chronic hypoxia-induced pulmonary hypertension in newborn piglets.     

ENZYMATIC ACTIVITIES RELATING TO THE RESPIRATION AND THE ONSET OF METAMORPHOSIS OF THE ASCIDIAN TADPOLE

The oxygen consumption of a single ascidian larva was measured. After hatching the consumption increases gradually. During the period of tail resorption it also increases gradually, but after the completion of tail resorption the consumption decreases conspicuously.
With the development of the larva after hatching, the activities of cytochrome oxidase and succinic dehydrogenase and of Janus green-reduction become detectable in the adhesive papillae, the proximal region of the tail, and the tail muscle. After the completion of tail resorption, these activities become indistinct.
These tissues underwent most profound morphological changes at the onset of metamorphosis. Soon after hatching, Janus green has no effect to induce metamorphosis. In larvae 4 hr after hatching, the shrinkage of adhesive papillae can be induced by Janus green-treatment. In 12 hr larvae, both the shrinkage of adhesive papillae and the tail resorption can be induced by Janus green. The enhancement of respiratory activities in the larvae after hatching may be related to the changes in the adhesive papillae and later to changes in the proximal region of the tail. Only when both of these changes occur can metamorphosis be induced.     

Interaction between noradrenaline or adrenaline and the beta 1-adrenergic receptor in the nervous system triggers early metamorphosis of larvae in the ascidian,Ciona savignyi

Molecular mechanisms underlying the metamorphosis of larvae, e.g., ligand and receptor interaction, have to be determined and roles for the nervous system in marine invertebrates are not well understood. We report here that treatment of swimming larvae of the ascidian Ciona savignyi with noradrenaline or adrenaline promoted morphological changes in early metamorphosis, e.g., tail resorption. Antagonists of the beta-adrenergic receptor, propranolol, and the beta(1)-adrenergic receptor, metoprolol, inhibited the noradrenaline-induced tail resorption, while an antagonist of the alpha-adrenergic receptor, phentolamine, and of the beta(2)- adrenergic receptor, butoxamine, had no inhibitory effects. In addition, a selective agonist of the beta-adrenergic receptor, isoproterenol, the concentration of which was lower than the effective concentration of the neurotransmitters, facilitated tail resorption. Immunohistochemical studies, using an anti-dopamine-hydroxylase antibody, showed that neurotransmitters such as noradrenaline and adrenaline localized around the brain vesicle of the larvae during metamorphosis. The beta(1)-adrenergic receptor stained with antibodies was localized on the nervous system. Temporal expression of the beta(1)-adrenergic receptor was intense in the nervous system in the larvae competent for metamorphosis. We propose that interactions between noradrenaline or adrenaline and the beta(1)-adrenergic receptor in the nervous system mediate the process of metamorphosis of Ciona larvae.     

Tissue-specific profile of DNA replication in the swimming larvae of Ciona intestinalis

The cell cycle is strictly regulated during development and its regulation is essential for organ formation and developmental timing. Here we observed the pattern of DNA replication in swimming larvae of an ascidian, Ciona intestinalis. Usually, Ciona swimming larvae obtain competence for metamorphosis at about 4-5 h after hatching, and these competent larvae initiate metamorphosis soon after they adhere to substrate with their papillae. In these larvae, three major tissues (epidermis, endoderm and mesenchyme) showed extensive DNA replication with distinct pattern and timing, suggesting tissue-specific cell cycle regulation. However, DNA replication did not continue in aged larvae which kept swimming for several days, suggesting that the cell cycle is arrested in these larvae at a certain time to prevent further growth of adult organ rudiments until the initiation of metamorphosis. Inhibition of the cell cycle by aphidicolin during the larval stage affects only the speed of metamorphosis, and not the formation of adult organ rudiments or the timing of the initiation of metamorphosis. However, after the completion of tail resorption, DNA replication is necessary for further metamorphic events. Our data showed that DNA synthesis in the larval trunk is not directly associated with the organization of adult organs, but it contributes to the speed of metamorphosis after settlement.     

