Distribution and molecular forms of brain natriuretic peptide in porcine heart and blood

Although brain natriuretic peptide (BNP) is a novel natriuretic peptide originally identified in porcine brain, recent investigation has verified the presence of BNP in porcine heart. In order to identify BNP as a circulating hormone, we analyzed the regional distribution and molecular form of immunoreactive (ir-) BNP in heart and blood. Tissue concentration of ir-BNP was high in atrium, but low in ventricle, in a manner similar to that of atrial natriuretic peptide (ANP). However, the concentration of ir-BNP in atrium was only about 1/50 that of ir-ANP. In plasma, ir-BNP was found at a concentration of 1-3 fmol/ml, which was about 1/20 that of ir-ANP. Both ir-BNP and ir-ANP were present as low molecular weight forms. Three forms of ir-BNP of about 3K daltons, including BNP-26, BNP-29 and BNP-32, are thought to circulate in blood.     

Atrial natriuretic peptide in the central nervous system of the rat

1. Studies of the presence of atrial natriuretic peptide immunoreactivity and receptor binding sites in the central nervous system have revealed unusual sites of interest. 2. As a result, numerous studies have appeared that indicate that brain atrial natriuretic peptide is implicated in the regulation of blood pressure, fluid and sodium balance, cerebral blood flow, brain microcirculation, blood-brain barrier function, and cerebrospinal fluid production. 3. Alteration of the atrial natriuretic peptide system in the brain could have important implications in hypertensive disease and disorders of water balance in the central nervous system.     

Concentration and molecular forms of brain natriuretic peptide in rat plasma and spinal cord

To characterize the biological functions of rat brain (B-type) natriuretic peptide (BNP), which has been shown to be present mainly in the heart and only faintly in the spinal cord, the concentration and molecular forms of BNP in plasma and spinal cord were determined. The concentration of immunoreactive (ir-) BNP was 2.00 fmol/ml in normal rat and 13.29 fmol/ml in morphine-treated rat, being respectively about 1/20 and 1/80 those of ir-atrial (A-type) natriuretic peptide (ANP). In morphine-treated rats, ir-BNP was shown to circulate mainly as BNP-45, which is identical to a major storage form found in cardiac atrium. In the spinal cord, BNP was also shown to be present as BNP-45, but its concentration was only 0.057 pmol/g, being about 1/60 that of spinal cord ANP. These results confirm that BNP mainly functions as a circulating hormone in the molecular form of BNP-45. Morphine stimulates secretion of ANP and BNP but by different ratios, suggesting different regulation systems for storage and secretion of ANP and BNP.     

Distribution of growth-blocking peptide in the insect central nervous tissue

Parasitization of the armyworm Pseudaletia separata by the endoparasitic wasp Cotesia kariyai inhibits larval growth and delays pupation, conditions necessary for proper maturation of the parasite larvae. Parasitization is correlated with an elevated level of a 25-amino-acid hormone-like peptide, growth-blocking peptide (GBP, ENFSGGCVAGYMRTPDGRCKPTFYQ). Injection of synthetic GBP into nonparasitized larvae dose dependently mimics the effects of parasitization by delaying the larval development. Here we studied the relationship between parasitization and both the production and distribution of GBP in central nervous tissues. We found that parasitization is correlated with an elevated expression of GBP mRNA, and increased concentrations of both proGBP and GBP in the host insect brain and subesophageal ganglion. The increase in proGBP precedes that of the mature GBP by about 12 h. In situ hybridization analysis using sections of parasitized and nonparasitized larval brains showed strong expression of GBP mRNA in perineural cells and/or class I neuroglia in the rind of both larval brains. The expression in parasitized larval brain-subesophageal ganglion is approximately two- to threefold higher than that in nonparasitized larvae. The presence of GBP in insect neural tissue, and its role in inhibiting growth, suggest an involvement in the regulation of neurosecretory cells.     

