Isolation and identification of human brain natriuretic peptides in cardiac atrium

By using a radioimmunoassay (RIA) system newly established for human brain natriuretic peptide (BNP), a high concentration of immunoreactive (ir-) human BNP (hBNP) has been found in cardiac atrium (1). Two molecular forms of ir-hBNP of 4K and 13-15K were isolated from atrial extracts by using anti-hBNP IgG immunoaffinity chromatography and reverse phase high performance liquid chromatography (HPLC). By microsequencing, the peptides were determined to be a pro-hBNP (gamma-hBNP) and its C-terminal 32-amino acid peptide (hBNP-32). Based on these results, in cardiac atrium, hBNP is found to be processed in a pathway similar to that of porcine BNP (pBNP) but distinct from that of rat BNP, although low MW hBNP-32 is a major form in contrast to pBNP which exists as a high MW gamma-pBNP.     

Isolation and sequence determination of human brain natriuretic peptide in human atrium

We isolated human brain natriuretic peptide (human BNP) from the human atrium. Sequence analysis has revealed that it is a 32-amino-acid peptide with the sequence S-P-K-M-V-Q-G-S-G-C-F-G-R-K-M-D-R-I-S-S-S-S-G-L-G-C-K-V-L-R-R-H, which is identical to the C-terminal sequence (77-108) of the human BNP precursor deduced from the cDNA sequence. The sequence of human BNP (77-108) is preceded by Pro75-Arg76 in the human BNP precursor, which is the same processing signal as Pro97-Arg98 of the precursor of atrial natriuretic peptide (ANP). The processing of the BNP precursor occurs in the cardiocyte, although that of the ANP precursor in the cardiocyte is unclear at present.     

Isolation and identification of C-type natriuretic peptide in chicken brain

C-type natriuretic peptide (CNP) has recently been identified in porcine brain as a third member of the mammalian natriuretic peptide family (1). Using a radioimmunoassay system for porcine CNP, we found a significant concentration of immunoreactive (ir-) CNP in chicken brain, from which a new peptide was isolated. By microsequence analysis, the amino acid sequence of the peptide was determined to be Gly-Leu-Ser-Arg-Ser-Cys-Phe- Gly-Val-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys. Based on its high homology to porcine CNP, the peptide was designated chicken C-type natriuretic peptide. Chicken CNP also elicits pharmacological effects highly similar to porcine CNP, suggesting that CNP functions as a neuropeptide in the chicken central nervous system.     

Isolation and sequence determination of rat cardiac natriuretic peptide

We have isolated a cardiac natriuretic peptide of 5K daltons from the rat atrium and determined its amino acid sequence. The 5K cardiac natriuretic peptide was elucidated to be a 45-amino acid peptide with the sequence of S-Q-D-S-A-F-R-I-Q-E-R-L-R-N-S-K-M-A-H-S-S-S-C-F-G-Q-K-I-D-R-I-G-A-V-S-R- L-G-C-D - G-L-R-L-F by sequencing the native peptide and its lysyl endopeptidase digests. The sequence of this peptide was identical to the amino acid sequence [51-95] of the rat brain natriuretic peptide (BNP) precursor deduced from the cDNA sequence. The 5K cardiac natriuretic peptide, or BNP[51-95], was identified as the major storage and secretory form derived from the BNP precursor in the rat heart.     

Molecular forms of atrial natriuretic peptides in dog atrium and plasma

The level of immunoreactive ANP (iANP) as determined by radioimmunoassay, exhibits a fairly even distribution throughout the canine atria. However, the maximal concentration was found in the appendages of both the left and right atrium, and the level was significantly higher on the left side in all analyzed localizations. Reverse-phase HPLC of atrial extracts, coupled with RIA, revealed the presence of three iANP fractions. The dominant fraction (65% of iANP) has an estimated molecular weight 16,300, showing that it corresponds to the hormone precursor. By contrast, in the plasma extract, 92% of iANP coelutes with synthetic human ANP99-126, thus confirming the structural identity of dog and human ANP.     

Atrial and brain natriuretic peptide in right atrium myocytes in the postreperfusion period in rat

We have studied the accumulation and excretion of atrial (ANP) and brain (BNP) natriuretic peptides in the early and late postreperfusion period (60 min and 60 days) in the myocardium of the right atrium in rats. The model of total ischemia proposed by Korpachev et al. (1982) was used. Immunocytochemical localization of peptides in cardiomyocytes was performed on ultrathin sections using the polyclonal antibodies. The intensities of accumulation (excretion) of ANP and BNP were analyzed by counting the immunolabeled granules (types A and B) with a transmission electron microscope. At 60 min and 60 days of the postreperfusion period, an increase in the synthesis and release of ANP and BNP was found. A more pronounced BNP reaction could be explained by the fact that, under normal conditions, the main hormone of the natriuretic peptide system regulating blood pressure is ANP, while BNP regulates blood pressure in cardiovascular pathology.     

