Nitric oxide-mediated cGMP synthesis in oligodendrocytes in the developing rat brain

We investigated the nature of cGMP-synthesizing cells in the developing rat forebrain using cGMP-immunocytochemistry in combination with in vitro incubation of brain slices. When brain slices of immature rats, aged between 1 and 4 weeks, were incubated with sodium nitroprusside (SNP), a nitric oxide (NO) donor compound, in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), small round cells with a few processes in and around the corpus callosum were visualized with the cGMP-antibody. The morphology and the distribution of the cGMP-positive cells were consistent with the criteria for oligodendrocytes. Furthermore, the cGMP-positive cells expressed 2′3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) and gelsolin, which are marker proteins for oligodendrocytes. Therefore, we concluded that the cGMP-positive cells were oligodendrocytes. A subpopulation of the oligodendrocyte was found to be cGMP-immunoreactive also when slices were incubated in the absence of SNP. Furthermore, incubation of the slice in the presence of L-NAME, an inhibitor of NO synthase, but in the absence of SNP abolished cGMP immunostaining. In addition, some populations of neurons and astrocytes in restricted brain areas produced cGMP in response to the incubation with SNP as previously reported, whereas both ameboid and ramified microglial cells did not respond to the treatment. Atrial natriuretic peptide, a stimulator of particulate guanylyl cyclase, enhanced cGMP synthesis in astrocytes in some brain regions but not in oligodendrocytes. These findings indicate that oligodendrocytes in the immature rat brain express soluble guanylyl cyclase. No cGMP-positive oligodendrocytes were found in the mature rat brain, suggesting that cGMP may mediate signals related to myelinogenesis in the rat brain. GLIA 19:286–297, 1997. © 1997 Wiley-Liss, Inc.     

C-type natriuretic Peptide stimulates prolactin secretion by a hypothalamic site of action

The most recently discovered member of the family of natriuretic peptides, C-type natriuretic peptide (CNP), exerts many pharmacologic actions similar to its structural homolog A-type natriuretic peptide (ANP). Like ANP it failed to significantly alter prolactin release from dispersed, rat anterior pituitary cells incubated under static or dynamic conditions. Unlike ANP, however, which inhibits prolactin secretion in vivo by a hypothalamic action, CNP injection into the third cerebroventricle significantly stimulated prolactin secretion in ovariectomized, conscious rats. The effect was highly significant 15 min after injection and transient, lasting 30 min in animals injected with 2 nmole CNP. In a companion group of rats, significant inhibition of plasma prolactin levels was observed after central administration of similar doses of ANP. These results suggest differing hypothalamic actions of the CNP and ANP perhaps mediated by multiple natriuretic peptide receptors present in the tissue. Further, they provide additional support for unique roles exerted within the central nervous system by these structural homologs.     

Dorsal root ganglion axon bifurcation tolerates increased cyclic GMP levels: the role of phosphodiesterase 2A and scavenger receptor Npr3

A cyclic GMP (cGMP) signaling pathway, comprising C‐type natriuretic peptide (CNP), its guanylate cyclase receptor Npr2, and cGMP‐dependent protein kinase I, is critical for the bifurcation of dorsal root ganglion (DRG) and cranial sensory ganglion axons when entering the mouse spinal cord and the hindbrain respectively. However, the identity and functional relevance of phosphodiesterases (PDEs) that degrade cGMP in DRG neurons are not completely understood. Here, we asked whether regulation of the intracellular cGMP concentration by PDEs modulates the branching of sensory axons. Real‐time imaging of cGMP with a genetically encoded fluorescent cGMP sensor, RT‐PCR screens, in situ hybridization, and immunohistology combined with the analysis of mutant mice identified PDE2A as the major enzyme for the degradation of CNP‐induced cGMP in embryonic DRG neurons. Tracking of PDE2A‐deficient DRG sensory axons in conjunction with cGMP measurements indicated that axon bifurcation tolerates increased cGMP concentrations. As we found that the natriuretic peptide scavenger receptor Npr3 is expressed by cells associated with dorsal roots but not in DRG neurons itself at early developmental stages, we analyzed axonal branching in the absence of Npr3. In Npr3‐deficient mice, the majority of sensory axons showed normal bifurcation, but a small population of axons (13%) was unable to form T‐like branches and generated turns in rostral or caudal directions only. Taken together, this study shows that sensory axon bifurcation is insensitive to increases of CNP‐induced cGMP levels and Npr3 does not have an important scavenging function in this axonal system.     

