* Departments of Molecular Signaling and Urology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Tamaho, Yamanashi, Japan; and the Department of Cellular Physiology, Institute of Nephrology, Niigata University, Niigata, Japan
Address correspondence to: Dr. Jian Yao, Department of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Tamaho, Yamanashi 409-3898, Japan. Phone/Fax: +81-55-273-8074; yao{at}yamanashi.ac.jp
This study investigated a potential role of nitric oxide (NO)in the regulation of gap junctional intercellular communication(GJIC). Incubation of mesangial cells (MC) with NO donor S-nitroso-N-acetylpenicillamine(SNAP) enhanced both basal and 8-bromo-cAMP–stimulatedGJIC as well as expression of gap junction protein connexin43(Cx43). This potentiating action of SNAP on Cx43 expressionwas mimicked by two other NO donors and significantly blockedby soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-]quinoxalin-1–1.Guanosine 3',5'-cyclic monophosphate (cGMP) analogue 8-bromo-cGMPexerted an effect similar to NO, whereas another cGMP analogue,8-pCPT-cGMP, which selectively activates cGMP-dependent kinasewithout affecting cGMP-inhibited phosphodiesterase (PDE3), hadno effect. Moreover, the synergistic action of NO on Cx43 expressionwas completely prevented by protein kinase A inhibitor H89 butnot by cGMP-dependent kinase inhibitor Rp-8-Br-PET-cGMP. Theseresults suggested a possible involvement of NO-cAMP interactionvia cGMP-mediated inhibition of PDE3. Indeed, PDE3 inhibitorcilostamide caused potentiation of 8-bromo-cAMP–elicitedelevations of Cx43 expression that is similar to the effectof SNAP, and an elevation of intracellular cAMP was detectedin SNAP-treated cells. With the use of genetically engineeredreporter MC that express secreted alkaline phosphatase underthe control of the cAMP response element, significant potentiationof cAMP-elicited activation of cAMP response element by SNAPwas found. This effect was abrogated in the presence of PDE3inhibitor cilostamide. Taken together, the results suggest thatNO is involved in the control of GJIC and Cx43 expression. Thiseffect of NO is due to activation of protein kinase A via cGMP-dependentinhibition of PDE3 activity.
Gap junctions (GJ) are clusters of transmembrane channels thatpermit the direct intercellular exchange of ions, secondarymessengers, and small signaling molecules. Intercellular communicationvia GJ is thought to play an important role in the control ofa variety of cellular functions, including cell growth, migration,differentiation, and electric coupling (1–4). In the cardiovascularsystem, cells are intensively interlinked by GJ channels (1,4).GJ in vascular cells provide a structural basis for coordinatedvasoconstriction or vasodilation and for extensive cross-talkbetween cells of the vascular wall (1,4). It has been reportedthat the vasodilative action of the endothelium-derived hyperpolarizingfactor (EDHF) is dependent on GJ between endothelial and smoothmuscle cells (SMC) (5,6) and that the propagation of vasomotorsignals along the vessels also requires GJ intercellular communication(GJIC) (1,7–9). The altered expression of GJ proteinsand its relevance to vascular diseases have also been reportedby many investigators (1,4,8,10–12). Therefore, the informationon the control of GJ channels in vascular cells is essentialfor understanding of the role of GJ in vascular pathophysiology.
Nitric oxide (NO) is an important molecule in the vascular system.Besides its vasodilative action, NO exerts multiple effectson SMC, including cell growth, migration, adhesion, and apoptosis.Because GJ channels are known to be important in many cellularfunctions, it is likely that GJ is involved in the NO-inducedalteration of cell behavior. Many effects of NO are caused byinduction of a second messenger molecule, guanosine 3',5'-cyclicmonophosphate (cGMP), which is produced after NO-mediated activationof soluble guanylate cyclase (sGC). Although cGMP-dependentkinase (PKG) is the major target of intracellular cGMP, cGMPalso interacts with other molecules, such as cGMP-gated ionchannels. In addition, the effects of cGMP could involve modulationof cAMP levels and protein kinase A (PKA) activity via stimulatingphosphodiesterase 2 or via inhibiting phosphodiesterase 3 (PDE3)activity (13–16). High concentrations of cGMP may alsodirectly stimulate PKA (17). cAMP is a well-characterized secondmessenger that promotes GJ protein expression and GJIC in avariety of cell types (18,19). We hypothesized that NO may affectGJ protein expression and GJIC via cGMP-mediated cross-talkwith cAMP signaling. The purpose of this study was to test thishypothesis.
