The action of isatin (2,3-dioxoindole) an endogenous indole on brain natriuretic and C-type natriuretic peptide-induced facilitation of memory consolidation in passive-avoidance learning in rats

Isatin is an endogenous indole which has been shown to counteract some of the effects of atrial natriuretic peptide (ANP) both in vitro and in vivo. The present study was designed to determine whether it could antagonise in vivo effects of the related peptides brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). The model used was consolidation of memory in a one-trial step-through passive-avoidance paradigm in the rat. Previous studies have shown that all three peptides (1 microg intracerebroventricular) can consolidate such learning and increase latency to entry a dark box. Isatin was given intraperitoneally at doses of 5, 10 and 50 mg/kg before the peptide, or a saline control. Both BNP and CNP significantly increased the latency of entry. Isatin alone had no effect. Isatin reduced the effect of both BNP and CNP; this was significant for its effect on BNP at 50 mg/kg and on CNP at both 10 and 50 mg/kg. These results show that isatin can inhibit behavioural effects of BNP and CNP as well as ANP.     

Distribution of natriuretic peptide precursor mRNAs in the rat brain

Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) represent members of a recently discovered neuropeptide family involved in central regulation of endocrine and autonomic functions. The present study employed an in situ hybridization approach to provide the first detailed comparative mapping of ANP, BNP, and CNP mRNAs in brain. Results indicate that ANP mRNA is highly expressed in anterior olfactory nuclei, limbic cortices, dorsal endopiriform nucleus, hippocampal subfield CA1, cortical amygdaloid nuclei, medial habenula, anteroventral periventricular and arcuate nuclei, periventricular stratum, zona incerta, mammillary nuclei, inferior olive, nucleus ambiguus, and pontine paragigantocellular nuclei. CNP mRNA is expressed at highest levels in olfactory nuclei, limbic cortices, dorsal endopiriform nucleus, hippocampal subfields CA1–3, anteroventral periventricular and arcuate nuclei, and numerous brainstem regions (including the pontine, lateral reticular, solitary tract, prepositus hypoglossal, and spinal trigeminal nuclei). Positive labeling for BNP mRNA was not observed in brain. The presence of both ANP and CNP mRNA in the same regions of distinct nuclei (e.g., the anteroventral periventricular and arcuate nuclei) suggests the potential for coexpression. Overall, the present data are consistent with a prominent role for both ANP and CNP in neuroendocrine regulation and central cardiovascular integration. The extensive localization of ANP and/or CNP mRNA in olfactory nuclei, limbic cortex, hippocampus, amygdala and diencephalic limbic relays further indicate a putative role for ANP and CNP as neuromodulators of olfactory/limbic information processing. © 1995 Wiley-Liss, Inc.     

The natriuretic peptide system in the brain: implications in the central control of cardiovascular and neuroendocrine functions.

The natriuretic peptide system consists of three endogenous ligands, i.e., atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), and at least three subtypes of receptors. All of the peptides and receptors exist in the central nervous system (CNS). ANPs in the brain are N-terminally truncated forms: ANP (4-28) and ANP (5-28). The primary structure of BNP varies considerably among species, whereas that of CNP is highly conserved. ANP, BNP, and CNP are distributed in discrete brain regions, although the distribution varies in different species. Few immunohistochemical studies have so far been performed on BNP and CNP. There are three subtypes of receptors: ANP-A and ANP-B, which are bioactive, and the C receptor, which does not seem to be directly related to bioactivity. In the rat, the major subtype of ANP receptor in the CNS is the ANP-B receptor, based on the results of Northern blotting. Since the ligand for ANP-B receptor is CNP, the CNP-ANP-B receptor system may be most important, at least in rat brain. It is still unknown whether or not a specific receptor for BNP exists in central or peripheral tissues. Further studies should clarify the exact localization of ANP, BNP, and CNP and the three receptor subtypes in the CNS. Although natriuretic peptides and their receptors are distributed widely in the CNS, the AV3V regions, basal medial hypothalamus, brainstem, and circumventricular organs are the most prominent sites. This suggests an important physiological role of the natriuretic peptide system in the central control of cardiovascular homeostasis. The natriuretic peptide system seems to be involved in the regulation of water and salt intake, blood pressure, and secretion of vasopressin in the direction of reducing body fluid and lowering blood pressure. Such actions of natriuretic peptides are antagonistic to the central actions of angiotensin II (AII). In fact, the distribution of ANP and AII and their receptors in the CNS overlaps considerably. It is highly likely, therefore, that the central natriuretic peptide system and the renin-angiotensin system play important roles in the central control of cardiovascular and body fluid homeostasis in opposite directions. The natriuretic peptide system may also be involved in neuroendocrine control and some other CNS functions, although the physiological significance of these actions is less clear at the present time. It is now clear that there is considerable plasticity in the regulation of natriuretic peptides and their receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure-activity studies on the effects of atrial natriuretic peptide, brain natriuretic peptide and their analogs on fear-motivated learning behavior in rats.

