1) Raeymaekers L, Eggermont JA, Wuytack F, Casteels R. Effects of cyclic nucleotide dependent
protein kinases on the endoplasmic reticulum Ca2+ pump of bovine pulmonary artery. Cell Calcium
1990; 11(4): 261-268.
2) Mundiña-Weilenmann C, Vittone L, Rinaldi G, Said M, de Cingolani GC, Mattiazzi A.
Endoplasmic reticulum contribution to the relaxant effect of cGMP- and cAMP-elevating agents in
feline aorta. Am J Physiol Heart Circ Physiol 2000; 278(6): H1856-65
This article is protected by copyright. All rights reserved.
Accepted Article
3) Robertson BE, Schubert R, Hescheler J, Nelson MT. cGMP-dependent protein kinase activates
Ca-activated K channels in cerebral artery smooth muscle cells. Am J Physiol 1993; 265(1 Pt 1):
C299-303.
4) Xin W, Li N, Cheng Q, Petkov GV. BK channel-mediated relaxation of urinary bladder smooth
muscle: a novel paradigm for phosphodiesterase type 4 regulation of bladder function. J Pharmacol
Exp Ther 2014; 349(1): 56-65.
5) Conti MA, Adelstein RS. The relationship between calmodulin binding and phosphorylation of
smooth muscle myosin kinase by the catalytic subunit of 3’:5’ cAMP-dependent protein kinase. J Biol
Chem 1981; 256(7): 3178–3181.
6) Rembold CM. Regulation of contraction and relaxation in arterial smooth muscle. Hypertension
1992; 20(2): 129–137.
7) Himpens B, Matthijs G, Somlyo AV, Butler TM, Somlyo AP. Cytoplasmic free calcium, myosin
light chain phosphorylation, and force in phasic and tonic smooth muscle. J Gen Physiol 1988; 92(6):
713-729.
8) Kitazawa T, Masuo M, Somlyo AP. G protein-mediated inhibition of myosin light-chain
phosphatase in vascular smooth muscle. Proc Natl Acad Sci U S A 1991; 88(20): 9307-9310.
9) Frazier EP, Mathy MJ, Peters SL, Michel MC. Does cyclic AMP mediate rat urinary bladder
relaxation by isoproterenol? J Pharmacol Exp Ther 2005; 313(1): 260-267.
10) Longhurst PA, Briscoe JA, Rosenberg DJ, Leggett RE. The role of cyclic nucleotides in
guinea-pig bladder contractility. Br J Pharmacol 1997; 121(8): 1665-1672.
This article is protected by copyright. All rights reserved.
Accepted Article
11) Morita T, Tsujii T, Dokita S. Regional difference in functional roles of cAMP and cGMP in lower
urinary tract smooth muscle contractility. Urol Int 1992; 49(4): 191-195.
12) Morita T, Ando M, Kihara K, Oshima H. Species differences in cAMP production and contractile
response induced by beta-adrenoceptor subtypes in urinary bladder smooth muscle. Neurourol Urodyn
1993; 12(2): 185-190
13) Michel MC, Sand C. Effect of pre-contraction on β-adrenoceptor-mediated relaxation of rat
urinary bladder. World J Urol 2009; 27(6): 711-715
14) Uchida H, Shishido K, Nomiya M, Yamaguchi O. Involvement of cyclic AMP-dependent and
-independent mechanisms in the relaxation of rat detrusor muscle via beta-adrenoceptors. Eur J
Pharmacol 2005; 518(2-3): 195-202.
15) Witte LP, de Haas N, Mammen M, et al. Muscarinic receptor subtypes and signalling involved in
the attenuation of isoprenaline-induced rat urinary bladder relaxation. Naunyn Schmiedebergs Arch
Pharmacol 2011; 384(6):555-563.
16) Schneider T, Fetscher C, Michel MC. Human Urinary Bladder Strip Relaxation by the
β-Adrenoceptor Agonist Isoprenaline: Methodological Considerations and Effects of Gender and Age.
Front Pharmacol 2011; 25(2): 11.
17) Truss MC, Uckert S, Stief CG, Kuczyk M, Jonas U. Cyclic nucleotide phosphodiesterase (PDE)
isoenzymes in the human detrusor smooth muscle. II. Identification and characterization. Urol Res
1996; 24(3): 123-128.