Oxidant stress from uncoupled nitric oxide synthase impairs vasodilation in fetal lambs with persistent pulmonary hypertension

Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased NO release and impaired pulmonary vasodilation. We investigated the hypothesis that increased superoxide (O(2)(*-)) release by an uncoupled endothelial nitric oxide synthase (eNOS) contributes to impaired pulmonary vasodilation in PPHN. We investigated the response of isolated pulmonary arteries to the NOS agonist ATP and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus and in sham-ligated controls in the presence or absence of the NOS antagonist nitro-L-arginine methyl ester (L-NAME) or the O(2)(*-) scavenger 4,5-dihydroxy-1,3-benzenedisulfonate (Tiron). ATP caused dose-dependent relaxation of pulmonary artery rings in control lambs but induced constriction of the rings in PPHN lambs. L-NAME, the NO precursor L-arginine, and Tiron restored the relaxation response of pulmonary artery rings to ATP in PPHN. Relaxation to NO was attenuated in arteries from PPHN lambs, and the response was improved by L-NAME and by Tiron. We also investigated the alteration in heat shock protein (HSP)90-eNOS interactions and release of NO and O(2)(*-) in response to ATP in the pulmonary artery endothelial cells (PAEC) from these lambs. Cultured PAEC and endothelium of freshly isolated pulmonary arteries from PPHN lambs released O(2)(*-) in response to ATP, and this was attenuated by the NOS antagonist L-NAME and superoxide dismutase (SOD). ATP stimulated HSP90-eNOS interactions in PAEC from control but not PPHN lambs. HSP90 immunoprecipitated from PPHN pulmonary arteries had increased nitrotyrosine signal. Oxidant stress from uncoupled eNOS contributes to impaired pulmonary vasodilation in PPHN induced by ductal ligation in fetal lambs.     

Nitric oxide inhibits metamorphosis in larvae of Crepidula fornicata, the slippershell snail

This paper concerns the role of nitric oxide (NO) in controlling metamorphosis in the marine gastropod Crepidula fornicata. Metamorphosis was stimulated by the nitric oxide synthase (NOS) inhibitors AGH (aminoguanidine hemisulfate) and SMIS (S-methylisothiourea sulfate) at concentrations of about 100-1000 micromol l(-1) and 50-200 micromol l(-1), respectively. Metamorphosis was not, however, induced by the NOS inhibitor l-NAME (l-N(G)-nitroarginine methyl ester) at even the highest concentration tested, 500 micromol l(-1). Moreover, pre-incubation with l-NAME at 20 and 80 micromol l(-1) did not increase the sensitivity of competent larvae to excess K(+), a potent inducer of metamorphosis in this species; we suggest that either l-NAME is ineffective in suppressing NO production in larvae of C. fornicata, or that it works only on the constitutive isoform of the enzyme. In contrast, metamorphosis was potentiated by the guanylate cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3, -a]quinoxalin-1-one) in response to a natural metamorphic inducer derived from conspecific adults. Because NO typically stimulates cGMP production through the activation of soluble guanylate cyclase, this result supports the hypothesis that NO acts as an endogenous inhibitor of metamorphosis in C. fornicata. The expression of NOS, shown by immunohistochemical techniques, was detected in the apical ganglion of young larvae but not in older larvae, further supporting the hypothesis that metamorphosis in C. fornicata is made possible by declines in the endogenous concentration of NO during development.     

Reactive oxygen species and anti-oxidant defenses in tail of tadpoles, Xenopus laevis

Tail regression in tadpoles is one of the most spectacular events in anuran metamorphosis. Reactive oxygen species and oxidative stress play an important role during this process. Presently, the cell- and tissue-specific localization of antioxidant enzymes such as superoxide dismutase (SOD) and catalase as well as neuronal and inducible nitric oxide synthase isoforms (nNOS and iNOS) responsible for production of nitric oxide (NO) were carried out during different stages of metamorphosis in tail of tadpole Xenopus laevis. NO also has profound effect on the mitochondrial function having its own nitric oxide NOS enzyme. Hence, in situ staining for NO and mitochondria also was investigated. The distribution of nNOS and iNOS was found to be stage specific, and the gene expression of nNOS was up-regulated by thyroxin treatment. In situ staining for NO and mitochondria shows co-localization, suggesting mitochondria being one of the sources of NO. SOD and catalase showed significant co-localization during earlier stages of metamorphosis, but before the tail regression begins, there was a significant decrease in activity as well as co-localization suggesting increased ROS accumulation. These findings are discussed in terms of putative functional importance of ROS and cytoplasmic as well as mitochondrial derived NO in programmed cell death in tail tissue.     

