Endothelin receptor antagonist prevents parathyroid cell proliferation of low calcium diet-induced hyperparathyroidism in rats.

Secondary hyperparathyroidism, one of the most frequently encountered disorders of the calcium homeostasis, is characterized by an increase in parathyroid epithelial (PT) cell number, which is crucial from a functional viewpoint. However, it is still unknown what factors are involved in PT cell proliferation. Endothelin-1 (ET-1), a vasoconstrictive peptide, has been shown to act as a mitogen in a variety of cell types. Rat PT cells are reported to synthesize ET-1 and possess its receptors. To test the hypothesis that ET-1 plays a role in PT cell proliferation, we used rat test subjects fed a low calcium diet for 8 weeks (low Ca rats). The number of the proliferating PT cells, measured by proliferating cell nuclear antigen immunostaining, was significantly increased, with striking immunoreactivity of ET-1 in the low Ca rats. An endothelin receptor antagonist, bosentan (100 mg/kg.day), prevented any increase in the proliferation of PT cells in the low Ca rats (14.3 +/- 2.7/1000 PT cells with no bosentan; 2.1 +/- 1.3 with bosentan; P < 0.01). These results indicate that ET-1 is involved in PT cell proliferation in vivo and suggest that blocking of ET receptors may become one of the important therapeutic strategies for preventing secondary hyperparathyroidism.

Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides.

The inhibition of specific transcription regulatory proteins is a new approach to control gene expression. The transcriptional activities of DNA-binding proteins can be inhibited by the use of double-stranded oligonucleotides that compete for the binding to their specific target sequences in promoters and enhancers. We used nicked (NDODN-kappaB) and circular (CDODN-kappaB) dumbbell DNA oligonucleotides containing a NF-kappaB binding site to analyze the inhibition of the NF-kappaB-dependent activation of the human immunodeficiency virus type-1 (HIV-1) enhancer. The dumbbell DNA oligonucleotides are stable, short segments of double-stranded DNA with closed nucleotide loops on each end, which confer resistance to exonucleases. The dumbbell and other oligonucleotides (decoys) with the NF-kappaB sequence were found to compete with the native strand for NF-kappaB binding. The circular dumbbell and double-stranded phosphorothioate oligonucleotides competed with the native strand for binding to the NF-kappaB binding proteins, while the nicked NF-kappaB dumbbell was a less effective competitor. In Jurkat T-cells, the dumbbell and other oligonucleotides were tested for their ability to block the activation of the plasmid HIV-NL4-3 Luc. The CDODN-kappaB strongly inhibits the specific transcriptional regulatory proteins, as compared with the NDODN-kappaB and the double stranded phosphodiester oligonucleotides. On the other hand, the double stranded phosphorothioate oligonucleotides could also block this activation, but the effect was non-specific. The circular (CDODN) dumbbell oligonucleotides may efficiently compete for the binding of specific transcription factors within cells, thus providing anti-HIV-1 or other therapeutic effects.

Endothelin-1 inhibits endothelin-converting enzyme-1 expression in cultured rat pulmonary endothelial cells.

BACKGROUND: The lung expresses large amounts of endothelin-converting enzyme-1 (ECE-1), which catalyzes a step in the biosynthesis of potent vasoactive endothelin-1 (ET-1) from the inactive intermediate big ET-1. Because there has been no report concerning a possible relationship between ET-1 and ECE-1, we investigated the effects of ET-1 on ECE-1 expression in cultured rat pulmonary endothelial cells. METHODS AND RESULTS: ECE-1 messenger RNA (mRNA) and protein expression in cultured endothelial cells were assayed by Northern and Western blotting, respectively. Incubation with ET-1 for 6 hours caused a significant decrease in ECE-1 mRNA expression. The action of ET-1 on ECE-1 mRNA expression was antagonized by pretreatment with BQ788, a specific ETB receptor antagonist, but not by pretreatment with BQ123, a specific ETA receptor antagonist. The expression of ECE-1 protein was also inhibited at 6 hours after incubation with ET-1. The effects of ET-1 on ECE-1 mRNA and protein expression were shown to be mimicked by ionomycin, a calcium ionophore, but not by 12-O-tetradecanoylphorbol 13-acetate, a protein kinase C activator. CONCLUSIONS: The present results demonstrate that ET-1 suppressed ECE-1 protein levels by inhibiting ECE-1 mRNA expression through the ETB receptor, suggesting the existence of a feedback action of ET-1 on ECE-1 in pulmonary endothelial cells.

The primary structure of two molecular species of porcine organ-common type acylphosphatase.

Electrophoretically homogeneous preparations of organ-common type acylphosphatase from porcine testis and brain were separated into two molecular species by reversed-phase liquid chromatography. From tryptic peptide map analysis, it was inferred that each of the two testis proteins is the same as the corresponding one of the two brain proteins. The complete primary structures of the two acylphosphatases from testis were then determined. The one molecular species consists of 100 amino acid residues: [sequence; see text] The other consists of 98 amino acid residues identical to the 3rd-100th residues of the above sequence and is also acetylated at the amino-terminal alanine. The 98-residue sequence has only 59% homology with porcine muscle acylphosphatase, but has 92% homology with human erythrocyte acylphosphatase. It was thus confirmed that the major acylphosphatases in testis, brain, and erythrocyte belong to the same organ-common type isoenzyme, distinct from the muscle type isoenzyme.

Distribution and classification of acylphosphatase isozymes.

Distributions of acylphosphatase isozymes among organs of several animal species were investigated. Organ extracts of pig and chicken were treated with isozyme-specific antibodies, subjected to electrophoresis on a polyacrylamide gel, then the gel was stained for acylphosphatase activity. Both animals showed three activity bands; one band was named common type isozyme because of its wide distribution in testis, muscle, brain, heart, spleen, kidney, liver, and erythrocyte, and the other two bands were named muscle type isozymes because of their localization in skeletal muscle. This classification was supported by selective and quantitative reactions of the isozymes to the isozyme-specific antibodies. Because the two bands of the muscle type have the same amino acid sequence and differ only in modifications on an -SH group, it is suggested that pig and chicken have only the two major types of acylphosphatase. This conclusion was supported by similar experiments on dog, human, rabbit, and pigeon.

Activity staining of acylphosphatase after gel electrophoresis.

Acylphosphatase is an ubiquitous enzyme found in a variety of mammalian and avian tissues. Two isozymes of different amino acid sequence have been found in human, chicken, pig, and horse. To survey the distribution of the acylphosphatase isozymes among animal species and tissues, we have developed an activity staining procedure for the enzyme after electrophoresis on a polyacrylamide gel. Tissue extracts of pigs were subjected to electrophoresis, then the gel was stained for acylphosphatase activity in a solution containing acetyl phosphate and lead nitrate. Three activity bands were observed: the slowest moving one, which coincided with that of purified testis acylphosphatase, was widely distributed in testis, muscle, brain, heart, spleen, kidney, liver, and erythrocyte; the other two bands, which coincided with monomer and dimer of purified muscle acylphosphatase, were relatively localized in skeletal muscle.