大鼠大脑微血管片段的分离纯化及鉴定

目的:分离大鼠大脑微血管并纯化去除完整的神经细胞,用于克隆血脑屏障上的特异表达基因.方法:采用液相合成法制备粒径200～500 nm的铁氧体磁珠,经两侧颈内动脉插管注入大鼠大脑半球.采用机械分离和酶消化相结合的方法解离脑组织,用筛网滤去组织块和大血管,再在磁场下分选标记磁珠的微血管片段,并从形态学、分子生物学和生物活性角度鉴定获得的脑微血管片段.结果:扫描电镜下没有发现微血管周围存在完整的神经细胞,但在部分区域有胶质的终足包裹.全脑组织微管相关蛋白2a、谷氨酰胺合成酶和CD31的RT-PCR产物均在相应位置出现阳性条带,分离纯化的脑微血管仅CD31阳性.微血管片段内皮细胞摄取的Rh123荧光强度显著低于传代培养的微血管内皮细胞荧光强度.结论:采用本方法可以获得高纯度的、不附带完整神经细胞的脑微血管片段.     

Structural and functional evolution of three cardiac natriuretic peptides

Natriuretic peptides (NPs) are a group of hormones playing important roles in cardiovascular and osmoregulatory systems in vertebrates. Among the NP subtypes, atrial NP (ANP), B-type NP (BNP), and ventricular NP (VNP) are circulating hormones expressed exclusively in the heart (cardiac NPs). The constitution of cardiac NPs is variable among species of vertebrates. In order to understand the evolutionary and functional significance of such variation, we performed a systematic survey of cardiac NP cDNAs in nine taxonomically diverse teleosts inhabiting environments of varying salinity. The discovery of the coexistence of the ANP, BNP, and VNP genes in the eel and rainbow trout suggested that the ancestral teleost had all three cardiac NPs. As the VNP cDNA was undetectable in ayu and six species of Neoteleostei, it is possible that VNP was lost before the divergence of Osmeroidei. The ANP gene was also undetectable in the medaka. Thus, only the BNP gene is universal in species examined in the present study. Synthetic medaka BNP preferentially activated two medaka GC-A-type receptors, suggesting that the three cardiac NPs share the same receptor. However, the regulation of BNP expression may be the most strict because ATTTA repeats in the 3'-untranslated region and the dibasic motif in the ring are conserved among teleosts and tetrapods. Linkage analyses in the rainbow trout located ANP, BNP, and VNP genes on the same chromosome, which suggested the generation of the VNP gene by tandem duplication as observed with ANP and BNP genes. If the duplication occurred before the divergence of tetrapods and teleosts, VNP may exist in the tetrapod lineage.     

Isolation and structures of xenopsin-related peptides from rat stomach, liver and brain

Using radioimmunoassay for detection, a mammalian counterpart to amphibian xenopsin (XP) was isolated and sequenced from pepsin-treated extracts of three different rat tissues and shown to be H-Phe-His-Pro-Lys-Arg-Pro-Trp-Ile-Leu-OH. This peptide, which shares six of the eight residues in XP, existed primarily in large molecular form(s) in the rat from which it could be liberated by the enzyme, pepsin. The XP-related sequence was differentially distributed through tissues, with concentrations ranging from ca. 80 pmol/g in diaphragm and skeletal muscle to ca. 800 pmol/g in stomach, liver and intestine. Like XP, the mammalian peptide potently crossreacted in a radioreceptor assay for neurotensin. These results prove the existence of radioreceptor-active XP-related sequences in multiple tissues of the rat.     

Atrial and brain natriuretic peptides share binding sites on cultured cells from the rat trachea

Summary We examined the distribution of binding sites for alpha-atrial natriuretic peptide (¹²⁵I-ANP_1–28) and the recently discovered porcine brain natriuretic peptide (¹²⁵I-pBNP) on immunocytochemically identified cells in dissociated culture preparations of the rat trachea. Specific binding sites for both ¹²⁵I-ANP_1–28 and ¹²⁵I-pBNP were evenly distributed over distinet subpopulations of smooth muscle myosin-like immunoreactive muscle cells, fibronectin-like immunoreactive fibroblasts and S-100-like immunoreactive glial cells. Neither keratin-like immunoreactive epithelial cells nor protein gene product 9.5-like immunoreactive paratracheal neurones expressed natriuretic peptide binding sites, although autoradiographically labelled glial cells were seen in close association with both neuronal cell bodies and neurites. The binding of each radiolabelled peptide was abolished by the inclusion of either excess (1 M) unlabelled rat ANP or excess unlabelled porcine BNP, suggesting that ANP and BNP share binding sites in the trachea. Furthermore, the ring-deleted analogue, Des-[Gln¹⁸, Ser¹⁹, Gly²⁰, Leu²¹, Gly²²]-ANF_4–23-NH₂, strongly competed for specific ¹²⁵I-ANP_1–28 and ¹²⁵I-pBNP binding sites in the tracheal cultures; this suggests that virtually all binding sites were of the clearance (ANP-C or ANF-R₂) receptor subtype.     