Localization of nitric oxide synthase-like immunoreactivity in the developing nervous system of the snail Ilyanassa obsoleta

Production of nitric oxide (NO), an evolutionarily conserved, intercellular signaling molecule, appears to be required for the maintenance of the larval state in the gastropod mollusc Ilyanassa obsoleta. Pharmacological inactivation of endogenous nitric oxide synthase (NOS), the enzyme that generates NO, can trigger metamorphosis in physiologically competent larvae of this species. Neuropils in the brains of these competent larvae display histochemical reactivity for NADPH diaphorase (NADPHd), an indication of neuronal NOS activity. The intensity of NADPHd staining is greatest in the neuropil of the apical ganglion (AG), a region of the brain that contains the apical sensory organ and that innervates the bilobed ciliated velum, the larval swimming and feeding organ. Once metamorphosis is initiated, the intensity of NADPHd staining in the AG and presumably, concomitant NO production, decline. The AG is finally lost by the end of larval metamorphosis, some 4 days after induction. To determine if the neurons of the AG are a source of larval NO, we conducted immunocytochemical studies on larval Ilyanassa with commercially available antibodies to mammalian neuronal NOS. We localized NOS-like immunoreactivity (NOS-IR) to 3 populations of cells in competent larvae: somata of the AG and putative sensory neurons in the edge of the mantle and foot. Immunocytochemistry on pre-competent larvae demonstrated that numbers of NOS-IR cells in the AG increase throughout the planktonic larval stage.     

Analysis of nitric oxide-cyclic guanosine monophosphate signaling during metamorphosis of the nudibranch Phestilla sibogae Bergh (Gastropoda: Opisthobranchia)

SUMMARY The gas nitric oxide (NO), and in some cases its downstream second messenger, cyclic guanosine monophosphate (cGMP) function in different taxa to regulate the timing of life-history transitions. Increased taxonomic sampling is required to foster conclusions about the evolution and function of NO/cGMP signaling during life-history transitions. We report on the function and localization of NO and cGMP signaling during metamorphosis of the nudibranch Phestilla sibogae . Pharmacological manipulation of NO or cGMP production in larvae modulated responses to a natural settlement cue from the coral Porites compressa in a manner that suggest inhibitory function for NO/cGMP signaling. However, these treatments were not sufficient to induce metamorphosis in the absence of cue, a result unique to this animal. We show that induction of metamorphosis in response to the settlement cue is associated with a reduction in NO production. We documented the expression of putative NO synthase (NOS) and the production of cGMP during larval development and observed no larval cells in which NOS and cGMP were both detected. The production of cGMP in a bilaterally symmetrical group of cells fated to occupy the distal tip of rhinophores is correlated with competence to respond to the coral settlement cue. These results suggest that endogenous NO and cGMP are involved in modulating responses of P. sibogae to a natural settlement cue. We discuss these results with respect to habitat selection and larval ecology.     

Tail development and regeneration throughout the life cycle of the four-toed salamander Hemidactylium scutatum

The salamander tail displays different functions and morphologies in the aquatic and terrestrial stages of species with complex life cycles. During metamorphosis the function of the tail changes; the larval tail functions in aquatic locomotion while the juvenile and adult tail exhibits tail autotomy and fat storage functions. Because tail injury is common in the aquatic environment, we hypothesized that mechanisms have evolved to prevent altered larval tail morphology from affecting normal juvenile tail morphology. The hypothesis that injury to the larval tail would not affect juvenile tail morphology was investigated by comparing tail development and regeneration in Hemidactylium scutatum (Caudata: Plethodontidae). The experimental design included larvae with uninjured tails and with cut tails to simulate natural predation. The morphological variables analyzed to compare normally developing and regenerating tails were 1) tail length, 2) number of caudal vertebrae, and 3) vertebral centrum length. Control and experimental groups do not differ in time to metamorphosis or snout-vent length. Tails of experimental individuals are shorter than controls, yet they display a significantly higher rate of tail growth and less resorption of tail tissue. Anterior to the site of tail injury, caudal vertebrae in juveniles display greater average centrum lengths. Results suggest that regenerative mechanisms are functioning not only to produce structures, but also to influence growth of existing structures. Further investigation of juvenile and adult stages as well as comparative analyses of tail morphology in salamanders with complex life cycles will enhance our understanding of amphibian development and of the evolution of amphibian life cycles. J Morphol 233:15–29, 1997. © 1997 Wiley-Liss, Inc.     