This article is protected by copyright. All rights reserved.
Accepted Article
18) Oger S, Behr-Roussel D, Gorny D, et al. Relaxation of phasic contractile activity of human
detrusor strips by cyclic nucleotide phosphodiesterase type 4 inhibition. Eur Urol 2007; 51(3):
772-780.
19) Kaiho Y, Nishiguchi J, Kwon DD, et al. The effects of a type 4 phosphodiesterase inhibitor and
the muscarinic cholinergic antagonist tolterodine tartrate on detrusor overactivity in female rats with
bladder outlet obstruction. BJU Int 2008; 101(5): 615-620.
20) Oger S, Behr-Roussel D, Gorny D, et al. Signalling pathways involved in sildenafil-induced
relaxation of human bladder dome smooth muscle. Br J Pharmacol 2010; 160(5): 1135-1143
21) Shahab N, Kajioka S, Seki N, Naito S. Functional role of muscarinic receptor subtypes in calcium
sensitization and their contribution to rho-kinase and protein kinase C pathways in contraction of
human detrusor smooth muscle. Urology 2012; 79(5): 1184.e7-13.
22) Zieba BJ, Artamonov MV, Jin L, et al. The cAMP-responsive Rap1 guanine nucleotide exchange
factor, Epac, induces smooth muscle relaxation by down-regulation of RhoA activity. J Biol Chem
2011; 286(19): 16681-16692.
23) Rajagopal S, Kumar DP, Mahavadi S, et al. Activation of G protein-coupled bile acid receptor,
TGR5, induces smooth muscle relaxation via both Epac- and PKA-mediated inhibition of RhoA/Rho
kinase pathway. Am J Physiol Gastrointest Liver Physiol 2013; 304(5): G527-535.
24) Roscioni SS, Maarsingh H, Elzinga CR, et al. Epac as a novel effector of airway smooth muscle
relaxation. J Cell Mol Med 2011; 15(7): 1551-1563.
This article is protected by copyright. All rights reserved.
Accepted Article
25) Nishimura J, Kolber M, van Breemen C. Norepinephrine and GTP-gamma-S increase
myofilament Ca2+ sensitivity in alpha-toxin permeabilized arterial smooth muscle. Biochem Biophys
Res Commun 1988; 157(2): 677-683.
26) Takahashi R, Nishimura J, Hirano K, Seki N, Naito S, Kanaide H. Ca2+ sensitization in
contraction of human bladder smooth muscle. J Urol 2004; 172(2): 748-752.
27) Shahab N, Kajioka S, Takahashi R, et al. Novel effect of 2-aminoethoxydiphenylborate through
inhibition of calcium sensitization induced by Rho kinase activation in human detrusor smooth
muscle. Eur J Pharmacol 2013; 708(1-3): 14-20.
28) Kitazawa T, Kobayashi S, Horiuti K, Somlyo AV, Somlyo AP. Receptor-coupled, permeabilized
smooth muscle. Role of the phosphatidylinositol cascade, G-proteins, and modulation of the
contractile response to Ca2+. J Biol Chem 1989; 264(10): 5339-5342.
29) Shahab N, Kajioka S, Takahashi-Yanaga F, et al. Obstruction enhances rho-kinase pathway and
diminishes protein kinase C pathway in carbachol-induced calcium sensitization in contraction of
α-toxin permeabilized guinea pig detrusor smooth muscle. Neurourol Urodyn 2012; 31(4): 593-599.
30) Uehata M, Ishizaki T, Satoh H, et al. Calcium sensitization of smooth muscle mediated by a
Rho-associated protein kinase in hypertension. Nature 1997; 389(6654):
990-994.
31) Toullec D1, Pianetti P, Coste H, et al. The bisindolylmaleimide GF 109203X is a potent and
selective inhibitor of protein kinase C. J Biol Chem 1991; 266(24): 15771-15781.
32) Christensen AE, Selheim F, de Rooii J, et al. AMP Analog Mapping of Epac1 and cAMP Kinase.
Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12
cell neurite extension. J Biol Chem 2003; 278(37): 35394-35402.
This article is protected by copyright. All rights reserved.