Distribution of the Aplysia cardioexcitatory peptide,NdWFamide, in the central and peripheral nervous systems of Aplysia

NdWFamide is an Aplysia cardioexcitatory tri-peptide containing D-tryptophan. To investigate the roles of this peptide, we examined the immunohistochemical distribution of NdWFamide-positive neurons in Aplysia tissues. All the ganglia of the central nervous system (CNS) contained NdWFamide-positive neurons. In particular, two left upper quadrant cells in the abdominal ganglion, and the anterior cells in the pleural ganglion showed extensive positive signals. NdWFamide-positive processes were observed in peripheral tissues, such as those of the cardio-vascular system, digestive tract, and sex-accessory organs, and in the connectives or neuropils in the CNS. NdWFamide-positive neurons were abundant in peripheral plexuses, such as the stomatogastric ring. To examine the NdWFamide contents of tissues, we fractionated peptidic extracts from the respective tissues by reversed-phase high-pressure liquid chromatography and then assayed the fractions by competitive enzyme-linked immunosorbent assay. A fraction corresponding to the retention time of synthetic NdWFamide contained the most immunoreactivity, indicating that the tissues contained NdWFamide. The prevalence of the NdWFamide content was roughly in the order: abdominal ganglion >heart >gill >blood vessels >digestive tract. In most of the tissues containing NdWFamide-positive nerves, NdWFamide modulated the motile activities of the tissues. Thus, NdWFamide seems to be a versatile neurotransmitter/modulator of Aplysia and probably regulates the physiological activities of this animal.     

Distribution of neuropeptide K-immunoreactivity in the rat central nervous system

The distribution of neuropeptide K (NPK), a 36-residue amidated peptide originally isolated from porcine brain, is described in the rat CNS by immunohistochemical methods. Antibodies were generated in rabbits to N-terminus and C-terminus regions of the peptide and the distribution of immunoreactive cell bodies and fibers was mapped in colchicine-treated and normal rat brains. Major areas of cell body staining included the medial habenular nucleus, the ventromedial nucleus of the hypothalamus, the interpeduncular nucleus, the lateral dorsal tegmental nucleus, the nucleus raphe pallidus, and the nucleus of the solitary tract. Some of the areas of dense NPK-fiber immunoreactivity included the ventral pallidum, the caudate-putamen, certain areas of the hypothalamus, the central and medial amygdaloid nuclei, the entopeduncular nucleus, the habenular nuclei, the substantia nigra pars reticulata, the caudal part of the spinal nucleus of the trigeminal nerve, the nucleus of the solitary tract and the dorsal horn of the spinal cord. A striking similarity exists between this pattern of immunoreactive staining and that described for substance P, suggesting that the tachykinin systems do not exist independently in the brain. The possible roles for multiple tachykinins in the brain are discussed.     

Carbonic anhydrase distributions in central and peripheral nervous system of the rat

Total and specific carbonic anhydrase activity was measured for 24 structures of the rat central and peripheral nervous system. In the CNS, white matter or regions containing largely white matter show a neuraxial distribution of enzyme activity; more cephalad structures display more activity. Gray matter regions do not show a rostrocaudal distribution and usually have lower activity than adjacent myelin-containing structures. PNS tissue shows neither the white-gray differences nor the rostrocaudal profile of CNS tissue. Subcellular fractionation of 18 regions of the CNS suggest that the predominance of membrane-bound carbonic anhydrase (60% of the total activity and independent of its magnitude) is a unique characteristic of all regions of the central nervous system.     

Distribution of galanin-like immunoreactivity in the pig, rat and human central nervous system

The distribution of galanin-like immunoreactivity in various regions of the central nervous system was assessed in three mammalian species, pig, rat, and human, by radioimmunoassay. Galanin concentrations were highest in the hypothalamus and pituitary region. In spinal cord, there was a rostrocaudal/dorsoventral gradient with highest levels observed in the sacral dorsal horn. Serial dilutions of porcine tissue extracts diluted parallel to the porcine standard curve, while the rat and human tissue extracts did not. In all tissues examined by high pressure liquid chromatography, the principal peak of immunoreactivity coeluted with the authentic porcine galanin standard and was decreased by trypsin cleavage. These results suggest a role for galanin in the central nervous system and support species differences in the structure of galanin.     

Distribution of tau proteins in the normal human central and peripheral nervous system