Mesangial cells (MC) are considered to be specialized SMC thatplay a pivotal role in the regulation of glomerular hemodynamics.One of the striking features of MC is that these cells possessextremely high density of GJ (20,21). This unique property indicatesa possibility that MC are actively involved in the functionof the juxtaglomerular apparatus (22). Like SMC, MC mainly expressGJ protein connexin 43 (Cx43) (20). We previously reported thatthe GJ in MC are critically involved in the transmission ofintercellular signal and in the coordination of MC contraction(23). However, information is still limited on the regulationof GJ function by pathophysiologic factors.
In this study, we investigated whether and how NO influencesGJ protein expression and GJIC in MC. Our present data revealthat NO is a potent stimulator of Cx43 expression and GJIC.We also demonstrate that this action of NO is due to the activationof PKA via cGMP-dependent inhibition of PDE3 activity.
Materials
cAMP Biotrak enzymeimmunoassay system was purchased from AmershamBiosciences (Buckinghamshire, UK). pCRE–secreted alkalinephosphatase (pCRE-SEAP) vector and Great EscAPe SEAP DetectionKit were obtained from BD Biosciences (Palo Alto, CA). TRITC-conjugatedswine anti-rabbit Ig was purchased from DAKO (Glostrup, Denmark),and cilostamide was from Wako (Osaka, Japan). All other reagentswere obtained from Sigma (St. Louis, MO).
MC
Establishment and characterization of rat MC were performedas described previously (23–25).
Stable Transfection
MC were transfected with pCRE-SEAP (BD Biosciences) togetherwith pcDNA3.1 (Invitrogen, Carlsbad, CA) that encodes a neomycinphosphotransferase gene using a calcium phosphate co-precipitationmethod (25). Stable transfectants were selected with 0.7 mg/mlG418, and a clone with the lowest background level and the highestSEAP inducibility was selected and used for studies.
Immunocytochemistry
Immunocytochemical staining for Cx43 was done as previouslyreported (23,24,26).
Measurement of GJIC
GJIC was assessed by transfer of the membrane-impermeant fluorescentdye, Lucifer Yellow, after single-cell microinjection with anautomated microinjection system (Zeiss Oberkochen, Jena, Germany),as described previously (23,24,26).
Western Blot Analysis
Equal amounts of cell lysates were separated by 10% SDS–polyacrylamidegels and electrotransferred onto 0.4-µM polyvinylidenedifluoride membranes. After blocking with 3% BSA in PBS, themembranes were incubated with anti-Cx43 antibody. After washingwith PBS-0.1% Tween 20, filters were probed with horseradishperoxidase–conjugated sheep anti-rabbit IgG or rabbitanti-mouse IgG. Immunoreactivity was detected by the enhancedchemiluminescence system (Amersham Biosciences, Buckinghamshire,UK). For assessing the amount of Cx43 protein loaded, filterswere treated with 2% SDS and 100 mM -mercaptoethanol in 62.5mM Tris-HCl (pH 6.8) for 30 min at 60°C and reprobed for-actin. Films were scanned, and the optical density of the bandswas measured with Scion Image. Data shown are representativeof at least three independent experiments with similar results.
Northern Blot Analysis
MC were treated with various agents for 12 h. Equal amountsof RNA (5 µg) extracted from cells were separated by electrophoresisand transferred onto nylon membranes (Hybond N+; Amersham Bioscience).The level of Cx43 mRNA was examined as described before (27),using the entire coding sequence of the rat Cx43 cDNA (28) asa probe (provided by Drs. G. Olbina and W. Eckhart, Molecularand Cell Biology Laboratory, The Salk Institute for BiologicStudies, San Diego, CA). The staining of 28S and 18S ribosomalRNA by ethidium bromide was used for loading controls.
cAMP Measurement
Confluent MC were exposed to 100 µM S-nitroso-N-acetylpenicillamine(SNAP) or left untreated for 30 min. After the stimulation,the cells were lysed and assayed for cAMP according to the manualinstruction for cAMP Biotrak Enzymeimmunoassay System. A partof cellular lysate was used for protein assay using Micro BCAProtein Assay Kit (Pierce, Rockford, IL).
SEAP Assay
Activity of SEAP was evaluated using the Great EscAPe DetectionKit following the protocol provided by the manufacturer. Inbrief, MC in 96-well plates were exposed to various stimulifor 24 h. Aliquots of supernatants were sampled from the culturesand centrifuged at 12,000 x g for 2 min. Fifteen microlitersof dilution buffer was mixed with 5 µl of sample, andthe mixture was incubated at 65°C for 30 min to eliminatethe endogenous alkaline phosphatase activity. Twenty microlitersof assay buffer was subsequently added to the mixture and incubatedfor an additional 10 min at room temperature. The CSPD substrateat a concentration of 1.25 mM was prepared by dilution with20x chemiluminescence enhancer, and 20 µl of the dilutedsubstrate was added to each sample, followed by 10 min at roomtemperature. The intensity of chemiluminescent signal was determinedby a luminometer (Gene Light 55; Microtech Nition, Chiba, Japan).
