Simultaneous measurement of atrial natriuretic polypeptide (ANP) messenger RNA and ANP in rat heart--evidence for a preferentially increased synthesis and secretion of ANP in left atrium of spontaneously hypertensive rats (SHR)

Tissue levels of atrial natriuretic polypeptide (ANP) messenger RNA (ANPmRNA) and ANP in the rat heart were measured simultaneously. In Wistar rats, ANPmRNA of the same size (approximately 0.95 kbp) was detected in all four chambers of the rat heart. The ANPmRNA level was the highest in the right atrium, and the left atrial level was slightly lower than the right atrial level. Ventricular levels were more than two orders of magnitude lower than atrial levels. Tissue ANP concentrations of four chambers were roughly parallel to ANPmRNA levels. In spontaneously hypertensive rats (SHR) with the elevated plasma ANP level, the ANPmRNA level in the left atrium was substantially increased. The left/right ratio of atrial ANPmRNA level in SHR (150%) was higher than that in control Wistar Kyoto rats (WKY) (90%). In contrast, the left/right ratio of atrial ANP concentration was decreased in SHR (44%) compared with that in WKY (84%). The ratio of ANP to ANPmRNA levels in the left atrium of SHR was about three times smaller than that in the right atrium of SHR, and those in bilateral atria of WKY. These results indicate that the biosynthesis and secretion of ANP from the left atrium is preferentially increased in SHR. Thus, simultaneous determination of ANPmRNA and ANP levels is a refined strategy of investigation for the biosynthesis, storage and secretion of ANP.     

Occurrence of a novel cardiac natriuretic peptide in rats

We established a specific radioimmunoassay for the ring structure of "iso-ANP" and detected iso-ANP[23-46]-like immunoreactivity (-LI) in the rat atrium (2.76 +/- 0.5 micrograms/g) and ventricle (13.9 +/- 5.7 ng/g). High performance-gel permeation chromatography revealed that iso-ANP[23-46]-LI in the rat heart was composed of two components with molecular weights of 10K and 5K. In reverse phase-high performance liquid chromatography, the retention times of these components were clearly different from that of synthetic iso-ANP. The 5K peptide was demonstrated to be present in the perfusate from isolated rat hearts and possessed binding ability to ANP receptors. This natriuretic peptide was, however, not detectable in other tissues including the brain. We conclude that the novel cardiac natriuretic peptide distinct from iso-ANP and ANP occurs in the rat heart and is secreted from the heart.     

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.     

Endothelin in human plasma and culture medium of aortic endothelial cells--detection and characterization with radioimmunoassay using monoclonal antibody

We have developed monoclonal (KY-ET-1-I) and polyclonal (ET-F5) antibodies against endothelin-1 (ET-1) and established sensitive radioimmunoassays (RIAs) with different specificities. The RIA with KY-ET-1-I detected ET-1, ET-2 and ET-3, while the RIA with ET-F5 recognized ET-3 very weakly. Using these RIAs, we have investigated the concentration and molecular forms of ET-1-like immunoreactivity (-LI) in culture medium of bovine aortic endothelial cells and human plasma. Culture medium of endothelial cells contained two major components compatible with big ET and ET-1. ET-1-LI was also detected in human plasma. ET-1-LI in human plasma consisted of apparent two components, the small molecular form emerging at the position of ET-1 and the large form with the peak eluting at the preceding fraction of the elution position of big ET. The concentration of the small form of ET in human plasma was about 5 pg/ml.     

Detection and characterization of endothelin-1-like immunoreactivity in rat plasma

Using two radioimmunoassays (RIAs) for endothelin-1 (ET-1) with and without a substantial cross-reactivity with ET-3, we have measured the plasma ET-1-like immunoreactivity (-LI) level in rat plasma. ET-1-LI was detected in plasma from male Wistar rats. ET-1-LI in rat plasma consisted of three components with molecular weights of 6K, 4K and 2.5K daltons by gel permeation chromatography. Two of the components were eluted at positions of big ET (4K) and synthetic ET-1 (2.5K). The remaining component was eluted at the preceding fraction (6K). No difference was observed in ET-1-LI of the small molecular form of ET (2.5K) between the two RIAs. Thus, there is little or no ET-3 in rat plasma, which has the sequence found originally in the rat genome. The concentration of the small molecular form of ET, presumably ET-1, in rat plasma was about 4 pg/ml.     

Immunohistochemical localization of atrial natriuretic polypeptide (ANP) in human atrial and ventricular myocardiocytes

To date, there have been few immunohistochemical investigations of atrial natriuretic polypeptide (ANP) in human cardiac tissue, especially the ventricles. In this study, myocardial tissue was obtained from two sources: the bilateral atria and ventricles at autopsy; and biopsy tissues from the right auricle and left ventricle of a patient with myocardial infarction undergoing surgery. These tissues were examined by the avidin-biotin immunoperoxidase technique using three kinds of primary ANP-antibodies. ANP-immunoreactivity was observed in the perinuclear region of myocytes of all tissues examined. The intensity of the reaction was stronger in atrial tissue, weaker in ventricular tissue. In the later tissue, the positive-staining myocytes were not part of the pulse-conducting system. Although the tissues we studied were not obtained from normal hearts, our data demonstrates that ANP-reactivity can be detected in ventricular myocytes outside the pulse-conducting system.     