In human brain, antibodies to tau proteins primarily label abnormal rather than normal structures. This might reflect altered immunoreactivity owing to post-mortem proteolysis, disease, or species differences. We addressed this issue by comparing the distribution of tau in bovine and human post-mortem nervous system tissues and in human neural cell lines, using new monoclonal antibodies (MAb) specific for phosphate-independent epitopes in bovine and human tau. In neocortex, hippocampus, and cerebellum, immunoreactive tau was widely expressed but segregated into the axon-neuropil domain of neurons. In spinal cord and peripheral nervous system, tau immunoreactivity was similarly segregated but less abundant. No immunoreactive tau was detected with our MAb in glial cells or in human neural cell lines that express neurofilament or glial filament proteins. Post-mortem delays in tissue denaturation of less than 24 hr did not affect the distribution of tau, but the method used to denature tissues did, i.e., microwave treatment preserved tau immunoreactivity more effectively than chemical fixatives such as Bouin's solution, and formalin-fixed tissue samples reacted poorly with our anti-tau MAb. We conclude that the distribution of tau proteins in human nervous system is similar to that described in perfusion-fixed experimental animals, and that visualization of normal immunoreactive tau in human tissues is critically dependent on the procedures used to denature post-mortem tissue samples. Furthermore, microenvironmental factors in different neuroanatomical sites may affect the regional expression of tau.     

Distribution of tripeptidyl-peptidase II in the central nervous system of rat

Tripeptidyl-peptidase II (TPP II) is a high molecular weight serine peptidase which removes tripeptides from a free N-terminus of longer peptides. Since it had previously been demonstrated that the enzyme can inactivate enkephalins and dynorphins in vitro by removing the N-terminal Tyr-Gly-Gly peptide, we wanted to see whether TPP II could be involved in this process also in vivo. Therefore, the localization of TPP II in different cerebral regions of rat was investigated by immunoblot analysis and activity measurements. It could be shown that TPP II is relatively evenly distributed in the central nervous system of rat. This indicates that the physiological role of the enzyme is probably not a specific degradation of enkephalins, but rather pertains to the general turnover of proteins.     

Immunoactive atrial natriuretic peptide in the rat eye: molecular forms in anterior uvea and retina

Acid extracts of rat anterior uvea and retina contain immunoactive atrial natriuretic peptide. Gel filtration chromatography demonstrates that the major forms of the uveal and retinal material have approximate molecular weights of 2400 and 1750 daltons respectively, similar in size to the hypothalamic form of the peptide but clearly distinguishable from the larger, cardiac form. When further analyzed by RP-HPLC, the immunoactive material from both ocular tissues elutes at a position similar to atrial natriuretic peptide-28, again distinguishing the ocular and cardiac forms of immunoactive material. These results suggest potential roles for atrial natriuretic peptide both in ocular vegetative physiology and in retinal function.     

Distribution and localization of pro-brain-derived neurotrophic factor-like immunoreactivity in the peripheral and central nervous system of the adult rat

The precursors for neurotrophins are proteolytically cleaved to form biologically active mature molecules which activate their receptors p75NTR and trks. A recent study showed that the precursor for nerve growth factor (NGF) can bind to p75NTR with a high affinity and induces apoptosis of neurons in vitro. Mutation in Val66Met of brain-derived neurotrophic factor (BDNF) results in reduction in hippocampal function in learning and in the dysfunction of intracellular BDNF sorting and secretion. To examine the functions of pro-neurotrophins in vivo, it is essential to know where they are expressed in the nervous system. In the present study, we have raised and characterized rabbit polyclonal antibodies against a peptide coding for the precursor region of the BDNF gene. The antibody specifically recognizes the precursor for BDNF by western blot. With the affinity purified precursor antibody, we have mapped the distribution and localization of the precursor for BDNF. The results showed that, like mature BDNF, pro-BDNF is localized to nerve terminals in the superficial layers of dorsal horn, trigeminal nuclei, nuclei tractus solitarius, amygdaloid complex, hippocampus, hypothalamus and some peripheral tissues. These results suggest that pro-BDNF, like mature BDNF, is anterogradely transported to nerve terminals and may have important functions in synaptic transmission in the spinal cord and brain.     

Distribution of glutamine synthetase in the rat central nervous system.

The results of a light microscopic immunohistochemical study of glutamine synthetase in rat nervous system are presented. In all sites studied the enzyme was confined to astrocytes. Except for trace amounts in ependymal cells, the enzyme was not observed in other cells of the nervous system including neurons, choroid plexus, third ventricular tanycytes, subependymal cells and mesodermally-derived elements. The intensity of astrocyte staining varied in different regions with the greatest degree noted in the hippocampus and cerebellar cortex while the least was noted in brain stem, deep cerebellar nuclei and spinal cord. The glutamine synthetase content correlated well with sites of suspected glutamergic activity in keeping with the view of a critical role of astrocytes in the regulation of the putative neurotransmitter glutamic acid.     