Guanylyl cyclase-B represents the predominant natriuretic peptide receptor expressed at exceptionally high levels in the pineal gland

The generation and function(s) of the signalling molecule cyclic GMP (cGMP) in brain are still poorly understood. One mechanism to raise intracellular cGMP levels is binding of C-type natriuretic peptide (CNP) to a membrane guanylyl cyclase (GC), termed GC-B. Here, we demonstrate an exceptionally strong expression of GC-B in the pineal gland. Crosslinking experiments performed with 125I-Tyr(0)-CNP and membranes from various rat tissues identified the receptor as a 130-kDa protein, expressed at highest levels in pineal membranes. Receptor autoradiography on brain sections confirmed a striking density of CNP binding sites in pineal tissue, whereas binding sites for the related atrial natriuretic peptide (ANP) predominate in other regions of the brain. Incubations of freshly dissected whole pineal glands in either the absence or presence of natriuretic peptides followed by immunohistochemical analyses of cGMP revealed strong accumulations of cGMP in response to CNP but not to ANP in the majority of pinealocytes. Stimulation of soluble GC (sGC) activity by use of sodium nitroprusside (SNP) resulted in a very similar pattern of cGMP immunostaining, indicating a co-expression at high levels of particulate and soluble forms of GC. These findings point to a major role of cGMP signalling in pinealocytes and suggest an important regulatory function for CNP.     

Localization of natriuretic peptide-activated guanylate cyclase mRNAs in the rat brain

Physiological actions of atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) are elaborated by membrane-bound natriuretic peptide receptors (NPRs). These receptors possess intracellular guanylate cyclase domains that mobilize cyclic guanosine monophosphate upon binding of peptide. Two distinct NPR subtypes have been described in brain: the NPR-A selectively binds ANP, whereas NPR-B exhibits high affinity for CNP. To define further the potential domains of ANP and CNP action in brain, the present study used in situ hybridization histochemistry to map NPR-A and NPR-B mRNA-expressing cell populations. Significant levels of neuronal NPR-A mRNA expression were observed only in the mitral cell layer of the olfactory bulb, medial habenula, subfornical organ, and area postrema. Expression of NPR-A mRNA was observed in forebrain white matter tracts, suggesting synthesis in glial cells. In contrast, NPR-B mRNA was widely expressed throughout the neuraxis. In the telencephalon, signal was abundant throughout limbic cortex and neocortex, olfactory bulb, hippocampus, and amygdala. Intense NPR-B mRNA hybridization was observed in preoptic-hypothalamic neuroendocrine circuits and in motor nuclei of cranial nerves. Intermediate expression of NPR-B mRNA was observed in brainstem nuclei controlling autonomic function. Labeling for NPR-B but not NPR-A mRNA was observed in pituicytes in the neural lobe of the pituitary and in scattered cells of the anterior pituitary. These results suggest that CNP is the primary biologically active natriuretic peptide in brain. In contrast with NPR-B, NPR-A appears to be expressed largely in restricted cell populations containing high levels of ANP and in circumventricular organs. These data implicate the NPR-A in autoregulation of ANP neurons and central registration of cardiac ANP release. © 1996 Wiley-Liss, Inc.     

C-type natriuretic peptide exerts effects opposing those of atrial natriuretic peptide on anxiety-related behaviour in rats

As evidence exists that C-type natriuretic peptide (CNP) exerts effects opposing those of atrial natriuretic peptide (ANP), we studied the behavioural properties of CNP after central infusion in rats by their performance in the elevated plus maze. Doses of 0.5 μg and 5 μg i.c.v. had distinct anxiogenic properties. Our data suggest opposing effects of CNP and ANP on anxiety-related behaviour in rats, which appear to be mediated via different receptor occupation and brain regions by a corticotropin-releasing hormone (CRH)-dependent mechanism.     

Presence of soluble and particulate guanylyl cyclase in the same hippocampal astrocytes

The localisation of particulate and soluble guanylyl cyclase was studied in hippocampal astrocytes. Counting the colocalisation of cGMP immunoreactivity with the astrocytic marker glial fibrillary acidic protein after stimulation of brain slices with sodium nitroprusside (0.1 mM) or atrial natriuretic peptide (100 nM), we were able to show that at least 67% of the hippocampal astrocytes contained both guanylyl cyclase isoforms. In addition, it was shown that a large number of atrial natriuretic peptide, brain-derived natriuretic peptide or sodium nitroprusside responsive cells contain the beta1-subunit of the soluble guanylyl cyclase. The results show that, in at least a subset of hippocampal astrocytes, soluble and particulate guanylyl cyclases are simultaneously present in the same cells.     

