The open reading frames (ORFs) TK0240, TK0474, and TK0882, annotated as agmatine ureohydrolase genes, were disrupted. Only the TK0882 gene disruptant showed a growth defect at 85°C and 93°C, and the growth was partially retrieved by the addition of spermidine. In the TK0882 gene disruptant, agmatine and N 1 -aminopropylagmatine accumulated in the cytoplasm. Recombinant TK0882 was purified to homogeneity, and its ureohydrolase characteristics were examined. It possessed a 43-fold-higher k cat /K m value for N 1 -aminopropylagmatine than for agmatine, suggesting that TK0882 functions mainly as N 1 -aminopropylagmatine ureohydrolase to produce spermidine. TK0147, annotated as spermidine/spermine synthase, was also studied. The TK0147 gene disruptant showed a remarkable growth defect at 85°C and 93°C. Moreover, large amounts of agmatine but smaller amounts of putrescine accumulated in the disruptant. Purified recombinant TK0147 possessed a 78-fold-higher k cat /K m value for agmatine than for putrescine, suggesting that TK0147 functions primarily as an aminopropyl transferase to produce N 1 -aminopropylagmatine. In T. kodakarensis, spermidine is produced mainly from agmatine via N 1 -aminopropylagmatine. Furthermore, spermine and N 4 -aminopropylspermine were detected in the TK0147 disruptant, indicating that TK0147 does not function to produce spermine and long-chain polyamines.
Significance Excitation–transcription (E-T) coupling can initiate and modulate essential physiological or pathological responses in cells, such as neurons and cardiac myocytes. Although vascular myocytes also exhibit E-T coupling in response to membrane depolarization, the underlying molecular mechanisms are unknown. Our study reveals that E-T coupling in vascular myocytes converts intracellular Ca 2+ signals into selective gene transcription related to chemotaxis, leukocyte adhesion, and inflammation. Our discovery identifies a mechanism for vascular remodeling as an adaptation to increased circumferential stretch.
An increase in intracellular Ca 2 concentration ([Ca 2 ] i ) activates Ca 2 -sensitive enzymes such as Ca 2 / calmodulin-dependent kinases (CaMK) and induces gene transcription in various types of cells. This signaling pathway is called excitation-transcription (E-T) coupling. Recently, we have revealed that a L-type Ca 2 channel/CaMK kinase (CaMKK) 2/CaMK1α complex located within caveolae in vascular smooth muscle cells (SMCs) can convert [Ca 2 ] i changes to gene transcription profiles that are related to chemotaxis. Although CaMK1α is expected to be the key molecular identity that can transport Ca 2 signals originated within caveolae to the nucleus, data sets directly proving this scheme are lacking. In this study, multicolor fluorescence imaging methods were utilized to address this question. Live cell imaging using mouse primary aortic SMCs revealed that CaMK1α can translocate from the cytosol to the nucleus; and that this movement was blocked by nifedipine or a CaMKK inhibitor, STO609. Experiments using two types of Ca 2 chelators, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), combined with caveolin-1 knockout (cav1-KO) mice showed that local Ca 2 events within caveolae are required to trigger this CaMK1α nuclear translocation. Importantly, overexpression of cav1 in isolated cav1-KO myocytes recovered the CaMK1α translocation. In SMCs freshly isolated from mesenteric arteries, CaMK1α was localized mainly within caveolae in the resting state. Membrane depolarization induced both nuclear translocation and phosphorylation of CaMK1α. These responses were inhibited by nifedipine, STO609, cav1-KO, or BAPTA. These new findings strongly suggest that CaMK1α can transduce Ca 2 signaling generated within or very near caveolae to the nucleus and thus, promote E-T coupling.
Ca 2+ signaling induces gene transcription and mediates cellular functions in diverse types of cells. In neurons, Ca 2+ influx through voltage-dependent Ca 2+ channels (VDCC) activates Ca 2+ /calmodulin-dependent protein kinases (CaMK), promotes gene transcription and constructs neural networks. This pathway is called excitation-transcription (E-T) coupling. E-T coupling is also reported in vascular smooth muscle cells (VSMC), but its molecular mechanisms and pathophysiological roles are unknown. In VSMCs, caveolin (cav)-1, an essential component of caveolae, forms Ca 2+ microdomains where VDCC and its effectors are accumulated and regulates vascular tone. In the present study, we hypothesized that prolonged VDCC activation in caveolae triggers E-T coupling in VSMC and causes vascular remodeling. When the mesenteric artery was depolarized for longer than 30 min, nuclear CREB phosphorylation and pro-inflammatory gene transcription were promoted. These responses were significantly reduced in cav-1 KO mice. Pharmacological inhibition or siRNA knockdown of CaMKK-CaMK pathway revealed the molecular pathway connecting Ca 2+ influx through VDCC in caveolae and CREB phosphorylation. These results suggest that excessive Ca 2+ signaling in caveolae causes E-T coupling in VSMC and triggers vascular remodeling by upregulating proinflammatory genes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.