Serotonin and Nitric Oxide Regulate Metamorphosis in the Marine Snail Ilyanassa obsoleta

Several neuroactive compounds have been implicated as playingroles in the circuitry that controls larval metamorphosis inmarine molluscs. For the caenogastropod Ilyanassa obsoleta,results of neuroanatomical studies suggest that the productionof nitric oxide (NO) increases throughout the planktonic stageand that NO production is necessary for the maintenance of thelarval state, especially as it becomes metamorphically competent.Bath application or injection of exogenous serotonin (5HT) caninitiate metamorphosis in competent larvae, and exogenous NOcan inhibit such serotonergically-induced metamorphosis. Inhibitionof endogenous nitric oxide synthase (NOS) can also trigger larvalmetamorphosis. The production of endogenous NO appears to decreaseconcurrently with the initiation of metamorphosis, but the specificinteractions between serotonergic and nitrergic neurons areunknown. Evidence in support of NO acting to up-regulate theenzyme guanylyl cyclase (GC) is still equivocal. Thus, we donot yet know if NO exerts its effects through the actions ofcyclic 3',5'-guanosine monophosphate (cGMP) or by a cGMP-independentmechanism. The ubiquity of nitrergic signalling and its significancefor developing molluscan embryos and larvae are still the subjectof speculation and require further investigation.     

The mechanism of bone resorption by cyclosporin: involvement of the NO-cGMP pathway

Treatment with cyclosporin A (CsA) following solid organ transplantations such as heart or liver generally results in bone loss. However, in vitro studies show that CsA inhibits bone resorption. Our previous in vivo animal studies demonstrated that the effects of nitric oxide (NO) on bone are biphasic; at high doses, NO increases bone resorption. In this study, we have examined in an in vitro setting to determine whether the bone loss caused by CsA administration is dependent on the NO-cyclic guanosine monophosphate (cGMP) pathway. Freshly isolated osteoclast-rich neonatal rat long bone marrow cells were added to 100 microM thick dentin sections that had been seeded with neonatal-rat calvarial osteoblasts. These co-cultures were maintained for 48 hrs in a basal medium with CsA (1, 5, and 10 microg/ml), both alone and with either L-Arginine (NO substrate; 10-3M), L-NAME (NO synthase enzyme inhibitor; 10-4M), or the combination of the two. The cultures were then fixed in cold 95% ethanol and stained with tartrate resistant acid phosphatase (TRAP) to identify osteoclasts and sites of osteoclastic resorption. Preparations were analyzed using an automated histomorphometry software package. Scanning electron microscopy affirmed that the areas identified by light microscopy as resorption sites contained osteoclastic lacunae. CsA inhibited bone resorption dose-dependently. CsA at 10 microg/ml produced a 90% inhibition of bone resorption (control = 5.5 -/+2.0%; CsA = 0.64 -/+ 0.09=). L-Arginine reversed this inhibition by 90% (Arg + CsA = 4.23 -/+ 1.57%; CsA = 0.64 -/+ 0.09%). The application of NOS inhibitor L-NAME inhibited bone resorption by 87% (Arg + CsA + L-NAME = 0.55 -/+ 0.14%; Arg + CsA = 4.23 -/+ 1.5%). We conclude that NO-cGMP pathway is involved in the CsA induced bone loss.     

Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase

Soluble guanylate cyclase (sGC) is an important downstream intracellular target of nitric oxide (NO) that is produced by endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). In this study, we demonstrate that sGC exists in a complex with eNOS and heat shock protein 90 (HSP90) in aortic endothelial cells. In addition, we show that in aortic smooth muscle cells, sGC forms a complex with HSP90. Formation of the sGC/eNOS/HSP90 complex is increased in response to eNOS-activating agonists in a manner that depends on HSP90 activity. In vitro binding assays with glutathione S-transferase fusion proteins that contain the alpha- or beta-subunit of sGC show that the sGC beta-subunit interacts directly with HSP90 and indirectly with eNOS. Confocal immunofluorescent studies confirm the subcellular colocalization of sGC and HSP90 in both endothelial and smooth muscle cells. Complex formation of sGC with HSP90 facilitates responses to NO donors in cultured cells (cGMP accumulation) as well as in anesthetized rats (hypotension). These complexes likely function to stabilize sGC as well as to provide directed intracellular transfer of NO from NOS to sGC, thus preventing inactivation of NO by superoxide anion and formation of peroxynitrite, which is a toxic molecule that has been implicated in the pathology of several vascular diseases.     

