Noboru Takami

Nitric oxide-cGMP-protein kinase G pathway negatively regulates vascular transient receptor potential channel TRPC6.

We investigated the inhibitory role of the nitric oxide (NO)–cGMP–protein kinase G (PKG) pathway on receptor‐activated TRPC6 channels in both a heterologous expression system (HEK293 cells) and A7r5 vascular myocytes. Cationic currents due to TRPC6 expression were strongly suppressed (by ∼70%) by a NO donor SNAP (100 μm) whether it was applied prior to muscarinic receptor stimulation with carbachol (CCh; 100 μm) or after G‐protein activation with intracellular perfusion of GTPγS (100 μm). A similar extent of suppression was also observed with a membrane‐permeable analogue of cGMP, 8Br‐cGMP (100 μm). The inhibitory effects of SNAP and 8Br‐cGMP on TRPC6 channel currents were strongly attenuated by the presence of inhibitors for guanylyl cyclase and PKG such as ODQ, KT5823 and DT3. Alanine substitution for the PKG phosphorylation candidate site at T69 but not at other sites (T14A, S28A, T193A, S321A) of TRPC6 similarly attenuated the inhibitory effects of SNAP and 8Br‐cGMP. SNAP also significantly reduced single TRPC6 channel activity recorded in the inside‐out configuration in a PKG‐dependent manner. SNAP‐induced PKG activation stimulated the incorporation of 32P into wild‐type and S321A‐mutant TRPC6 proteins immunoprecipitated by TRPC6‐specific antibody, but this was greatly attenuated in the T69A mutant. SNAP or 8Br‐cGMP strongly suppressed TRPC6‐like cation currents and membrane depolarization evoked by Arg8‐vasopressin in A7r5 myocytes. These results strongly suggest that TRPC6 channels can be negatively regulated by the NO–cGMP–PKG pathway, probably via T69 phosphorylation of the N‐terminal. This mechanism may be physiologically important in vascular tissues where NO is constantly released from vascular endothelial cells or nitrergic nerves.

Brefeldin A arrests the intracellular transport of a precursor of complement C3 before its conversion site in rat hepatocytes

The effects of brefeldin A on intracellular transport and posttranslational modification of complement C3 (C3) were studied in primary culture of rat hepatocytes. In the control culture C3 was synthesized as a pre‐cursor (pro‐C3), which was processed to the mature form with α‐ and β‐subunits before its discharge into the medium. In the presence of brefeldin A the secretion of C3 was strongly blocked, resulting in accumulation of pro‐C3. However, after a prolonged interval the mature form of C3 was finally secreted. The results indicate that brefeldin A impedes translocation of pro‐C3 to the Golgi complex where pro‐C3 is converted to the mature form, but not its proteolytic processing, in contrast to the effects of monensin and weakly basic amines.

Putative mechanisms of action of probucol on high-density lipoprotein apolipoprotein A-I and its isoproteins kinetics in rabbits

Probucol is a widely prescribed lipid-lowering agent, the major effects of which are to lower cholesterol in both low- and high-density lipoproteins (LDL and HDL, respectively). The mechanism of action of probucol on HDL apolipoprotein (apo) A-I kinetics was investigated in rabbits, with or without cholesterol feeding. 125I-labeled HDL was injected intravenously, and blood samples were taken periodically for 6 days. Kinetic parameters were calculated from the apo A-I-specific radioactivity decay curves. Fractional catabolic rate (FCR) and synthetic rate (SR) of apo A-I in rabbits fed a normal chow and normal chow with 1% probucol were similar. Apo A-I FCR of the rabbits fed 0.5% cholesterol was significantly increased but there were no changes in SR, compared to findings in the normal chow-fed group. Apo A-I FCR of the rabbits fed 1% probucol with 0.5% cholesterol (both 1 month and 2 months) was significantly increased compared to findings in rabbits fed the normal chow as well as 0.5% cholesterol diet group, while SR of apo A-I was significantly reduced in the former groups. Kinetics at 1 month after discontinuation of 1% probucol (under cholesterol feeding) showed a similar FCR of HDL-apo A-I to that of the rabbits fed 0.5% cholesterol, but the SR of apo A-I remained lower. Apo A-I isoproteins kinetics assessed by autoradiography of isoelectric focusing slab gels showed that the synthesis of proapo A-I was significantly reduced in the 1% probucol with 0.5% cholesterol administered, compared to the 0.5% cholesterol group. Thus, the action of probucol on HDL apo A-I kinetics was only prominent in case of higher serum cholesterol levels. The decreased HDL or apo A-I seen with probucol was apparently the result of an increase in FCR and a decrease in SR of HDL-apo A-I. A decreased synthesis of apo A-I remained evident even 1 month after discontinuing probucol. The action of probucol on the intracellular synthetic processes of apo A-I was revealed by the reduced synthesis of proapo A-I.

Selective cell-surface expression of dipeptidyl peptidase IV with mutations at the active site sequence

The cell surface expression of dipeptidyl peptidase IV (DPPIV) was examined in COS-1 cells transfected with its cDNA with or without mutations at the active site sequence Gly-Trp-Ser-Tyr-Gly (positions 629-633). Mutants with substitution of Trp630----Glu or Ser631----Ala were expressed on the cell surface as normally as the wild-type DPPIV, although the mutant with Ala631 had no enzyme activity. In contrast, any single substitutions of Gly at positions 629 and 633 resulted in no surface expression of the mutants, which were, instead, detected within the cells. When Tyr632 was substituted, one mutant (Tyr----Phe) was expressed on the surface, whereas the others (Tyr----Gly or Leu) were intracellularly retained. These results indicate that the surface expression of DPPIV is critically influenced by mutations at the active site sequence.