Accepted Article
33) Rehmann H, Schwede F, Doskeland SO, et al. Ligand-mediated Activation of the
cAMP-responsive Guanine Nucleotide Exchange Factor Epac. J Biol Chem 2003; 278(40):
38548-38556.
34) Morgado M, Cairrão E, Santos-Silva AJ, Verde I. Cyclic nucleotide-dependent relaxation
pathways in vascular smooth muscle. Cell Mol Life Sci 2012; 69(2): 247-266.
35) Somlyo AP, Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 1994;
372(6503): 231-236.
36) Azam MA, Yoshioka K, Ohkura S, et al. Ca2+-independent, inhibitory effects of cyclic adenosine
5'-monophosphate on Ca2+ regulation of phosphoinositide 3-kinase C2alpha, Rho, and myosin
phosphatase in vascular smooth muscle. J Pharmacol Exp Ther 2007; 320(2): 907-916.
37) Arrighi N, Bodei S, Zani D, et al. Different muscarinic receptor subtypes modulate proliferation
of primary human detrusor smooth muscle cells via Akt/PI3K and map kinases. Pharmacol Res 2013;
74: 1-6.
38) Asano M, Suzuki Y, Hidaka H. Effects of various calmodulin antagonists on contraction of rabbit
aortic strips. J Pharmacol Exp Ther 1982; 220(1): 191-196.
39) Grassie ME, Sutherland C, Ulke-Lemée A, et al. Cross-talk between Rho-associated kinase and
cyclic nucleotide-dependent kinase signaling pathways in the regulation of smooth muscle myosin
light chain phosphatase. J Biol Chem 2012; 287(43): 36356-36369.
40) Ehlert FJ1, Ahn S, Pak KJ, et al. Neuronally released acetylcholine acts on the M2 muscarinic
receptor to oppose the relaxant effect of isoproterenol on cholinergic contractions in mouse urinary
bladder. Pharmacol Exp Ther 2007; 322(2): 631-637.
This article is protected by copyright. All rights reserved.
Accepted Article
41) Yamaguchi O, Marui E, Kakizaki H, et al. Phase III, randomised, double-blind,
placebo-controlled study of the β3-adrenoceptor agonist mirabegron, 50 mg once daily, in Japanese
patients with overactive bladder. BJU Int 2014; 113(6): 951-960.
42) de Rooij J, Zwartkruis FJ, Verheijen MH, et al. Epac is a Rap1 guanine-nucleotide-exchange
factor directly activated by cyclic AMP. Nature 1998; 396(6710): 474-477.
43) Kawasaki H, Springett GM, Mochizuki N, et al. A family of cAMP-binding proteins that directly
activate Rap1. Science 1998; 282(5397): 2275-2279.
44) Holz GG, Kang G, Harbeck M, Roe MW, Chepurny OG. Cell physiology of cAMP sensor Epac.
J Physiol 2006; 577(Pt 1): 5-15.
ACKNOWLEDGEMENTS
This work was supported by a Grant-Aid for Special Purposes (23592371 and 26462445) from the
Japan Society for the Promotion of Science. We thank Prof. Alexaj Verkhratsky, University of
Manchester, for proofreading the manuscript and useful discussion. Noriko Hakota and Eriko
Gunshima assisted with experiments. Dr. Hirofumi Koga, Dr. Hiroyuki Nomura and Mrs. Ikumi
Takeuchi cooperated to obtain tissue from patients at Harasanshin Hospital. The English in this
document has been checked by at least two professional editors, both native speakers of English. For a
certificate, please see: http://www.textcheck.com/certificate/X06oxy
This article is protected by copyright. All rights reserved.
Accepted Article
FIGURE LEGENDS
Figure 1
Effects of cAMP on contraction induced by cumulative [Ca2+]i addition in α-toxin permeabilized
human detrusor smooth muscle.
Representative traces obtained from α-toxin permeabilized human detrusor smooth muscle following
the cumulative addition of Ca2+ in the absence (A) and presence (B) of 100 μM cAMP are shown. The
mean concentration-response curves for tension force to [Ca2+]i without (open circle) and with (filled
circle) 100 μM cAMP are shown in C. The tension force is expressed in a relative manner; the tension
force obtained at 3 μM [Ca2+]i is normalised as 100%. Error bars indicate means ± SD. Asterisks
indicate a significant difference (P < 0.05; N = 3, n = 6).