Arabinogalactan-Proteins from Primary and Mature Roots of Radish (Raphanus sativus L.)

Organ-specific variations in blood group H-like activity were observed in developing radish plants. A temporary increase in serological activity was found to occur in the roots at the earlier stages of development. Arabinogalactan-proteins (AGPs) were isolated from primary and mature roots, and investigated for changes in their physicochemical properties, structure, and serological activities. These root AGPs were composed mainly of l-arabinose and d-galactose but were distinguishable from each other in their contents of l-fucose as well as of protein and hydroxyproline. The structures of the carbohydrate moieties of the root AGPs were essentially similar to those of AGPs isolated from seeds and mature leaves in that they consisted of consecutive (1→3)-linked β-d-galactosyl backbone chains having side chains of (1→6)-linked β-d-galactosyl residues, to which α-l-arabinofuranosyl residues were attached in the outer regions. One prominent feature of the primary root AGPs was that they contained appreciable amounts of l-fucose, which was presumably responsible for expression of the serological activity. In their immunological reactions with rabbit anti-radish leaf AGP antibody, the root AGPs were shown to share common antigenic determinant(s) with those of seed and leaf AGPs.     

Immunohistochemical localization of atrial natriuretic polypeptide (ANP) in human atrial and ventricular myocardiocytes

Summary To date, there have been few immunohistochemical investigations of atrial natriuretic polypeptide (ANP) in human cardiac tissue, especially the ventricles. In this study, myocardial tissue was obtained from two sources: the bilateral atria and ventricles at autopsy; and biopsy tissues from the right auricle and left ventricle of a patient with myocardial infarction undergoing surgery. These tissues were examined by the avidin-biotin immunoperoxidase technique using three kinds of primary ANP-antibodies. ANP-immunoreactivity was observed in the perinuclear region of myocytes of all tissues examined. The intensity of the reaction was stronger in atrial tissue, weaker in ventricular tissue. In the later tissue, the positive-staining myocytes were not part of the pulse-conducting system. Although the tissues we studied were not obtained from normal hearts, our data demonstrates that ANP-reactivity can be detected in ventricular myocytes outside the pulse-conducting system.     

Acute pulmonary alveolar hypoxia increases lung and plasma endothelin-1 levels in conscious rats.

To investigate the effect of pulmonary alveolar hypoxia on the synthesis and release of endothelin (ET)-1, ET-1-like immunoreactivity (-LI) levels of the lung and plasma were measured in conscious unrestrained rats under hypoxic conditions. Sixty-min exposure to alveolar hypoxia (10% O2 or 5% O2) increased the ET-1-LI level in the lung. The plasma ET-1-LI level in hypoxic rats also increased significantly. The increase of plasma and lung ET-1-LI levels were parallel to the severity of hypoxia. These results demonstrates that acute pulmonary alveolar hypoxia increases lung and plasma ET-1-LI levels in conscious unrestrained rats, suggesting a possible physiological or pathophysiological significance of ET in alveolar hypoxia.     

Establishment of five human myeloma cell lines

Summary Five human myeloma cell lines, KMM-1, KMS-5, KMS-11, KMS-12- PE, and KMS-12-BM, have been established at Kawasaki Medical School since 1980. As the KMS-12-PE and KMS-12-BM lines were obtained from the same patient, these five cell lines have been derived from four patients with multiple myeloma. The five myeloma cell lines are stably growing at present in RPMI 1640 medium supplemented with 10% fetal bovine serum. They can also grow in a defined culture medium without serum. That these cell lines were, human myeloma cells was confirmed by the following findings. Ultranstructually, all five cell lines showed features characteristic of plasma cells. KMM-1 and KMS-11 cells secreted lambda and kappa chains into the culture medium, respectively, but the other cell lines produced no immunoglobulins. KMM-1 expressed cytoplasmic lambda antigen, KMS-5 showed cytoplasmic delta, and KMS-11 expressed surface kappa, whereas KMS-12-PE and KMS-12-BM cells showed no surface or cytoplasmic immunoglobulins. Regarding reaction with a monoclonal plasma cell antibody (PCA-1), four of the five lines were positive, the exception being KMS-5. Another monoclonal antibody (CD38), which also recognizes plasma cells, reponded to KMM-1, KMS-12-PE, and KSM-12-BM. KMS-5 cells expressed acute lymphoblastic leukemia antigens (CALLA). These data suggest that such lines as KMM-1, KMS-11, KMS-12-PE, and KMS-12-BM represent later stages of B-cell differentiation, and that KMS-5 represents a relatively early stage of B-cell differentiation. All the cell lines lacked Epstein-Barr virus nuclear antigen, showed abnormal karyotypes of human origin, and differed from each other in the isozyme patterns examined. Only KMS-5 was tumorigenic when transplanted subcutaneously into nude mice.