Statistical Analyses
Values are expressed as either means ± SEM or means ±SD. Comparison with control was done with one-way ANOVA andthe Dunnett test. Comparison of two populations was made byt test. P < 0.05 was considered to be statistically significant.
NO Induces Cx43 Expression
Incubation of MC with NO donor SNAP for 24 h resulted in a dose-dependentincrement of Cx43 protein (Figure 1A). This effect of SNAP wasmuch more enhanced in the presence of a cell-permeable analogueof cAMP, 8-bromo-cAMP (500 µM). The enhancement of Cx43expression by SNAP was observed at a wide range of concentrationsfrom 1 to 250 µM. 8-Bromo-cAMP also induced a dose-dependentelevation of Cx43, and its effect was dramatically potentiatedwhen it was added together with 100 µM SNAP (Figure 1B).A time-course analysis revealed that the marked elevation ofCx43 occurred within 16 h after exposure to these stimuli (Figure 1C).
Figure 1. Effects of nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) on basal and cAMP-elicited expression of connexin43 (Cx43). (A) Dose-dependent induction of Cx43 expression by SNAP. Rat mesangial cells (MC) were exposed to various concentrations of SNAP in the presence (+) or absence (–) of 500 µM 8-bromo-cAMP for 24 h. Cellular protein was extracted and subjected to Western blot analysis using either an anti-Cx43 antibody (top row) or an anti–-actin antibody (bottom row). (B) Potentiation of 8-bromo-cAMP–elicited expression of Cx43 by SNAP. MC were treated for 24 h with various concentrations of 8-bromo-cAMP in the presence or absence of SNAP (100 µM), and expression of Cx43 was evaluated. (C) Time-dependent effects of SNAP and 8-bromo-cAMP on Cx43 expression. MC were treated with 100 µM SNAP, 250 µM 8-bromo-cAMP, or both for indicated durations, and Cx43 expression was examined.
The action of SNAP on Cx43 expression was mimicked by otherstructurally unrelated NO donors, (Z)-1-[-2-(aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate(DETA-NONOate) and sodium nitroprusside (SNP; Figure 2A), suggestingthat the common product of these agents, NO but not other metabolites,was responsible for the elevated expression of Cx43.
Figure 2. Effects of various inhibitors on the action of SNAP. (A) Prevention of the potentiating effects of NO donors by 1H-[1,2,4]oxadiazolo[4,3,-]quinoxalin-1–1 (ODQ). MC were treated either with or without 25 µM ODQ for 15 min and exposed to three different NO donors [100 µM SNAP, 100 µM (Z)-1-[-2-(aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (NONOate) and 200 µM sodium nitroprusside (SNP)] together with 250 µM 8-bromo-cAMP for 24 h. The levels of Cx43 and -actin were determined by Western blot analysis. (B) Lack of involvement of guanosine 3',5'-cyclic monophosphate (cGMP)-dependent kinase (PKG) in the effect of SNAP. MC were treated with SNAP (100 µM), 8-bromo-cGMP (500 µM), or 8-pCPT-cGMP (500 µM) alone or in combination with 8-bromo-cAMP (250 µM) for 24 h. For analyzing a role of PKG, cells were pretreated with a specific PKG inhibitor, Rp-8-Br-PET-cGMP (Rp-cGMP; 50 µM) for 15 min and then exposed to SNAP and 8-bromo-cAMP for 24 h in the presence of the PKG inhibitor. (C) Blockade of the potentiating effect of SNAP on Cx43 expression by protein kinase A (PKA) inhibitor. MC were treated with various cAMP-elevating agents (250 µM 8-bromo-cAMP; 5 µM 3-isobutyl-1-methylxanthine [IBMX] or 1 µM forskolin) alone or in combination with 100 µM SNAP for 24 h. For examining the role of PKA, cells were pretreated with PKA inhibitor H89 (10 µM) for 15 min before the exposure of SNAP plus 8-bromo-cAMP for 24 h with the PKA inhibitor. (D) Densitometric analysis of the main data shown in A, B, and C. The results are expressed as percentages of the basal Cx43 (mean ± SEM, n = 3 to 5). #P < 0.01 versus basal control; *P < 0.01 versus single stimulus.
