Kiichiro Nakajima

Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells

The effects of synthetic rat adrenomedullin (rAM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma, on receptor binding and cAMP generation were studied in cultured rat vascular smooth muscle cells (VSMC). A binding study using [125I]rAM revealed the presence of a single class of high‐affinity (K d1.3 × 10−8 M) binding sites for rAM in VSMC. The apparent K i of rat calcitonin gene‐related peptide (rCGRP) was 3 × 10−7 M. Affinity labeling of VSMC membranes with [125I]rAM revealed two distinct labeled bands with apparent molecular weights of 120 and 70 kDa, both of which were abolished by excess unlabeled rAM or rCGRP. rAM stimulated cAMP formation with an approximate EC50 of 10−8 M, the effect of which was additive with isoproterenol, but not with rCGRP. The rAM‐induced cAMP response was unaffected by propranalol, indomethacin, or quinaerine, but inhibited by a CGRP receptor antagonist, human CGRP[8–37]. These data suggest that VSMC possesses specific AM receptors functionally coupled to adenylate cyclase with which CGRP interacts.

Improvement in the oxidative folding of endothelin-1 by a lys-Arg extension at the amino terminus: Implication of a salt bridge between Arg−1 and Asp8

An amino-terminal extension of endothelin-l by the lys-Arg dipeptide in the prosequence (KR-ET-1) greatly increased the ratio of native-type to non-native-type disulfide isomer (96/4 versus 71/29) during the oxidative folding reaction. This improvement was completely abolished by substituting Asn for Asp at position 8 (D8N-KR-ET-1), whereas most of it was maintained with similar carboxamide analogues replaced at Glu10 or Asp18. Structure analyses by circular dichroism spectroscopy revealed that (i) in the carboxylate state, the α-helical content of the native-type isomer of KR-ET-l is higher than that of the native-type isomer of ET-1, while such a variation is not observed in the corresponding non-native-type isomer of KR-ET-l; and (ii) the enhanced α-helicity resulting from the Lys-Arg extension is largely diminished in D8N-KR-ET-l. From these results and our previous findings that the helical structure in KR-ET-l is stabilized by a particular salt bridge between the extended Arg−1 basic moiety and either the Asp8 or Glu10 acidic side chain in Et-1 [Aumelas, A. et al., Biochemistry, 34 (1995) 4546], we conclude that the formation of a specific salt bridge between the side chains of Arg−1 and Asp8 in KR-ET-1 is critical for the predominant generation of the native-type disulfide isomer, probably because it stabilizes the helical structure of parental ET-1.

Switching of turn conformation in an aspartate anion peptide fragment by NH . . . O- hydrogen bonds.

Aspartic acid protease model peptides Z–Phe–Asp(COOH)–Thr–Gly–Ser–Ala–NHCy (1) and AdCO–Asp(COOH)–Val–Gly–NHBzl (3), and their aspartate anions (NEt4)[Z–Phe–Asp(COO−)–Thr–Gly–Ser–Ala–NHCy] (2) and (NEt4)[AdCO–Asp(COO−)–Val–Gly–NHBzl] (4), having an invariant primary sequence of the Asp–X(Thr,Ser)—Gly fragment, were synthesized and characterized by 1H‐NMR, CD, and infrared (IR) spectroscopies. NMR structure analyses indicate that the Asp Oδ atoms of the aspartate peptide 2 are intramolecularly hydrogen‐bonded with Gly, Ser, Ala NH, and Ser OH, supporting the rigid β‐turn‐like conformation in acetonitrile solution. The tripeptide in the aspartic acid 3 forms an inverse γ‐turn structure, which is converted to a β‐turn‐like conformation because of the formation of the intramolecular NH · · · O− hydrogen bonds with the Asp Oδ in 4. Such a conformational change is not detected between dipeptides AdCO–Asp(COOH)–Va–NHAd (5) and (NEt4)[AdCO–Asp(COO−)–Val–NHAd] (6). The pKa value of side‐chain carboxylic acid (5.0) for 3 exhibits a lower shift (0.3 unit) from that of 5 in aqueous polyethyleneglycol lauryl ether micellar solution. NMR structure analyses for 3 in an aqueous micellar solution indicate that the preorganized turn structure, which readily forms the NH · · · O− hydrogen bonds, lowers the pKa value and that resulting hydrogen bonds stabilize the rigid conformation in the aspartate anion state. We found that the formation of the NH · · · O− hydrogen bonds involved in the hairpin turn is correlated with the protonation and deprotonation state of the Asp side chain in the conserved amino acid fragments.