Our previous studies have demonstrated that rat atrial natriuretic peptide (rANP 1-28) and porcine brain natriuretic peptide (pBNP 1-32) administered into the lateral brain facilitate the consolidation of a passive avoidance response and delay the extinction of an active avoidance response in fear-motivated learning in rats. To study the structure-activity relationships in the same learning processes, the effects of several fragments related to ANP and BNP were investigated following their intracerebroventricular administration to rats. The following peptides were studied: rANP 1-28, rANP 5-28, rANP 5-27, rANP 7-23 (ring), rANP 17-23, hANP 10-28, hANP 15-28, hANP 20-28, hANP 1-28, pBNP 1-32 and pBNP 7-32. The peptides were used in equimolar concentration. Two of the peptides studied, ANP 20-28 and ANP 17-23, were ineffective on the extinction of active avoidance behavior and on the consolidation of passive avoidance learning. They exhibited similar actions. The results showed that small fragments of ANP and BNP can carry the biological activity of ANP and BNP on the central nervous system (CNS). It is likely that the biological active center for ANP lies between amino acids 15 and 23 and it is suspected that the ring structure is not absolutely important for the CNS activity.     

Natriuretic Peptides Stimulate Cyclic GMP Production in an Immortalized LHRH Neuronal Cell Line

The role of cyclic 3',5'-guanosine monophosphate (cGMP) as a second messenger in LHRH neurons is not well understood. Recent studies involving nitric oxide, a direct activator of soluble guanylate cyclase (GC), have implicated cGMP in the regulation of LHRH secretion both in vivo and in vitro . Evidence for the membrane-bound form of GC in LHRH neurons has thus far not been reported. In polymerase chain reaction screening of various cell lines for the natriuretic peptide receptors—which represent GCs—we identified both GC-A and GC-B cDNAs by southern blot hybridization in reverse transcribed and amplified extracts of the GT1-7 cell line, an immortalized LHRH neuronal cell line. Subsequent experiments demonstrated that all of the natriuretic peptides elevated cGMP production with a rank order of potency: CNP > ANP > BNP. Time course studies revealed a rapid intracellular accumulation of cGMP following exposure to CNP with a peak at 2.5 min. CNP was some 200-fold more potent than the NO donor, sodium nitroprusside, in stimulating cGMP accumulation in these cells. These data show for the first time the presence of functional mGCs on LHRH cells, and suggest that the natriuretic peptides may also participate in the regulation of LHRH activity.     

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.     

Natriuretic peptides inhibit apoptosis and prolong the survival of serum-deprived PC12 cells

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were investigated to determine effects on apoptotic DNA fragmentation and survival in serum-deprived PC12 cells. Both peptides caused prolonged cGMP (but not cAMP) elevations lasting for > or = 6 h. The cGMP elevations were 10-, 50- and 68-fold for ANP and 26-, 100- and 148-fold for BNP at 1, 10 and 100 nM, respectively. BNP caused dose-dependent increases in cell survival rates during 3 days of serum deprivation. BNP (1 nM) increased 24 h survival rate from 36% to 67%. ANP (1 nM), BNP (1 nM) and 8-bromo-cGMP (0.1 mM) inhibited by 74.8%, 46.7% and 86.8%, respectively, the apoptotic DNA fragmentation in serum-deprived PC12 cells, measured by our recently developed quantitative technique using capillary electrophoresis with laser-induced fluorescence detector (CE-LIF). The data suggest prolonged cGMP elevations caused by ANP or BNP inhibit apoptotic DNA fragmentation and prolong the survival of serum-deprived PC12 cells.     

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.     