Figure 2
Effects of cyclic adenosine monophosphate (cAMP), forskolin (FSK) and rolipram on
Ca2+-induced contraction in α-toxin permeabilized human detrusor smooth muscle (DSM).
Representative traces demonstrate the effects of 100 μM cAMP (A), 10 μM FSK (B), and 10 μM
rolipram (C) on 1 μM [Ca2+]i-induced contraction in intact human detrusor smooth muscle. Graph
summarises the relaxation effect of cAMP, FSK and rolipram. The maximum tension force at 1 μM
[Ca2+]i was normalised as 100%. Columns represent means ± SD (N = 3 - 5, n = 6 - 20) (D).
This article is protected by copyright. All rights reserved.
Accepted Article
Figure 3
Effects of cAMP on Ca2+ sensitisation induced by guanosine-5'-triphosphate (GTP) plus
carbachol (CCh) in the absence and presence of AF-DX116 or
4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) in α-toxin permeabilized human
detrusor smooth muscle.
Representative traces show the effects of 100 μM cAMP addition on Ca2+ sensitisation induced by
GTP plus CCh in the absence or presence of 1 μM AF-DX (A) and 1 μM 4-DAMP (B) in α-toxin
permeabilized human detrusor smooth muscle. Graph summarises the relaxation effect of cAMP with
(N = 3, n = 11) or without AF-DX (N = 3, n = 11) and 4-DAMP (N = 3, n = 10). Columns represent
means ± S.E. Only P values indicative of significance are presented.
Figure 4
Effects of cAMP on Ca2+ sensitisation induced by GTP plus carbachol (CCh) in the absence and
presence of Y-27632 or GF109203X in α-toxin permeabilized human detrusor smooth muscle.
Representative traces show the effects of 100 μM cAMP on Ca2+ sensitisation induced by 100 μM
GTP and 10 μM CCh in the absence or presence of 10 μM Y-27632 (Aa) or GF-109203X (Ba) in
α-toxin permeabilized human detrusor smooth muscle. Each graph shows the relationship between the
relaxation effect of 100 μM cAMP and the developed tension force induced by Ca2+ sensitisation (100
μM GTP and 10 μM CCh) obtained from each skinned fibre. The developed tension force induced by
GTP and CCh, and the cAMP-induced relaxation values are expressed in a relative manner; Ca2+ (1
μM)-induced tension force is normalised as 1.0. The relative values before (open circle) and after
This article is protected by copyright. All rights reserved.
Accepted Article
(filled circle) application of 10 μM Y-27632 (Ab) or 10 μM GF-109203X (Bb) obtained from the
same strip were plotted and connected with lines since Ca2+ sensitisation-enhancement by GTP plus
CCh varies among individual detrusor strips (21). Inner graphs demonstrate the relative value average
before and after application of Y-27632 or GF-109203X. Asterisks indicate significant differences
within the same strip, as compared with its own control. (Y-27632, N = 3, n = 8, P = 0.0011;
GF-109203X, N = 3, n = 6, P = 0.015).
Figure 5
Effects of cAMP on tension force induced by sphingosylphosphorylcholine (SPC), phorbol 12,13
dibutyrate (PDBu) or calyculin A in α-toxin permeabilized human detrusor smooth muscle.
Representative traces show the effects of 100 μM cAMP on tension force activated by 1 μM SPC (A),
1 μM PDBu (B) or 1 μM calyculin A (C) at 1 μM [Ca2+]i in α-toxin permeabilized human detrusor
smooth muscle. (D) The graph summarises normalised cAMP-induced relaxation following
augmentation by 1 μM SPC (N = 5, n = 21), 1 μM PDBu (N = 4, n = 16) or 1 μM calyculin A (N = 2,
n = 8). Columns represent means ± SE.
Figure 6
Effects of cAMP on Ca2+-induced contraction in the presence of a protein kinase A (PKA)
inhibitor, calmodulin antagonist, or PI3K inhibitor in α-toxin permeabilized human detrusor
smooth muscle.
This article is protected by copyright. All rights reserved.
