Takao Masaki

Three distinct human endothelin-related genes were cloned by screening a genomic DNA library under a low hybridization stringency with a synthetic oligonucleotide probe encoding a portion of the endothelin sequence. Genomic Southern blot analysis with the same oligonucleotide probe showed three corresponding chromosomal loci not only in the human genome but also in porcine and rat genomes. The nucleotide sequences of the three human genes were highly conserved within the regions encoding the 21-residue (mature) endothelins, in spite of the fact that the immediately upstream exon sequences, which encode a part of the propeptides, retained little similarity. Moreover, each of the human genes predicted a putative 21-residue peptide, similar to but distinct from each other: (i) the "classical" endothelin (ET-1), (ii) [Trp6,Leu7]endothelin (ET-2), and (iii) [Thr2,Phe4,Thr5,Tyr6, Lys7,Tyr14]endothelin (ET-3). Synthetic ET-1, ET-2, and ET-3 were prepared according to the deduced amino acid sequences, and the biological activities were assayed by contraction of isolated porcine coronary artery strips and by intravenous injection to anesthetized rats. All these synthetic peptides produced strong vasoconstrictor and pressor responses. However, the quantitative profiles of the pharmacological activities were considerably different among the three isopeptides, suggesting the possible existence of endothelin receptor subtypes.

The identification of mutations in the Tau gene in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has made it possible to express human tau protein with pathogenic mutations in transgenic animals. Here we report on the production and characterization of a line of mice transgenic for the 383 aa isoform of human tau with the P301S mutation. At 5-6 months of age, homozygous animals from this line developed a neurological phenotype dominated by a severe paraparesis. According to light microscopy, many nerve cells in brain and spinal cord were strongly immunoreactive for hyperphosphorylated tau. According to electron microscopy, abundant filaments made of hyperphosphorylated tau protein were present. The majority of filaments resembled the half-twisted ribbons described previously in cases of FTDP-17, with a minority of filaments resembling the paired helical filaments of Alzheimer's disease. Sarkosyl-insoluble tau from brains and spinal cords of transgenic mice ran as a hyperphosphorylated 64 kDa band, the same apparent molecular mass as that of the 383 aa tau isoform in the human tauopathies. Perchloric acid-soluble tau was also phosphorylated at many sites, with the notable exception of serine 214. In the spinal cord, neurodegeneration was present, as indicated by a 49% reduction in the number of motor neurons. No evidence for apoptosis was obtained, despite the extensive colocalization of hyperphosphorylated tau protein with activated MAP kinase family members. The latter may be involved in the hyperphosphorylation of tau.

Endothelin-1 is a 21-amino acid potent vasoconstrictor peptide produced by vascular endothelial cells. We have cloned the whole length of the human preproendothelin-1 (PPET-1) gene and the corresponding cDNA and determined the complete nucleotide sequences. The 2026-nucleotide human mRNA for PPET-1 (excluding the polY(A) tail) is encoded in five exons distributed over 6836 base pairs of the genome. The 5'-flanking region of the gene contains (i) octanucleotide sequences for the phorbol ester-responsive elements, also known as the binding elements for FOS.JUN complex; (ii) consensus motifs for the binding site of nuclear factor 1, which may mediate the induction described previously of PPET-1 mRNA by transforming growth factor-beta; (iii) hexanucleotide sequences for the acute phase reactant regulatory elements that may be involved in the induction of endothelin-1 under acute physical stress in vivo. Further, the 3'-nontranslated sequence of human PPET-1 mRNA contains three AUUUA motifs, which may mediate selective translation-dependent destabilization of the mRNA. Northern blot analysis in cultured endothelial cells from human umbilical veins shows that PPET-1 mRNA is in fact rapidly induced by the active phorbol ester 12-O-tetradecanoylphorbol 13-acetate within 10 min. Analysis of mRNA life span by using actinomycin D demonstrates that PPET-1 mRNA has a short intracellular half-life of about 15 min and is superinduced by cycloheximide. This superinduction is found to be due to the stabilization of the mRNA by cycloheximide, as in the case of other known AUUUA-containing mRNAs. These findings suggest that the regulation of expression of PPET-1 mRNA may be mediated in part by these sequence elements.

Endothelin is a potent endothelium-derived vasoconstrictor peptide recently characterized from porcine and human vascular endothelial cells. Here we provide evidence that endothelin activates the dihydropyridine-sensitive, voltage-dependent Ca2+ channel in porcine coronary artery smooth muscle. The vasoconstrictor action of endothelin is efficiently antagonized by low doses of the dihydropyridine Ca2+-channel blocker nicardipine. Endothelin augments the Ca2+-induced contraction in a high-K+ depolarizing solution, markedly enhances high-threshold Ca2+-channel current on the whole-cell patch clamp recording, and causes a sustained increase in the intracellular Ca2+ that is largely dependent on extracellular Ca2+. These findings suggest that endothelin exerts its vasoconstrictor effect by either directly or indirectly activating the voltage-dependent Ca2+ channel.