Atsuro Miyata

Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38)

A novel neuropeptide with 38 residues (PACAP38) was isolated from ovine hypothalamic tissues using the pituitary adenylate cyclase activation in rat pituitary cell cultures as a parameter of the biological activity (Miyata et al, Biochem. Biophys. Res. Commun. 164, 567-574, 1989). From the side fractions obtained during the purification of PACAP38, a shorter form peptide with 27 residues corresponding to the N-terminal 27 amino acids of PACAP38 and amidated C-terminus was isolated and named as PACAP27. Synthetic PACAP27 showed a biological activity of adenylate cyclase stimulation comparable to PACAP38. Moreover PACAP27 which shows a considerable homology with vasoactive intestinal polypeptide (VIP) demonstrated a similar vasodepressor activity as VIP, but the adenylate cyclase stimulating activity was about 1000 times greater than VIP.

Molecular forms of atrial natriuretic polypeptides in mammalian tissues and plasma

A highly sensitive radioimmunoassay detecting for all the atrial natriuretic polypeptides isolated so far from human and rat (hANPs and rANPs) has been established by using an antiserum raised against alpha-hANP, since the antiserum recognizes the subsequence flanked by two cysteine residues (positions 7 and 23) in alpha-hANP and crossreacts with human as well as rat ANPs. By using the radioimmunoassay combined with gel chromatography or high performance liquid chromatography, it was revealed that ANP immunoreactivity in human atria is composed of alpha-(28 residues), beta-(56 residues: alpha-hANP dimer) and gamma-hANP(126 residues) in various ratios. In rat, porcine and bovine atrial tissues, however, gamma-rANP(126 residues) was found to be a major component. In contrast with atrial ANP, plasma ANP was found to be predominantly alpha-form in rat.

Molecular cloning of human platelet thromboxane A synthase

Complementary DNA coding for thromboxane A synthase was amplified by polymerase chain reaction using primers synthesized according to the partial amino acid sequences of human platelet thromboxane A synthase (Nüsing, R., Schneider-Voss, S., and Ullrich, V. (1990) Arch. Biochem. Biophys. 280, 325-330) and cloned into pBluescript SK II(-). The primary structure of human platelet enzyme was deduced from the nucleotide sequence of the cDNA. The enzyme is composed of 533 amino acids with a molecular weight of 60,487. The primary structure of the enzyme exhibited a 34-36% homology to the amino acid sequences of cytochrome P450s classified in the P450 III gene family. The highly conserved cysteine-containing sequence involved in the heme-binding site of P450 was found near the carboxyl terminus (residues 472-492). The size of the major thromboxane A synthase mRNA from human platelets and human erythroleukemia cells was estimated to be approximately 2.2 kilobases by RNA blot analysis.

Plasma Adrenomedullin Concentrations and Cardiac and Arterial Hypertrophy in Hypertension

It has been reported that plasma concentrations of adrenomedullin (AM), a novel vasodilator peptide, are higher in patients with essential hypertension than those in normotensive subjects. To clarify the clinical significance of increased levels of AM in patients with essential hypertension, in this study we examined the relationship between plasma concentrations of AM and the structure of the left ventricle or carotid artery. Plasma AM concentrations; renin activity; and norepinephrine, epinephrine, and creatinine concentrations in 50 patients with untreated essential hypertension without renal dysfunction and heart failure were measured. We also measured the mean wall thickness of the left ventricle and left ventricular mass index by M-mode echocardiography and intimal-medial thickness and arterial distensibility of the carotid artery by ultrasonography. Hypertensive patients were divided into two groups: hypertensives with and those without left ventricular hypertrophy. Plasma AM concentrations in hypertensive patients with left ventricular hypertrophy were significantly higher than in hypertensive patients without left ventricular hypertrophy (7.87+/-2.70 vs 5.74+/-1.65 fmol/mL, P<.01). In all hypertensive patients, plasma AM concentrations were not correlated with blood pressure, plasma renin activity, plasma norepinephrine, plasma epinephrine, or plasma creatinine concentration. Plasma AM concentrations were positively correlated with left ventricular mass index or mean wall thickness (r=.37, P=.009; r=.40, P=.004, respectively) and inversely correlated with carotid artery distensibility (r=-.33, P=.02), whereas plasma AM concentrations were not correlated with intimal-medial thickness. These results suggest that the observed elevation of plasma AM in patients with essential hypertension with normal renal function may be partly related to cardiac hypertrophy and decreased carotid artery distensibility.

Abundant expression of thromboxane synthase in rat macrophages

The cloned cDNA for rat thromboxane (TX) synthase with a size of 1851 bp contained a 1599‐bp open reading frame which encoded a 533‐amino acid protein sharing 79.7% identity with human TX synthase. RNA blot analysis was carried out with rat cells and tissues. Rat peritoneal macrophages most abundantly expressed mRNA for TX synthase, and its level was not changed by in vivo stimulation of casein. Bone marrow, spleen, lung and thymus also expressed the TX synthase gene. These findings suggest the possibility that TXA2 plays a role in the immune system.

Immunoaffinity purification and cDNA cloning of human platelet prostaglandin endoperoxide synthase (cyclooxygenase).