Accepted Article
Representative traces show the effects of 100 μM cAMP on 1 μM Ca2+-induced contraction in the
presence or absence of 10 μM H-89 (A), 100 μM W-7 (B) or 10 μM LY-294002 (C) in α-toxin
permeabilized human detrusor smooth muscle. Graph shows the relative cAMP-induced relaxation
value following 10 μM H-89 (slashed column; N=4, n=8), 100 μM W-7 (dotted column; N = 4, n = 8)
or 10 μM LY-294002 (open column; N = 3, n = 6). Relaxation without any reagents (control; filled
column) is normalised as 100%. Columns represent means ± SE. Only P values indicative of
significance are presented.
Figure 7
Effects of cAMP analogues on Ca2+-induced contraction in α-toxin permeabilized human
detrusor smooth muscle.
Representative traces show the effects of 100 μM cAMP (Aa), 100 μM 6Bnz-cAMP (Ab) and 100
μM 8pCPT-2’-O-Me-cAMP (Ac) on 1 μM Ca2+-induced contraction, and the effects of 100 μM
6Bnz-cAMP (Ba) and 100 μM 8pCPT-2’-O-Me-cAMP (Bb) on Ca2+ sensitisation (100 μM GTP plus
10 μM CCh at fixed 1 μM [Ca2+]i) in α-toxin permeabilized human detrusor smooth muscle. Columns
show the relaxation effects of cAMP (filled column), 6Bnz-cAMP (slashed column) and
8pCPT-2’-O-Me-cAMP (open column) in C. Columns represent means ± SE. The relaxation effect of
6Bnz-cAMP was insignificant compared with the relaxation effect of cAMP (P = 0.32 on 1 μM
Ca2+-induced contraction; N = 5, n = 18, P = 0.075 on Ca2+ sensitisation; N = 4, n = 11). In contrast,
the relaxation effect of 8pCPT-2’-O-Me-cAMP was significantly different compared with the
relaxation effect of cAMP (P < 0.0001 on 1 μM Ca2+-induced contraction; N = 4, n = 17, P = 0.00017
on Ca2+ sensitisation; N = 5, n = 19).
This article is protected by copyright. All rights reserved.
Accepted Article
Figure 8
Western blot examination of exchange protein activated by cAMP (Epac)1, Epac2, Ras-related
protein 1 (Rap1) and Ras-related C3 botulinum toxin substrate 1 (Rac1) in human detrusor
smooth muscle. Various mouse organs (brain, pancreas, kidney and detrusor smooth muscle) were
used as positive and negative controls. Arrows indicate the expected molecular weights (MW) of
target proteins.
Figure 9
Schematic diagram illustrating plausible mechanisms underlying the relationship between cAMP and
a Ca2+ sensitisation-related kinase.; FSK, forskolin
This article is protected by copyright. All rights reserved.
Accepted Article
Table 1
chemical reagents
principle action
A-23187
Ca2+ ionophore
AF-DX116
selective muscarinic receptor 2 (M2) inhibitor
6-Bnz-cAMP
selective protein kinase A (PKA) activator
calyculin A
myosin light chain phosphatase (MLCP) inhibitor
carbachol (CCh)
selective muscarinic receptor agonist
cyclopiazonic acid (CPA)
inhibitor of endo-(or sarco-)plasmic reticulum Ca2+-ATPase
4-DAMP
selective muscarinic receptor 3 (M3) inhibitor
dibutyryl-cAMP
membrane-permeable cAMP
forskolin
adenylyl cyclase activator
GF-109203X
selective protein kinase C (PKC) inhibitor
H-89
selective protein kinase A (PKA) inhibitor
LY-294002
selective phosphoinositide 3 kinase (PI3K) inhibitor
8-pCPT-2’-O-Me-cAMP
selective Epac activator
phorbol 12, 13-dibutyrate (PDBu)
selective protein kinase C (PKC) activator
rolipram
selective phosphodiesterase (PDE) IV inhibitor
sphingosylphosphorylcholine (SPC)
selective rho kniase (ROK) activator
W-7
selective calmodulin inhibitor
Y-27632
selective rho kniase (ROK) inhibitor
This article is protected by copyright. All rights reserved.
Accepted Article
This article is protected by copyright. All rights reserved.
Accepted Article
This article is protected by copyright. All rights reserved.
Accepted Article
This article is protected by copyright. All rights reserved.
Accepted Article
This article is protected by copyright. All rights reserved.
...
論文の公開元へ