Molecular forms of immunoactive atrial natriuretic peptide in the rat hypothalamus and atrium

Acid extracts of rat hypothalamus and atrium were prepared by a procedure previously shown to minimize proteolytic degradation of peptides. The majority of the immunoactive material in the atrial extracts had a molecular weight of approximately 9,000 to 15,000 daltons, while that in the hypothalamic extracts had a molecular weight of about 1,500 to 1,800 daltons. The major molecular weight forms of atrial natriuretic peptide from each extract were further distinguishable when analyzed by RP-HPLC. These results suggest that small peptides such as atriopeptins I, II, and III, may not be authentic post-translational processing products in the atrium, and that the hypothalamus and atrium may differentially cleave pro-atrial natriuretic peptide to form tissue-specific products.     

Bioactive atrial natriuretic factor-like peptides in rat anterior pituitary

The presence of biologically active atrial natriuretic factor (ANF)-like peptides was demonstrated in rat anterior pituitary. ANF-like immunoreactivity was detected in rat anterior pituitary by specific radioimmunoassay and was extracted from rat anterior pituitary homogenates by heat-activated Vycor glass beads; extracts were purified by reverse-phase high performance liquid chromatography. Two peaks containing ANF immunoreactive material were obtained. The first peak was eluted from the C18 mu Bondapak column at a position similar to the 28-amino acid carboxy terminal peptide (Ser99-Tyr126)-ANF of prohormone. The second peak had the same pattern of elution as the 126-amino acid prohormone, (Asn1-Tyr126)-ANF. The biological activity of the smaller molecular weight peptide (28 amino acid) was assessed by its inhibitory effect on 10(-8) M ACTH-stimulated aldosterone secretion in rat zona glomerulosa cell suspension. This ANF-like material also displaced I125-labelled ANF from rat glomerular receptors with a potency similar to synthetic (Arg101-Tyr126)-ANF. Immunocytochemical localization revealed a distribution of ANF-stained cells similar in pattern and location to that of gonadotrophs. These results suggest the existence of biologically active ANF-like peptides and ANF prohormone within the anterior pituitary. However, their role remains to be elucidated.     

Nature of atrial natriuretic polypeptide in rat brain

Using reverse phase high performance liquid chromatography (RP-HPLC) coupled with two radioimmunoassays for atrial natriuretic polypeptide (ANP) with different specificities, we investigated the nature of alpha-rat ANP-like immunoreactivity (alpha-rANP-LI) with a low molecular weight in the rat brain. Two major peaks with alpha-rANP-LI in the extract from rat whole brains were eluted in the vicinity of the elution position of synthetic alpha-rANP, a 28-amino acid polypeptide. These two components co-migrated with synthetic alpha-rANP (4-28) and alpha-rANP (5-28), respectively. The peak corresponding to alpha-rANP (4-28) was the highest, and only a little alpha-rANP-LI was detected at the elution position of alpha-rANP. An identical profile in RP-HPLC was also observed in the extract from the rat hypothalamus. These results indicate that the major components of alpha-rANP-LI with a low molecular weight in the rat brain are alpha-rANP (4-28) and alpha-rANP (5-28).     

Porcine brain natriuretic peptide-like immunoreactivity in rat tissues

The presence of immunoreactive porcine brain natriuretic peptide in rat tissues was studied with a specific radioimmunoassay for porcine brain natriuretic peptide-26. The cross-reactivity of the antiserum used was less than 0.001% with rat atrial natriuretic peptide, rat brain natriuretic peptide-32 and rat brain natriuretic peptide-45. Immunoreactive porcine brain natriuretic peptide was detectable in various tissues of the rat, and high concentrations of immunoreactive porcine brain natriuretic peptide were found in the brain and cardiac atrium, with the highest level in the hypothalamus (159±30 fmol/gram wet tissue, mean±SEM, n=4). Reverse phase high performance liquid chromatography showed that the immunoreactive porcine brain natriuretic peptide of the whole brain and heart extracts eluted mainly at an identical position to synthetic porcine brain natriuretic peptide-26. These findings indicate that porcine brain natriuretic peptide-like substance, distinct from rat brain natriuretic peptide, is present in high concentrations in the rat brain and cardiac atrium.     