Sequence requirements for proteolytic cleavage of precursors with paired basic amino acids

When expressed in COS cells, human prorenin was secreted into the medium without being processed to an active renin. Co-expression of furin, a mammalian homologue of the yeast KEX2 gene product, did not affect proteolytic processing of prorenin. A mutant proreninR-4 constructed by site-directed mutagenesis of Pro (-4) to Arg was not cleaved by an endoprotease in the COS cell. However, proreninR-4 was detectably cleaved to yield the active renin upon co-transfection with furin DNA, indicating that Arg at position -4 is important for recognition and processing by furin in addition to the absolute requirement for paired basic amino acids. Another mutant precursor in which Leu (+1) of proreninR-4 was replaced with Ser was found to be much more efficiently processed than proreninR-4, regardless of co-expression of furin. The results suggest that not only a basic amino acid at position -4 but also Leu at position +1 significantly affect the processing of prorenin catalyzed by the COS cell endoprotease or furin.

Intracellular processing of complement pro-C3 and proalbumin is inhibited by rat α1-protease inhibitor variant (Met352→Arg) in transfected cells

Complement C3, when its cDNA was transfected into COS-1 cells, was synthesized as a precursor, pro-C3, which was intracellularly processed into the alpha and beta subunits, although not completely. A cDNA for rat alpha 1-protease inhibitor (alpha 1-PI) was mutated in vitro to encode its variant with the modified active site (Met352----Arg). In cells co-transfected with the mutant alpha 1-PI cDNA and the C3 cDNA, pro-C3 expressed was secreted without being processed into the subunits. Co-transfection of the mutant alpha 1-PI cDNA and the albumin cDNA also resulted in the inhibition of intracellular conversion of proalbumin into serum-type albumin. No inhibition of the processing of each preform was observed in cells co-transfected with the normal alpha 1-PI cDNA. Taken together, the results indicate that the alpha 1-PI variant (Met352----Arg) expressed inhibits specifically an intracellular enzyme which is involved in the proteolytic processing of both pro-C3 and proalbumin.

Selective processing of proalbumin determined by site-specific mutagenesis

Rat proalbumin is cleaved at the dibasic pair Arg-Arg and converted into a mature form with Glu at the NH2 terminus. In the present study site-directed mutagenesis of the albumin cDNA was designed to generate proalbumin variants in which Glu1 was substituted with various amino acid residues. The expression plasmids constructed were transfected into COS-1 cells, and the intracellular processing of proalbumins expressed was examined by labeling experiments. Substitution of Glu1----Ser allowed the expressed proalbumin to be processed as observed for the wild-type precursor. However, replacement of Glu1 with a hydrophobic residue (Val, Leu or Ile) resulted in no processing of proalbumin, despite retaining the same cleavage signal Arg-Arg as above. The results indicate that the residue at position 1 adjacent to the dibasic pair is also important for recognition by the proalbumin-processing enzyme.

Biosynthesis and processing of pro-C3, a precursor of the third component of complement in rat hepatocytes: effect of secretion-blocking agents

Biosynthesis and intracellular processing of the third component (C3) of complement were studied in cultured rat hepatocytes. In the control cells, the complement C3 was synthesized as a pro‐form, a single polypeptide chain comprising both the α‐ and β‐subunits. Although the cleavage of the pro‐form into the subunits was not clearly demonstrable within the cells during pulse‐chase periods, all the secreted C3 was the mature processed form. The cells were treated with secretion‐blocking agents with different modes of action, colchicine and monensin. Colchicine caused an accumulation of the processed C3 within the cells, whereas monensin blocked the secretion without a significant accumulation of the processed form. The results indicate that the conversion of the C3 pro‐form into the subunits takes place in the secretory vesicles just before the secretion.

Proalbumin is processed to serum albumin in COS-1 cells transfected with cDNA for rat albumin

Synthesis and processing of rat albumin were investigated in COS-1 cells transiently expressing rat albumin. Analysis using isoelectric focusing revealed that serum-type albumin, which is indistinguishable from the counterpart isolated from rat hepatocyte cuture medium, was secreted from the transfected COS-1 cells, indicating that proalbumin is effectively converted into serum albumin in the COS-1 cells, if not completely. Furthermore methylamine was found to cause the diminution of serum albumin released from the cells, substantiating that the proteolytical conversion of proalbumin occurs in the Golgi complex before discharge from the COS-1 cells.

Macrophage Chemotactic Response to Elastin-Derived VGVAPG and VGVPG Permutations: A Structure-Activity Relationship and Receptor Binding Assay

Elastin, an insoluble component of the extracellular matrix, is considered as the core protein and insoluble part of the elastic fiber, which confers elasticity to extensible tissues such as the arterial walls, lungs, skin and elastic fibers. Elastin has unique repeating sequences in the hydrophobic region, including the pentapeptide VPGVG and the hexapeptide VGVAPG. Elastin in itself is considered biologically inactive, but when degraded renders elastin fragments or elastin peptides which interact with a variety of cell types to modulate cellular behavior. VGVAPG, for example has been found to induce Chemotaxis in monocytes [1], fibroblasts [2] and cancer cells [3]. It is known that binding of extracellular matrix components to the cell membrane is mediated by specific receptor proteins that promote cellular attachment and display biological activity, such as Chemotaxis. Our research was conducted according to this working hypothesis as prompted by our previous observation that the polyhexapeptide (VGVAPG)n was able to induce chemotactic response to macrophages, while the polypentapeptide (VPGVG)n failed to do so. This study was conducted with the aim of evaluating the potency of the hexapeptide VGVAPG and pentapeptide VGVPG permutations in inducing Chemotaxis, identifying the receptor involved in the chemotactic activity, investigating whether VGVAPG could act as a ligand for the macrophage receptor, and determining the structure-activity relationship involved in this biological activity.