Clinical significance of natriuretic peptides in patients with aneurysmal subarachnoid hemorrhage]

Hyponatremia and hypovolemia following aneurysmal subarachnoid hemorrhage (SAH) might be speculated by exaggerated secretion of natriuretic peptides and resulted ischemic sequela caused by cerebral vasospasm. We measured serum concentration of natriuretic peptides and investigated their influence on post-SAH hyponatremia. Among 49 patients of SAH, their plasma concentration of the natriuretic peptides (atrial natriuretic peptide: ANP and brain natriuretic peptide: BNP) were measured at the day of ictus and 7th day of SAH. The correlation between concentration of natriuretic peptides and location of aneurysm, severity of SAH, incidence of hyponatremia and symptomatic vasospasm were elucidated. The plasma concentration of ANP did not alter on admission and 7th day post SAH, whereas that of BNP increased in the patients with moribund SAH and those with ruptured A-com aneurysm. The initial increase of BNP following SAH could be attributed to direct damage of SAH on the hypothalamus. Hyponatremia and symptomatic vasospasm tended to occur in the patients who had persistent increase of plasma BNP concentration during one week post SAH. Therapeutic intervention to maintain normonatremia by fluid-management decreased occurrence of symptomatic vasospasm, even though patients with increased plasma BNP concentration. It might be concluded that increased secretion of BNP following SAH is caused by direct effect to the hypothalamus and prolonged hyper secretion of BNP resulted hyponatremia, hypovolemia and exaggerated symptomatic vasospasm.     

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.     

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.     

Contents of corticotropin-releasing hormone and arginine vasopressin immunoreactivity in the spleen and thymus during a chronic inflammatory stress

We have previously found that proopiomelanocortin (POMC) mRNA and levels of adrenocorticotropin (ACTH) and β-endorphin peptides are increased in the spleen and thymus of rats with adjuvant-induced arthritis (AA), an immunologically mediated inflammatory disease. To determine whether alterations in immune tissue POMC during AA are also accompanied by changes in immune tissue corticotropin-releasing hormone immunoreactivity (ir-CRH) and arginine vasopressin (AVP), we measured ir-CRH and AVP by radioimmunoassays in spleen and thymic extracts 14 days following injection of adjuvant. Ir-CRH was detectable in all extracts of spleen and thymus. Total contents of ir-CRH in the spleen and thymus were not altered following arthritis, although a significant decrease was observed in splenic extracts from arthritic rats (40.0 ± 4.2 fmol/g tissue) compared to controls (69.5 ± 8.4 fmol/g tissue) when contents were expressed as amount per weight of tissue. Low levels of AVP were also detected in immune tissues, with contents significantly increased in spleens from arthritic animals (17.4 ± 1.6 fmol/g tissue) compared to controls (10.6 ± 1.9 fmol/g) but thymic contents of AVP were not altered by arthritis (10.6 ± 1.3 fmol/g) compared to controls (9.2 ± 0.7 fmol/g). Control levels of AVP were significantly higher in spleens and thymuses from female rats (53 ± 5 and 25 ± 4 fmol/g tissue, respectively) compared to males. G-50 chromatography revealed that the principal form of splenic ir-CRH is CRH(1–41), although in non-arthritic animals some ir-CRH eluted in a position indicating a slightly larger form. Therefore the responses of ir-CRH contents in tissues of the immune system differ from those observed for POMC mRNA and POMC peptide products during this chronic inflammatory stress.     

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.     

Effects of atrial and brain natriuretic peptides upon cyclic GMP levels, potassium transport, and receptor binding in rat astrocytes

The ability of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) to alter cyclic GMP levels and NaKCl cotransport in rat neocortical astrocytes was determined. At concentrations of 10(-9)-10(-6) M, rat ANP99-126 (rANF), rat ANP102-126 (auriculin B), and rat ANP103-126 (atriopeptin III) stimulated 6- to 100-fold increases in cyclic GMP levels. Porcine BNP (pBNP) and rat BNP (rBNP) were 20%-90% as effective as rANF over most of this concentration range, although 10(-6) M pBNP produced a greater effect than rANF. NaKCl cotransport as measured by bumetanide-sensitive 86Rb+ influx was not altered by exposure of astrocytes to 10(-6)M rANF, pBNP, or rBNP. Both pBNP and rBNP, as well as rat ANP103-123 (atriopeptin I) and des[gl18, ser19, gly20, leu21, gly22] ANF4-23-NH2 (C-ANF4-23) strongly competed for specific 125I-rANF binding sites in astrocyte membranes with affinities ranging from 0.03 to 0.4 nM, suggesting that virtually all binding sites measured at subnanomolar concentrations of 125I-rANF were of the ANP-C (ANF-R2) receptor subtype. These receptors are thought to serve a clearance function (Maack et al.: Science 238:675-678, 1987) and may be linked to a guanylate cyclase activity that is chemically and pharmacologically distinct from that coupled to ANP-A (ANF-R1) receptors (Féthiere et al.: Mol Pharmacol 35:584-592, 1989). ANP receptors on astrocytes may function in limiting the access of ANP and BNP to neurons involved in body fluid and cardiovascular regulation.     