The influence of nitric oxide synthase inhibitor L-NAME on bones of male rats with streptozotocin-induced diabetes

The pathophysiological processes underlying the development of diabetic osteopenia has not hitherto been elucidated. Induction of streptozotocin diabetes leads in our experiments to decrease of bone density, ash, mineral content and to thinner cortical width compared to control male rats. In order to investigate the pathogenetic role of bone resorption by osteoclasts in streptozotocin-induced diabetes, we determined the circulating levels of tartrate-resistant acid phosphatase (TRAP), a biochemical marker for bone resorption. Plasma TRAP values in diabetic rats did not differ from their corresponding controls. Streptozotocin diabetes by itself did not have any effect on the weight of seminal vesicles which are highly testosterone-dependent. Low doses of nitric oxide cause bone resorption, but higher doses of NO inhibit bone resorbing activity. We examined the effect of L-NAME (inhibitor of nitric oxide production) after six weeks of administration to diabetic rats. There was no further significant loss of bone mineral density, ash and mineral content or tibia weight in diabetic rats treated with L-NAME. L-NAME itself did not decrease bone metabolism. In our study no evidence of an increased bone resorption was found. Our results have indicated that a predominance of bone resorption over bone formation is not involved in the pathogenesis of diabetes-associated osteopenia. Inhibition of NO neither increased osteoclastic activity (TRAP) nor induced osteopenia in L-NAME-treated rats. This suggests a possibility that NO is not involved in the pathogenesis of diabetic osteopenia.     

Protein nitration as footprint of oxidative stress-related nitric oxide signaling pathways in developing Ciona intestinalis

Developmental processes in the ascidian Ciona intestinalis depend on a complex interplay of events including, during metamorphosis, a caspase-dependent apoptosis which is regulated by the nitric oxide (NO)-cGMP signaling pathway. Herein we disclose an alternate NO-mediated signaling pathway during Ciona development which appears to be critically dependent on local redox control. Evidence in support of this conclusion includes: (a) inhibitors of NO synthase (NOS) and scavengers of NO-derived nitrating agents markedly decrease the rate of Ciona metamorphosis; (b) an NO donor or peroxynitrite caused an opposite effect; (c) increased protein nitration is observed at larva stage. Integrated proteomic and immunochemical methodologies identified nitrated tyrosine residues in ERK and snail. Overall, these results point to protein nitration as a hitherto overlooked NO-dependent regulatory mechanism in Ciona which is specifically triggered by elevated ROS production during developmental processes.     

Decreased association of HSP90 impairs endothelial nitric oxide synthase in fetal lambs with persistent pulmonary hypertension

Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased nitric oxide (NO) release and impaired pulmonary vasodilation. We investigated the hypothesis that decreased association of heat shock protein 90 (HSP90) with endothelial NO synthase (eNOS) impairs NO release and vasodilation in PPHN. The responses to the NOS agonist ATP were investigated in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus, and in sham ligation controls. ATP caused dose-dependent vasodilation in control pulmonary resistance arteries, and this response was attenuated in PPHN vessels. The response of control pulmonary arteries to ATP was attenuated by NG-nitro-l-arginine methyl ester (l-NAME), a NOS antagonist, and geldanamycin, an inhibitor of HSP90-eNOS interaction. The attenuated response to ATP observed in PPHN was improved by pretreatment of vessels with l-NAME or 4,5-dihydroxy-1,3-benzene-disulfonate, a superoxide scavenger. Pulmonary arteries from PPHN lambs had decreased basal levels of HSP90 in association with eNOS. Association of HSP90 with eNOS and NO release increased in response to ATP in control pulmonary artery endothelial cells, but not in cells from PPHN lambs. Decreased HSP90-eNOS interactions may contribute to the impaired NO release and vasodilation observed in the ductal ligation model of PPHN.