Nitric oxide targets oligodendrocytes and promotes their morphological differentiation

In the central nervous system, nitric oxide (NO) transmits signals from one neurone to another, or from neurones to astrocytes or blood vessels, but the possibility of oligodendrocytes being physiological NO targets has been largely ignored. By exploiting immunocytochemistry for cGMP, the second messenger generated on activation of NO receptors, oligodendrocytes were found to respond to both exogenous and endogenous NO in cerebellar slices from rats aged 8 days to adulthood. Atrial natriuretic peptide, which acts on membrane‐associated guanylyl cyclase‐coupled receptors, also raised oligodendrocyte cGMP in cerebellar slices. The main endogenous source of NO accessing oligodendrocytes appeared to be the neuronal NO synthase isoform, which was active even under basal conditions and in a manner that was independent of glutamate receptors. Oligodendrocytes in brainstem slices were also shown to be potential NO targets. In contrast, in the optic nerve, oligodendrocyte cGMP was raised by natriuretic peptides but not NO. When cultures of cerebral cortex were continuously exposed to low NO concentrations (estimated as 40–90 pM), oligodendrocytes responded with a striking increase in arborization. This stimulation of oligodendrocyte growth could be replicated by low concentrations of 8‐bromo‐cGMP (maximum effect at 1 µM). It is concluded that oligodendrocytes are probably widespread targets for physiological NO (or natriuretic peptide) signals, with the resulting rise in cGMP serving to enhance their growth and maturation. NO might help coordinate the myelination of axons to the ongoing level of neuronal activity during development and could potentially contribute to adaptive changes in myelination in the adult. GLIA 2015;63:383–399     

脑C型利钠利尿肽的mRNA转录及其血浆表达水平在大鼠创伤性脑水肿中的改变

目的:观察创伤性脑水肿对脑组织中C型利钠利尿肽(CNP)mRNA转录水平的影响及与其血浆中CNP水平的相关性。方法:35只S-D大鼠随机分入对照组、创伤组。采用冲击加速机制复制重度脑创伤。创伤后1天组的脑CNP mRNA转录水平采用逆转录-多聚酶链反应法(RT-PCR)行半定量测定。并同时采用酶免疫分析法(EIA),观测伤后多时点的体内血浆CNP水平。结果:创伤后1天脑水肿期内,挫伤皮质,同侧丘脑,对侧皮质,对侧丘脑和延髓等部位,与对照组相比,其CNP mRNA转录水平无显著改变(P>0.05)。然而,创伤后1天与3天组血浆的CNP水平均较对照组有明显升高(P<0.05)。结论:尽管CNP一直被认为是一种神经肽,但可能并未参与创伤性脑水肿的病理过程。而全身循环中升高的血浆CNP水平可能为机体对重度脑创伤的反应之一,其作用主要通过循环系统发挥。     

Hypothalamic effects of C-type natriuretic Peptide on luteinizing hormone secretion

Previous studies have demonstrated hypothalamic sites of action of A-type natriuretic peptide (ANP) in the inhibition of luteinizing hormone (LH) secretion, acting at least in part, via an opiatergic mechanism. C-type natriuretic peptide (CNP) was identified recently and is thought to be the predominant brain form of the family of natriuretic peptides. Third cerebroventricular injection of CNP in doses of either 0.1, 1.0 or 2.0 nmole significantly inhibited, in a dose-related fashion, plasma LH levels when compared to levels present in saline-injected controls. When compared to the LH-inhibiting action of ANP, CNP appeared more potent (effective at lower doses) and efficacious (longer duration of action for the maximum effective doses). The LH-inhibiting effect of CNP was blocked by prior treatment with the δ-opioid receptor antagonist naltrindole (50 μg), suggesting an enkephalinergic mechanism of action. CNP in log doses ranging from 0.01 to 1,000 nM did not significantly alter LH release from dispersed pituitary cells harvested from random cycle female rats, either under static or dynamic (perifusion) incubation conditions. These results indicate that CNP, like ANP, acts at the hypothalamic level to alter LH secretion and suggest that CNP may be the preferential neuroactive members of this family of peptides.     

Intravenous C-type natriuretic peptide augments behavioral and endocrine effects of cholecystokinin tetrapeptide in healthy men.

