Purification and characterization of recombinant human macrophage colony-stimulating factor expressed in Chinese hamster ovary cells.

We expressed human macrophage colony-stimulating factor (M-CSF) in Chinese hamster ovary (CHO) cells by introducing an expression plasmid coding for a 554-amino-acid M-CSF precursor and dihydrofolate reductase (DHFR) gene, and by amplifying the sequence. A cell line was obtained that secreted approximately 200,000 units/ml after 6 days in culture. The expressed recombinant human M-CSF (rhM-CSF) primarily consisted of two molecular species, a main 80-90 kD M-CSF as a homodimer and a molecular form higher than 150 kD. Purification of a main rhM-CSF gave an apparently homogeneous protein disulfide-bonded from 42-kD subunits, but one of the purified rhM-CSFs was composed of two subunit species with molecular masses of 44 and 42 kD. These purified rhM-CSFs had substantially the same specific activity (1 to 4 x 10(7) units/mg protein). Deglycosylation experiments with the latter rhM-CSF using chemical (trifluoromethanesulfonic acid) and enzymatic methods found a terminal neuraminic acid in addition to N- and O-glycosylation, but the two subunit species did not coalesce into a single molecule.

Solution-phase synthesis of porcine brain natriuretic peptide (pBNP) using S-trimethylacetamidomethylcysteine.

The hexadodecapeptide corresponding to the entire amino acid sequence of porcine brain natriuretic peptide (pBNP) was synthesized by assembling four segments in solution, followed by HF deprotection and subsequent oxidation to establish an intramolecular disulfide bridge. The synthesis using the newly developed S-trimethylacetamidomethylcysteine [Cys(Tacm)] derivative gave a better yield than that using the S-2,4,6-trimethylbenzylcysteine [Cys(Tmb)] derivative. The chick rectum relaxant activity of the synthetic pBNP was 2.9 times more potent than that of alpha-rat atrial natriuretic peptide (alpha-rANP).

Solution-phase synthesis of alpha-rat atrial natriuretic peptide (alpha-rANP).

alpha-Rat atrial natriuretic peptide (alpha-rANP) was synthesized by assembling five peptide fragments in solution, followed by HF-dimethylselenide-m-cresol deprotection and subsequent air-oxidation. Synthetic alpha-rANP exhibited more potent diuretic and natriuretic activity in rats than synthetic alpha-hANP.

Solution-phase synthesis of alpha-human atrial natriuretic peptide (alpha-hANP).

Alpha-human atrial natriuretic peptide (alpha-hANP) was synthesized by assembling six peptide fragments in solution followed by deprotection with HF and subsequent air-oxidation. The trimethylbenzyl group was employed as an S-protecting group of cysteine. The HF-dimethylselenide-m-cresol system was employed as a final deprotecting reagent and, at the same time, as a reducing reagent of Met(O). Synthetic alpha-hANP elicited potent diuretic and natriuretic activity in rats.

Purification and some properties of the fast migrating alkaline phosphatase in FL-amnion cells (the Kasahara isoenzyme) and its cDNA cloning.

One of two main FL-amnion cell alkaline phosphatase (AP), the fast migrating one (FL-APF) has been reported to be identical to Kasahara isoenzyme (K.I.), which occurs preferentially in sera of patients with primary hepatoma. We purified FL-APF of which the apparent molecular weight was 135,000 by gel filtration, and that of the subunit was 62,000 on SDS/PAGE, indicating homodimeric structure of FL-AL-APF. FL-APF was found to react with monoclonal antibody against adult intestinal AP, but not with monoclonal antibody to placental AP. We isolated FL-APF cDNA clone from FL-amnion cells, of which cDNA was 2525 base pairs in length. Nucleotide sequence of the coding region and the 3' untranslated region was identical to the sequence of human adult intestinal AP cDNA. But the untranslated region of the 5' end of the isolated clone was slightly longer than that of intestinal AP. Hence, FL-APF (K.I.) may occur by altered glycosylation of intestinal AP.

Specific binding activities and cyclic GMP responses by atrial natriuretic polypeptide in kidney epithelial cell line (LLC-PK1).

Receptor binding activities and cyclic GMP responses by alpha-human atrial natriuretic polypeptide (alpha-hANP) and its fragments were studied in a kidney epithelial cell line (LLC-PK1). Binding of 125I-alpha-hANP to the cells at 0 degrees C was saturable, time-dependent and reversible, indicating the presence of a single class of binding sites. alpha-hANP (7-23)NH2 fragment inhibited most effectively the specific binding of 125I-alpha-hANP to the LLC-PK1 cells, followed by alpha-hANP (17-28) and alpha-hANP (8-22), while alpha-hANP (1-6) and alpha-hANP (24-28) did not. alpha-hANP stimulated the formation of cyclic GMP in the LLC-PK1 cells dose-dependently. Although no fragments of alpha-hANP used were effective for cyclic GMP formation in the LLC-PK1 cells, alpha-hANP (7-23) NH2 antagonized the action of alpha-hANP on cyclic GMP formation. These data suggest that the LLC-PK1 cells retain specific receptors for atrial natriuretic polypeptide (ANP) and respond to ANP by stimulating cyclic GMP formation, and therefore this cell line may be useful for studying the mechanism of action for ANP in renal tubular cells.

Alpha-human atrial natriuretic polypeptide is released from the heart and circulates in the body.