Binding of the carboxyl-terminal fragments of human parathyroid hormone to rat osteoblastic cells ROS 17/2.8

We have recently demonstrated that the carboxyl-terminal (C-terminal) PTH fragments increase or decrease alkaline phosphatase activity in dexamethasone-treated ROS 17/2.8 cells, depending on the length of deletion of amino-terminal amino acids of the PTH molecule, and interact with amino-terminal (N-terminal) PTH fragment [Acta Endocrinol 128:367]. In the present study, we examined individual and combined inhibitory effects of N-terminal and a series of truncated C-terminal PTH fragments [PTH (1-34), (35-84), (53-84) and (71-84)] on the binding of intact PTH molecule [PTH (1-84)] to ROS 17/2.8 cells.The C-terminal PTH fragments, as well as the N-terminal PTH fragment, partially inhibited the binding of [35S]-labeled PTH (1-84) to the cells. The inhibitory effect of C-terminal PTH fragments was reduced along with the deletion of aminoterminal amino acids of the PTH molecule, but still retained in the shortest C-terminal PTH fragment, PTH (71-84). When added together, PTH (1-34) reinforced the inhibitory effect of each C-terminal PTH fragment. The combination of PTH (1-34) and the complementary C-terminal PTH fragment, PTH (35-84), resulted in inhibition of [35S] PTH (1-84) binding to the level obtained by addition of the same concentration of unlabeled PTH (1-84).These findings suggested that the region relatively close to the C-terminal end of the PTH molecule might be essential for the binding of C-terminal PTH fragment could be responsible for the modification of the binding affinity of the peptide to the receptor and the action of the peptide.

The [53–63] amino acid portion of the parathyroid hormone molecule is essential for the stimulatory effect of carboxyl-terminal PTH fragments on alkaline phosphatase activity in dexamethasone-treated rat osteoblastic osteosarcoma cells, ROS 17/2.8

The effect of a series of truncated carboxyl-terminal parathyroid hormone (PTH) fragments on alkaline phosphatase (ALP) activity was further examined in dexamethasone-treated rat osteoblastic osteosarcoma cells, ROS 17/2.8. As we previously reported, dexamethasone-induced ALP activity was inhibited not only by hPTH(1-84) and aminoterminal PTH fragment hPTH(1-34), but also by carboxyl-terminal PTH fragment hPTH(69-84). The longer carboxyl-terminal PTH fragment hPTH(53-84) stimulated ALP activity, and the shorter carboxyl-terminal PTH fragment hPTH(71-84) did not affect ALP activity.The longest newly synthesized carboxyl-terminal PTH fragment hPTH(35-84), which is complementary to amino-terminal PTH fragment hPTH(1-34), stimulated ALP activity as potently as hPTH(53-84), but not more potently than hPTH(53-84). Another newly synthesized carboxyl-terminal PTH fragment hPTH(64-84), which has an intermediate peptide length between hPTH (53-84) and hPTH(69-84), inhibited ALP activity as potently as hPTH(69-84).These results suggest that the 35-52 amino acid portion of the PTH molecule might not be crucial for the stimulatory effect of carboxyl-terminal PTH fragments on ALP activity, and that the 53–63 portion, but not the 64–68 portion, of the PTH molecule might be essential for the stimulatory effect of carboxyl-terminal PTH fragments on ALP activity. Furthermore, the importance of the 69th and 70th amino acid of the PTH molecule for the inhibitory effect of carboxyl-terminal PTH fragments on ALP activity was confirmed.