Given the anxiogenic effects of the type-B natriuretic peptide receptor agonist C-type natriuretic peptide (CNP) in rodents, we investigated the influence of CNP pretreatment upon the behavioral and endocrine action of the panicogen cholecystokinin tetrapeptide (CCK-4) in healthy men. In a randomized double-blind balanced design, 20 male volunteers were given an intravenous infusion of 300 microg of CNP vs. placebo followed by 25 microg of CCK-4. The behavior was assessed using panic, anxiety, and dissociation questionaires before the infusion and after the CCK-4 stimulus. Furthermore, the stress-sensitive hormones adrenocorticotropic hormone (ACTH), cortisol, and prolactin were measured. CNP pretreatment enhanced the anxiogenic and prodissociative effects of CCK-4 and significantly augmented the ACTH surge after CCK-4. However, no effect of CNP was seen upon panic symptoms. Our preliminary data support a role of type-B natriuretic peptide receptors in anxiety modulation in normal man.     

Nocturnal accumulation of cyclic 3',5'-guanosine monophosphate (cGMP) in the chick pineal organ is dependent on activation of guanylyl cyclase-B

The role of cGMP in the avian pineal is not well understood. Although the light-sensitive secretion of melatonin is a well-known output of the circadian oscillator, pharmacologically elevated cGMP levels do not result in altered melatonin secretory amplitude or phase. This suggests that pineal cGMP signalling does not couple the endogenous circadian oscillator to the expression of melatonin rhythms. Nonetheless, the free-running rhythm of cGMP signalling implies a link to the circadian oscillator in chick pinealocytes. As the circadian rhythm of cGMP levels in vitro is not altered by pharmacological inhibition of phosphodiesterase activity, we infer that the synthesis, rather than the degradation of cGMP, is under circadian control. In vitro experiments with the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine as well as with an inhibitor of the NO-sensitive soluble guanylyl cyclase (sGC), showed that the NOS-sGC pathway does not play a major role in the circadian control of cGMP generation. In organ culture experiments, we demonstrated that C-type natriuretic peptide (CNP), but not atrial natriuretic peptide (ANP), elevated daytime levels of cGMP. As CNP acts on the membrane guanylyl cyclase isoform B (GC-B), which is expressed at very high levels in mammalian pineals, we investigated the effect of the membrane GC-specific inhibitor HS-142 on chick pineal cGMP levels. CNP-induced daytime cGMP levels were reduced by HS-142. More importantly, 'spontaneously' high nocturnal levels of cGMP in vitro were reduced to daytime levels by acute addition of HS-142. These data implicate endogenous nocturnal CNP release and subsequent activation of GC-B as the major input driving cGMP synthesis in the chick pineal organ.     

C-type natriuretic peptide is a Schwann cell-derived factor for development and function of sensory neurones

Cyclic GMP (cGMP) is known to play important roles for neuronal development and neurite pathfinding. However, the regulatory mechanism that governs the synthesis of cGMP in the nervous system is not well defined. In the present study, we examined the role of C-type natriuretic peptide (CNP), which increases intracellular cGMP upon binding to its receptor, guanylyl cyclase (GC)-B, in the peripheral nervous system. Immunohistochemistry revealed that CNP is demonstrated in Schwann cells, whereas GC-B mRNA is highly expressed in dorsal root ganglion (DRG) neurones. In cultured DRG neurones, GC-B was demonstrated in dendrites of TrkA-positive cells, where it co-exists with cGMP-dependent protein kinase I (cGKI), the major intracellular mediator of cGMP actions. Addition of CNP in the culture medium increased the density of fine neurites, which was accompanied by the increase in phosphorylation of vasodilator-stimulated phosphoprotein, a cGKI substrate. Furthermore, in mice deficient for the CNP gene (CNP-KO), the numbers of TrkA-positive DRG neurones were diminished. Likewise, there were much less cGKI-positive neurones in DRG and cGKI-positive fibres in the dorsal spinal cord of CNP-KO than wild-type mice. Finally, the bone deformity-rescued CNP-KO mice displayed a decreased response to formalin-induced pain compared to wild-type. Taken together, these results suggest that CNP is derived from Schwann cells and plays an important role for the development and function of nociceptive sensory neurones.     