In order to clarify whether or not atrial natriuretic polypeptides are hormones in man, we have measured plasma alpha-human atrial natriuretic polypeptide (alpha-hANP)-like immunoreactivity (alpha-hANP-LI) with or without extraction procedure. alpha-hANP-LI was detected in plasma extracts from all 5 normal subjects and 7 patients with heart diseases. The alpha-hANP-LI concentration in normal peripheral plasma was 37.7 +/- 7.0 pg/ml (mean +/- SE). Plasma concentrations of alpha-hANP-LI in the coronary sinus obtained by cardiac catheterization were 3 to 10 times higher than those in the peripheral vein, inferior vena cava, right atrium, pulmonary artery and aorta. High performance gel permeation chromatography coupled with a radioimmunoassay (RIA) for alpha-hANP revealed that alpha-hANP-LI in normal peripheral plasma eluted at the position corresponding to that of authentic alpha-hANP without detectable amounts of high molecular weight forms. alpha-hANP-LI extracted from plasma taken from the coronary sinus of two patients also showed a single peak of alpha-hANP-LI co-eluting with alpha-hANP. In contrast, not only alpha-hANP but gamma-hANP and beta-hANP, high molecular weight forms, were present in the human atrial tissue. These results indicate that alpha-hANP is the predominant form of alpha-hANP-LI in human plasma and that this form generated in the atrial cardiocytes is preferentially released from these cells and circulates in the body.

Existence of atrial natriuretic polypeptide in kidney.

Using a specific radioimmunoassay (RIA) for alpha-atrial natriuretic polypeptide (alpha-ANP), we have demonstrated the presence of alpha-rat ANP-like immunoreactivity (alpha-rANP-LI) in the rat kidney which is considered to be a target organ for atrial natriuretic polypeptides released from the heart. Most of alpha-rANP-LI was localized in the cortex. High performance gel permeation chromatography coupled with the RIA revealed that renal alpha-rANP-LI was eluted at the position of a low molecular weight form corresponding to alpha-rANP without detectable amounts of high molecular weight forms. This is in contrast to the observation that gamma-rANP, a high molecular weight form of 13k daltons, is the dominant form of alpha-rANP-LI in the rat atrium. In water-deprived rats, the concentration and content of alpha-rANP-LI in the kidney showed a significant decrease compared with control rats. In addition, the alpha-rANP-LI concentration and content in this organ revealed a substantial decrease after perfusion with physiological saline. These results indicate the existence of atrial natriuretic polypeptide (ANP) in the kidney and suggest that part of renal ANP may originate from the heart.

Occurrence of atrial natriuretic polypeptide in brain.

In order to determine whether or not atrial natriuretic polypeptides (ANPs) exist in the brain, we have studied extracts from the rat brain using a specific radioimmunoassay (RIA) for alpha-atrial natriuretic polypeptide. The presence and widespread distribution of alpha-rat ANP-like immunoreactivity (alpha-rANP-LI) have been demonstrated in the rat brain. The highest concentration of alpha-rANP-LI (20-22 ng/g) is in the hypothalamus and the septum. Moderate concentrations of alpha-rANP-LI (2-8 ng/g) are also found in the midbrain, cerebral cortex, olfactory bulb, thalamus, pons-medulla and hippocampus. High performance gel permeation chromatography coupled with the RIA revealed that alpha-rANP-LI found in the rat brain consists of three components eluting at the positions of gamma-rat ANP (gamma-rANP), beta-rat ANP (beta-rANP) and alpha-rANP, respectively. Among these a low molecular weight form of alpha-rANP-LI emerging at the elution position corresponding to alpha-rANP is predominant in the rat brain. This is in contrast to the finding that gamma-rANP, a high molecular weight form of 13k daltons is the dominant form of alpha-rANP-LI in the rat atrium. These results clearly show that there exists a widespread neural system containing ANPs in the brain.

Accelerated natriuresis induced by synthetic atrial natriuretic polypeptide in spontaneously hypertensive rats.

Effects of synthetic alpha human atrial natriuretic polypeptide (alpha-hANP) on diuresis, natriuresis and mean arterial blood pressure (MAP) were compared between 4-5 month-old male spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) under ether anesthesia. In both groups, the peptide injected (0.5 micrograms/100g body weight, i.v.) caused potent (about ten fold), rapid and short-acting (for 15 min) increases in sodium (Na+) and chloride excretions and also an increase in urine flow and potassium excretion with lesser magnitude. Although ratios of the maximum response to basal value were much the same, net increases in urine flow and Na+ output were significantly greater in SHR than in WKY. As to the effect on MAP, a rapid (within 2 min) fall observed in the two groups. These results suggest that the atrial natriuretic peptide may be involved in the altered regulatory mechanism of fluid and electrolyte balance in SHR models with genetic hypertension.

Radioimmunoassay for alpha-human and rat atrial natriuretic polypeptide.

Using a synthetic common carboxy-terminal fragment of alpha-human atrial natriuretic polypeptide (alpha-hANP) and alpha-rat atrial natriuretic polypeptide (alpha-rANP), we have produced an antiserum for alpha-ANP(17-28) and established a radioimmunoassay (RIA) for alpha-ANP that recognizes alpha-hANP and alpha-rANP equally. High performance gel permeation chromatography coupled with the RIA revealed that alpha-hANP-like immunoreactivity (alpha-hANP-LI) in the human atrium consists of three major components, gamma-hANP, alpha-hANP and another. On the other hand, alpha-rANP-LI in the rat atrium comprised at least two components, 13K alpha-rANP-LI and 3K-5K alpha-rANP-LI, which were presumably gamma-rANP and beta-rANP, respectively. Thus, considerable amounts of gamma-hANP and gamma-rANP are present in human right auricles and rat atria, respectively. The RIA established in this study provides a useful tool to investigate the pathophysiological significance of alpha-ANP and related peptides in cardiovascular disorders and shows that gamma-ANP is not only a precursor of alpha-ANP but acts as an important hormone.