The cDNA for prostaglandin endoperoxide synthase (cyclooxygenase) was cloned from human platelets by the polymerase chain reaction amplification method, and the primary structure of the enzyme was deduced from the nucleotide sequence. The enzyme was composed of 599 amino acids including 23-amino acid signal sequence, and the calculated molecular weight of the mature protein was 65,995. The enzyme was immunoaffinity-purified from human platelets. The N-terminal amino acid sequence determined by Edman degradation was Ala-Asp-Pro-Gly-Ala-Pro-Thr-Pro-, and the result confirmed the primary structure of the enzyme, which was deduced from the cDNA sequence.

Increased release of atrial natriuretic polypeptides in rats with DOCA-salt hypertension

This study compared atrial and plasma concentrations of immunoreactive alpha-rat atrial natriuretic polypeptide (i alpha-rANP) in rats given tap water (control), a 1% saline solution (salt), deoxycorticosterone acetate (DOCA) and DOCA plus 1% saline solution (DOCA-salt) after 1 and 8 weeks of treatment. DOCA (100 mg/kg) was given by implanting a piece of silicon rubber impregnated with DOCA subcutaneously. Atrial i alpha-rANP increased, while plasma i alpha-rANP decreased with time in all groups. Atrial concentration of i alpha-rANP was significantly lower in the DOCA-salt group than in the other groups at 1 week, and was reduced in the DOCA and DOCA-salt groups as compared to the control group at 8 weeks. On the other hand, plasma concentration of i alpha-rANP was significantly higher in the DOCA and the DOCA-salt groups than in the control group at 1 week; the DOCA and DOCA-salt group values were also higher than the control and salt group values at 8 weeks. Atrial concentration of i alpha-rANP was inversely correlated with systolic blood pressure in the all rats at 1 week (r = 0.48, p less than 0.001) and at 8 weeks (r = 0.33, p less than 0.05). Plasma concentration of i alpha-rANP was positively correlated with systolic blood pressure at 8 weeks (r = 0.37, p less than 0.05). In addition, there was a significant positive correlation between plasma/atrial ratio of i alpha-rANP concentration and systolic blood pressure at either stage (r = 0.41, p less than 0.01 at 1 week; r = 0.40, p less than 0.01 at 8 weeks). Thus, it seems likely that the release of ANPs is increased in response to expansion of extracellular fluid volume or elevation of blood pressure, modifying the development of hypertension in DOCA-salt rats.

A unique proenkephalin-converting enzyme purified from bovine adrenal chromaffin granules

Abstract A unique proenkephalin converting enzyme specifically generating enkephalin was partially purified from lysates of adrenal chromaffin granules. The enzyme, whose molecular weight is estimated as ca. 220,000, is thiol-dependent protease, with optimal pH at around 5.5. The enzyme converts proenkephalin to enkephalins by cleaving specifically at the sites of consecutive basic amino acid residues. The enzyme also converts BAM-12P, an adrenal “big” Met-enkephalin, to Met-enkephalin in a similar manner. During the enzyme reaction, formation of [Arg 6 ]-Met-enkephalin was not observed. Additionally, [Arg 6 ]-enkephalins were not converted to enkephalins by the enzyme. Consequently, the enzyme was proved to be a unique converting enzyme distinct from either trypsin-like or carboxypeptidase B-like proteases.

Molecular forms of atrial natriuretic polypeptides circulating in human plasma

Molecular forms of atrial natriuretic polypeptides circulating in human plasma were analyzed by chromatographic separation, coupled with radioimmunoassay. Analyses were done with human plasma samples taken from 24 human subjects, divided into groups of healthy volunteers, patients with renal disease, and patients with heart disease, with 8 subjects in each group. Although alpha-hANP was found as a major circulating form in most of the plasma specimens (16 cases), beta-hANP accompanied by alpha-hANP was found in 6 cases including 2 healthy volunteers. In addition, hANP-immunoreactive macromolecule, which is likely a bound form of hANP, was found to some extent in all the specimens tested. Diversity of human plasma in ANP molecular distribution was discussed in connection with a radioimmunoassay for plasma ANP.

N-arachidonoyl glycine induces macrophage apoptosis via GPR18

N-arachidonoyl glycine (NAGly), a member of lipoamino acids, was reported to exhibit anti-inflammatory effects in experimental ear edema or peritonitis. However the underlying mechanisms have not been clarified so far. In this study, we attempt to investigate the effects of NAGly on macrophages, including the relevant signaling pathways. NAGly potently induced apoptosis in mouse macrophage-derived cell line, RAW264.7. Pretreatment with inhibitors for MEK and p38 MAPK prevented the apoptosis induced by NAGly, although NAGly activated ERK1/2, p38 MAPK and JNK. Further, we focused on implication of GPR18, one of the orphan G protein-coupled receptors, because NAGly has been reported as a candidate ligand for GPR18. Pretreatment with pertussis toxin or siRNA to knock down the expression of GPR18 significantly attenuated the apoptosis induced by NAGly. In mouse peritoneal macrophages, the expression of GPR18 mRNA was elevated in proinflammatory stimulated macrophages but not in anti-inflammatory stimulated macrophages; consistently, NAGly remarkably reduced cell viability of the former, as compared to the latter. These results suggest that NAGly might be involved in function of macrophages through GPR18.