Localization and age-related changes of nitric oxide- and ANP-mediated cyclic-GMP synthesis in rat cervical spinal cord: an immunocytochemical study

An immunocytochemical technique was used to study the localization and developmental aspects of cyclic GMP (cGMP)-synthesizing structures in the cervical spinal cord of 2-week and 3-month-old Lewis rats in response to the nitric oxide (NO) donor sodium nitroprusside (SNP) and/or atrial natriuretic peptide (ANP). By using cell-specific markers, the cell structures involved were investigated. To visualize cGMP, a combined technique of low- and high-power magnification, using a confocal laser scanning microscope was used. NOS-mediated cGMP synthesis was observed in the cervical spinal cord in laminae I, II and III in 14-day-old rats, which activity was mainly absent at the age of 3 months. The involvement of NO in the NMDA-mediated increase in cGMP immunostaining (cGMP-IS) was demonstrated by the absence of cGMP-IS in slices incubated in the presence of NMDA together with the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). This NO-mediated effect of NMDA on cGMP-IS was completely absent in the 3-month-old rats. ANP-mediated cGMP synthesis resulted in an increase in cGMP in laminae I and II, which was generally similar at both ages. Astrocytes in both white and gray matter were found to be cGMP-IS in the basal, NO- and ANP-stimulated conditions. Using confocal laser microscopy, NO-mediated cGMP synthesis was observed in large cholinergic terminals nearby motor neurons in the ventral horn. An extensive colocalization between NO-stimulated cGMP synthesis and parvalbumin-positive (GABAergic) neurons and fibers was observed in all laminae. In the ANP-stimulated condition, a colocalization with parvalbumin structures was found in laminae II and III. No NO- or ANP-mediated cGMP synthesis was found in fibers immunopositive for the presynaptic glutamate transporter, serotonin, or tyrosine hydroxylase.     

Natriuretic peptides regulate the expression of tissue factor and PAI-1 in endothelial cells

In the present study, we demonstrate that brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) interact with angiotensin II (Ang II) in regulative blood coagulation and fibrinolysis by suppressing the expressions of both tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) induced by Ang II. The expressions of TF and PAI-1 mRNA were analyzed by northern blotting methods, and the activities of TF on the surface of rat aortic endothelial cells (RAECs) and PAI-1 in the culture media were respectively measured by chromogenic assay. Both BNP and CNP suppressed the expressions of TF and PAI-1 mRNA induced by Ang II in a time- and concentration-dependent manner via cGMP cascade, which suppressions were accompanied by respective decrease in activities of TF and PAI-1. However, neither the expression of tissue factor pathway inhibitor (TFPI) nor tissue-type plasminogen activator (TPA) mRNA was affected by the treatment of BNP and CNP.     

The C-Type Natriuretic Peptide Receptor is the Predominant Natriuretic Peptide Receptor mRNA Expressed in Rat Hypothalamus

The natriuretic peptide receptors (NPR) are membrane-bound guanylate cyclases with extracellular binding domains specific for particular members of the natriuretic peptide family. NPR-A binds atrial natriuretic peptide (ANP) with high affinity, whereas the NPR-B appears to be specific for C-type natriuretic peptide (CNP). Previous data indicating extensive overlap between localization of ANP and CNP in hypothalamic neuroendocrine circuits suggest the importance of determining whether specificity of natriuretic peptide action may be conferred via receptor type present on target cells. To address this issue, we used in situ hybridization histochemistry to localize NPR-A and NPR-B mRNA in the hypothalamus. NPR-A mRNA was not found in substantial abundance in any hypothalamic nucleus; however, detectable NPR-A signal was observed in other brain regions, including the subfornical organ and medial habenula. In contrast, NPR-B mRNA was expressed throughout the hypothalamus, including neurons of the magnocellular and parvocellular paraventricular, the arcuate, and the supraoptic nuclei. Expression was also seen in other nuclei essential to neuroendocrine control, including the median preoptic, anteroventral periventricular, tuberomammillary, ventromedial and suprachiasmatic nuclei. NPR-B mRNA was also observed in the neural lobe of the pituitary gland, suggesting expression by pituicytes. The results suggest that NPR-B is the primary natriuretic peptide receptor in hypothalamus, and by inference indicate that CNP is the primary active natriuretic peptide in neuroendocrine regulation.     