Quantitative distribution of calcitonin gene-related peptide in the rat central nervous system

Using an antiserum directed against human calcitonin gene-related peptide (hCGRP), which fully cross reacts with rat CGRP, a sensitive radioimmunoassay was developed. The antiserum was characterized by displacement curve characteristics and high performance liquid chromatography. The assay was applied to rat brain tissue and the concentration of CGRP for 48 microdissected brain areas is presented. Highest levels (1000–4500 fmol/mg protein) were found in the central amygdaloid, caudate putamen, and spinal trigeminal nerve nucleus and tract, substantia gelatinosa, and the dorsal horn of the spinal cord. Moderate levels (200–600 fmol/mg protein) were found in the bed nucleus of the stria terminalis, the subfornical organ, the paraventricular, arcuate, dorsomedial, dorsal parabrachial, ambiguus and tractus solitarii nuclei and in the median eminence. These results coincide with those previously obtained by immunohistochemistry. The widespread distribution in the brain suggests involvement of CGRP in a variety of behavioral functions.     

Cloning and characterization of two forms of C-type natriuretic peptide receptor in rat brain.

Two similar membrane bound guanylate cyclases (GC-A and GC-B) are known as natriuretic peptide receptors, but have not been well characterized yet. In this study, we have isolated two forms of GC-B cDNA clones along with GC-A cDNA clones from rat brain. The two forms of rat GC-B differ from each other only by 75bp deletion at 3'-flanking region of the putative transmembrane domain, the shorter form lacking the nucleotide binding site by the deletion. Expression of these cDNAs on mammalian cells revealed that (1) GC-B is a specific receptor for CNP whereas GC-A is stimulated effectively both by ANP and BNP, and (2) the two forms of GC-B possess practically the same high binding affinity for CNP while the shorter form could not induce cGMP production by the binding of CNP. These data indicate that in rat brain is present the non-functional receptor for CNP caused by the short deletion.     

Distribution of catch-relaxing peptide (CARP)-like immunoreactive neurons in the central and peripheral nervous system of Helix pomatia

Immunocytochemistry was performed on the nervous system of Helix by the use of an antibody raised against a myotropic neuropeptide, the catch-relaxing peptide (CARP), isolated from Mytilus edulis. In each ganglion of the central nervous system of Helix pomatia, numerous CARP-immunoreactive cell bodies and a dense immunoreactive fiber system could be observed with a dominancy in the cerebral and pedal ganglia. The majority of the immunoreactive neurons are unipolar, although multipolar neurons also occur. In the neuropil areas, CARP-immunoreactive fibers show extensive arborization, which may indicate a central role of CARP. CARP-immunoreactive elements could be observed in each investigated peripheral nerve and peripheral areas, namely in the intestine, heart, aorta, buccal mass, lips, and foot. However, CARP-immunoreactive cell bodies could only be demonstrated in the intestine and the foot musculature. Thin varicose CARP-immunoreactive fibers were observed over both muscle and gland cells in the different peripheral organs, suggesting a peripheral role of CARP. In vivo CARP injection into the body cavity (10^-3, 10^-4, 10^-5 M) altered the general behavioral state of the animals and induced the relaxation of the musculature of the whole body wall indicating that CARP has a significant role in the regulation of muscle contraction.     

Immunofluorescence studies of neurofilaments in the rat and human peripheral and central nervous system

Localization of antisera to neurofilament antigens derived from rat peripheral nerve was carried out in tissues of rat and human peripheral and central nervous systems by indirect immunofluorescence. Unfixed and chloroform-methanol-fixed frozen sections of tissues were incubated in purified IgG of the experimental rabbit antisera and subsequently exposed to goat anti-rabbit IgG conjugated with fluorescein isothiocyanate. Control studies were conducted on identical tissue preparations incubated in the same concentrations of nonspecific rabbit IgG or in experimental rabbit IgG absorbed with extracts of rat peripheral nerve containing neurofilament antigen. Extensive immunofluorescence was observed in rat and human peripheral and central nervous systems. The distribution and configuration of immunofluorescence corresponded to neurofilament-rich structural components of these tissues. Prominent immunofluorescence was also noted in neuronal cell bodies of spinal sensory ganglia, especially in perikarya of the large neuronal type. Immunofluorescence of the central nervous system was located predominantly in myelinated axons of the white matter in cerebrum, cerebellum, brain stem, and spinal cord. Less intense immunofluorescence was also seen in neuronal perikarya and in short thin linear processes of grey matter.