Effects of short-term dexamethasone treatment during pregnancy on the development of the immune system and the hypothalamo-pituitary adrenal axis in the rat

The effects of glucocorticoid (GC) treatment on the mature immune and neuroendocrine system are known to be reversible. However, prenatal GC exposure may have irreversible consequences on the development of the newborn. In this study, possible long-lasting effects of short-term prenatal GC treatment were examined on the developing thymus, spleen and hypothalamo-pituitary adrenal axis (HPA axis). Female rats were given dexamethasone (DEX, 400 μg i.p.) on day 17 and 19 of pregnancy and offspring was studied at several time intervals (1–20 days) after birth, for examination of thymus, spleen, hypothalamus and blood plasma. Examination of thymus and spleen revealed that prenatal exposure to DEX resulted in decreased T cell numbers in thymus and spleen on day 1 after birth. Thymus regeneration after DEX exposure both during pregnancy and in adult life was completed after 24 days. However, the kinetics of regeneration of the thymi after prenatal DEX exposure were different from that seen after DEX in adult life. Whereas DEX treatment during pregnancy resulted in an increased ratio of CD4⁺/CD8⁻ thymocytes over CD4⁻/CD8⁺ thymocytes compared to control groups on day 7 and day 20 after birth (time X treatment interaction; P < 0.05), DEX treatment in adult life did not change this ratio. T cell numbers in the spleen were significantly decreased at all neonatal ages studied. Regarding the hypothalamus, prenatal exposure to DEX altered the pattern of neonatal changes in peptide expression in corticotropin-releasing hormone neurons, with a selective reduction in CRH storage in the median eminence (7 and 9 days after birth) and an increase in AVP storage (9 and 20 days after birth). The ratio of AVP over CRH was significantly increased at all developmental ages studied. No effects were seen on basal ACTH and corticosterone levels in plasma. In conclusion, the kinetics of thymus regeneration after DEX exposure during pregnancy were different from that seen after DEX exposure in adult life. Prenatal DEX exposure also seemed to delay the migration of T cells into the spleen. Furthermore, prenatal DEX treatment exerted major effects on hypothalamic CRH neurons that maintained for at least 20 days after birth, which points towards an enhanced stress responsiveness of the HPA axis in later life.     

The thymus gland in elderly patients with myasthenia gravis.

This study was undertaken to determine the amount and histologic appearance of the persistent thymic tissue removed post mortem from 20 patients over 60 years of age with myasthenia gravis. One patient died several days after thymectomy. No recognizable thymic tissue on gross examination was seen in any patient. On microscopic examination, 11 patients had no thymic tissue. The other nine patients, including the one with thymectomy, all showed marked involution of the thymus. No germinal centers were seen. The thymus glands of two additional patients, still alive after thymectomy at ages 62 and 70, showed similar findings. Marked involution of the thymus also was found in each of six elderly controls. Thymectomy is not likely to be effective treatment of the elderly myasthenic patient.     

Autoreactivity between lymphocytes and thymus cells in myasthenia gravis.

Thymus cell preparations from four of five myasthenia gravis patients with thymic hyperplasia and from one additional patient with thymic involution stimulated autologous peripheral blood lymphocytes. No autostimulation was observed in two patients with thymoma. Autostimulation as associated with an increase in the fraction of B lymphocytes in the thymus.     

Behavioral effects of atrial and brain natriuretic peptides in rats.

The effects of intracerebroventricular administration of rat atrial natriuretic peptide (rANP-1-28) and porcine brain natriuretic peptide-32 (pBNP-32) on passive and active avoidance behavior and on electroconvulsive shock-induced amnesia were studied in rats. The dose range for both peptides was selected to lie between 0.016 and 0.32 nmol. The two peptides were found to facilitate consolidation of the passive avoidance response, to delay extinction of the active avoidance response, and to prevent electroconvulsive shock-induced amnesia in a similar way. It is suggested that some modulatory functions in the central nervous system of the rat, so far attributed to ANP, may in fact involve a dual control by both ANP and BNP, and there is no difference in the biological activity of the two peptides as far as fear-motivated learning behavior is concerned.