Opposing Neuroendocrine Actions of the Natriuretic Peptides: C-Type and A-Type Natriuretic Peptides do not Interact with the same Hypothalamic Cells Controlling Prolactin Secretion

The two major members of the family of natriuretic peptides (NPs) in brain, A-type natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) exert opposing actions on the neuroendocrine regulation of prolactin (PRL) secretion. We have targeted for compromise and destruction cells within the diencephalon which bear receptors for ANP (NPR-A receptors), CNP (NPR-B receptors), or both peptides (NPR-C receptors) using novel cytotoxin cell targeting methodology in order to determine if the neuroendocrine effects of these two peptides are exerted on similar cell systems. In animals pretreated with ANP conjugated to the cytotoxic A chain of ricin, central administration of a dose of ANP which is known to inhibit PRL secretion did not alter PRL levels in plasma; however, subsequent administration of CNP elicited the stimulation of PRL secretion. In rats pretreated with CNP-ricin A chain conjugate, a treatment we hypothesize targets for destruction CNP responsive cells, ANP injection did inhibit PRL secretion, while the stimulatory effect of CNP was absent. These results suggest that the neuroendocrine effects of these two natriuretic peptides on PRL secretion are expressed on different cellular elements of the hypothalamo-pituitary axis. Furthermore, they reveal that neither peptide acts directly on the tuberoinfundibular dopamine system since pretreatment with either cytotoxin conjugate failed to alter basal PRL levels. Thus ANP and CNP do not appear to express opposing actions on the same cell systems, suggesting the recruitment of each peptide individually by differing, unique stimuli for PRL release.     

Detection of membrane-bound guanylate cyclase activity in rat C6 glioma cells at different growth states following activation by natriuretic peptides

We studied the activity and the ultracytochemical localization of membrane-bound guanylate cyclase (GC) after stimulation with rat atrial natriuretic peptide (rANP), porcine brain natriuretic peptide (pBNP), rat brain natriuretic peptide (rBNP), or porcine C-type natriuretic peptide (CNP) in rat C6 glioma cells during proliferation or following exposure of confluent cells to dibutyryl cyclic AMP (db-cAMP) or retinoic acid (RA). Under our experimental conditions all peptides were activators of GC as demonstrated by the accumulation of cGMP within cells. During proliferation of C6 cells, the amounts of cGMP remained approximately constant. However, at subconfluency, confluency and postconfluency, the GC reaction product was located at different sites in C6 cells. At subconfluency, GC reaction product was on membranes of protoplasmic extensions, at postconfluency, GC reaction product was in association with membranes of cell bodies, and at confluency, both localizations of GC reaction product were detected. Incubation of confluent cells in culture medium containing db-cAMP or RA induced the appearance of long and slender protoplasmic extensions. Under these conditions, the GC reaction product was localized exclusively to these processes. These data suggest that GC is differently located depending on the state of growth of glial cells, and that in differentiating glial cells GC is preferentially located in cell processes.     

Alpha-helical-corticotropin-releasing hormone reverses anxiogenic effects of C-type natriuretic peptide in rats

Previously we have shown that atrial natriuretic peptide (ANP) has anxiolytic-like properties after intraperitoneal, intracerebroventricular and intraamygdala infusion in rats. Since C-type natriuretic peptide (CNP) exerts endocrine and behavioral effects opposing those of ANP, we characterized the behavioral properties of CNP after icv infusion in rats by their performance in the elevated plus maze with and without the corticotropin-releasing hormone (CRH) antagonist alpha-helical-CRH (alpha-CRH). Low CNP doses of 0.05 microg icv or 0.1 microg icv did not significantly influence the behavior of rats in the plus maze. At higher doses (0.5 microg, 2 microg, 5 microg icv) CNP had distinct anxiogenic properties. Our hypothesis that corticotropin-releasing hormone (CRH) is involved, which elicits anxiety-like behavior, was examined by icv coadministration of alpha-CRH, an antagonist at CRH-1 and CRH-2-receptors. Icv alpha-CRH alone had no intrinsic anxiolytic properties at a dose of 25 microg. The anxiogenic effects of 2 microg CNP icv seen in the plus maze were entirely blocked by alpha-CRH. Directly after exposition ACTH and corticosterone levels did not differ between the groups, but after 30 min ACTH levels were significantly higher in the CNP-treated group compared to alpha-CRH/CNP-treated animals. Corticosterone was found significantly lowered in the alpha-CRH/saline group compared to the CNP treated group but not compared to saline controls. Our data suggest opposing effects of CNP and ANP on anxiety-related behavior and neuroendocrine regulation in rats, which appear to be mediated via different receptor occupation and brain regions, and by a CRH-dependent mechanism.