Hyperthermic effect of centrally administered natriuretic peptides in the rat.

The effects of atrial natriuretic peptide (ANP-28), brain natriuretic peptide (BNP-32) and C-type natriuretic peptide (CNP-22) on body temperature were investigated in rats. Intracerebroventricular administration of each peptide in doses of 400 or 1000 ng caused a dose-related elevation in colon temperature 30 and 60 min after injection. A 40 ng dose of ANP-28 was also hyperthermic at 60 min. An intramuscular (i.m.) injection of noraminophenazone (a cyclooxygenase inhibitor) abolished the natriuretic peptide-induced hyperthermia. The results show that natriuretic peptides may participate in thermoregulatory processes in the central nervous system, and that their hyperthermic effect may be mediated via a cyclooxygenase-involved pathway.     

Cocaine regulates TRH-related peptides in rat brain

Cocaine administration has previously been reported to alter the levels of prepro-TRH mRNA and TRH (pGlu-His-Pro-NH₂) in the limbic system of rats (J. Neurochem. 60 (1993) 1151). We have now demonstrated that a previously unrecognized family of TRH-like peptides is involved in the actions of cocaine. We treated young adult male Sprague-Dawley rats (five per group, 250 g body weight at sacrifice) for 2 weeks with either twice daily injections of saline (control group), twice daily injections of 15 mg/kg cocaine until sacrifice (chronic group), single injection of 15 mg/kg cocaine 2 h prior to sacrifice (acute group) or chronic cocaine injections replaced by saline injections 72 h prior to sacrifice (withdrawal group (WD)). Twelve different brain regions were dissected and immunoreactivity for TRH (TRH-IR), EEP (pGlu-Glu-Pro-NH₂; EEP-IR) and related peptides were measured by radioimmunoassay (RIA). High pressure liquid chromatography (HPLC) revealed that in many brain regions EEP-IR and TRH-IR consisted of a mixture of TRH, and other TRH-like peptides including EEP, pGlu-Val-Pro-NH₂ (Val²-TRH), pGlu-Tyr-Pro-NH₂ (Tyr²-TRH), pGlu-Leu-Pro-NH₂ (Leu²-TRH), and pGlu-Phe-Pro-NH₂ (Phe²-TRH). Following i.p. injection, these TRH-like peptides readily crossed the blood–brain barrier but cleared very slowly from brain tissues.

Acute cocaine produced a 4.1-fold increase in Val²-TRH level in medulla while Val²-TRH and Tyr²-TRH, increased 6.2- and 2.9-fold, respectively in pyriform cortex PYR. TRH and Leu²-TRH, decreased 47 and 93%, respectively in the nucleus accumbens (AM) while other EEP-IR peaks decreased 50–100% consistent with the significant decrease in total EEP-IR in the AMs following acute cocaine treatment. Because 2 h is too short a time to alter levels of neuropeptides via changes in the rate of biosynthesis, the acute cocaine-induced elevation or reduction in TRH and related peptides is most likely due to suppression or stimulation, respectively, of the corresponding peptide secretion rate. Because TRH and TRH-like peptides have antidepressant, analeptic and euphorigenic properties, we conclude that these endogenous substances are potential mediators of both the cocaine “high” and withdrawal symptoms.     

Distribution of gastrin and CCK-like peptides in rat brain

Summary The distribution of gastrin and CCK-like peptides in the rat brain was studied by immunocytochemistry using an antiserum reacting equally well with both groups of peptides. Immunoreactive nerve cell bodies were detected in all cortical areas, in the hippocampus where they were particularly numerous, in the mesencephalic central gray and in the medulla oblongata. After colchicine treatment immunoreactive material appeared also in cell bodies of the magnocellular hypothalamic system. Immunoreactive nerve fibers were widely distributed in the brain. Particularly dense accumulations were seen in the hippocampus near the ventral surface of the brain, in the caudate nucleus, in the interpeduncular nucleus, the parabrachial nucleus, the dorsal part of the medulla oblongata and in the dorsal horn of the spinal cord. In the hypothalamus immunoreactive nerve fibers were observed in all nuclei, being most frequent in the ventromedial, dorsal and lateral hypothalamic nuclei. A rich supply of nerve fibers was seen in the outer zone of the median eminence and in the neurohypophysis. From previous immunochemical analysis it appears that the peptide demonstrated in most parts of the brain is identical with CCK-8. In the neurosecretory cell bodies of the hypothalamus, the median eminence and the neurohypophysis, however, the immunoreactive material is probably identical with gastrin.