Effect of NO on Cx43 Expression Is Mediated not by PKG but by PKA
Most of the biologic actions of NO are supposed to be mediatedby cGMP signals (16). We therefore examined the role of thecGMP pathway in Cx43 expression. First, we tested whether aninhibitor of sGC, 1H-[1,2,4]oxadiazolo[4,3,-]quinoxalin-1–1(ODQ), diminishes the effect of NO on Cx43 expression. As shownin Figure 2A, ODQ at 25 µM unexpectedly increased thebasal level of Cx43 for reasons presently unknown. Even so,ODQ substantially prevented the potentiating actions of threedifferent NO donors (SNAP, NONOate, and SNP). Second, we testedwhether the stable, lipophilic analogues of cGMP (8-bromo-cGMPand 8-pCPT-cGMP) can mimic the promoting action of SNAP on Cx43expression. As demonstrated in Figure 2B, 8-bromo-cGMP but not8-pCPT-cGMP (which selectively activates PKG but does not affectcGMP-inhibited phosphodiesterase) exerted the effect similarto SNAP on Cx43. Of note, both agents at the same concentrationused (500 µM) elicited comparable activation of PKG, asevaluated by the phosphorylation levels of vasodilator-stimulatedphosphoprotein at serine 239 (a site preferentially phosphorylatedby PKG) (29) (data not shown). Thus, it is unlikely that thepotentiating action of SNAP on Cx43 expression was mediatedby PKG activation. Indeed, pretreatment of cells with PKG-specificinhibitor Rp-8-Br-PET-cGMP (50 µM) did not attenuate theeffect of SNAP (Figure 2B).
Given that cAMP is a well-known second messenger in promotingboth Cx43 expression and GJIC (14,18,19), a role of the cAMPsignaling in the action of NO was examined. We first testedwhether SNAP can enhance Cx43 expression triggered by cAMP-elevatingagents other than 8-bromo-cAMP. As shown in Figure 2C, SNAP(100 µM) significantly potentiated the expression of Cx43triggered by low concentrations of 3-isobutyl-1-methylxanthine(a nonspecific PDE inhibitor; 5 µM) and forskolin (a directactivator of adenylyl cyclase; 1 µM). Because cAMP-dependenteffects are usually mediated by PKA, we further investigatedthe role of PKA in the expression of Cx43. As demonstrated inFigure 2C, pretreatment of cells with PKA inhibitor H89 (10µM) completely blocked the synergistic effect of SNAPand 8-bromo-cAMP on Cx43 expression.
The densitometric analysis of the main findings shown in Figures 1and 2 is summarized in Figure 2D. NO donor SNAP significantlyenhanced both basal and 8-Br-cAMP–elicited Cx43 expression.This action of SNAP was mimicked by 8-Br-cGMP and completedblocked by PKA inhibitor H89.
It should be noted that treatment of cells with NO and 8-bromo-cAMPnot only increased the amount of Cx43 but also altered the statusof Cx43 phosphorylation. In control cells, mainly nonphosphorylatedCx43 was observed. It appeared as one band in Western blots,whereas in the treated cells, Cx43 existed in both phosphorylatedand nonphosphorylated forms, which appeared as double or triplebands in Western blot. The slower migrating band is consideredto correspond to a phosphorylated form of Cx43.
PDE3 Inhibitor Mimics the Effect of NO
One possible mechanism by which NO stimulates PKA activationis an increase in cAMP concentration caused by cGMP-mediatedinhibition of PDE3 (13–15). To examine this possibility,we compared the effect of SNAP with that of a specific PDE3inhibitor, cilostamide. As shown in Figure 3, treatment of cilostamide(20 µM) increased Cx43 expression. In combination with250 µM 8-bromo-cAMP, a synergistic effect was observed.In contrast, PDE4 inhibitor rolipram (20 µM) had no effecton the expression of Cx43.
Figure 3. Effect of phosphodiesterase (PDE) inhibitors on Cx43 expression. MC were treated with 8-bromo-cAMP (250 µM), SNAP (100 µM), cilostamide (20 µM), or rolipram (20 µM) alone or in combination for 24 h. Expression of Cx43 was examined by Western blot analysis. Similar results were obtained by two additional experiments.
Several previous studies have documented that PDE3 and PDE4inhibitors work synergistically in modulating multiple cellbehavior (30,31). We tested whether these agents also act synergisticallyin inducing Cx43 expression. As shown in Figure 3, the combinationof PDE3 and PDE4 inhibitors markedly induced the expressionof Cx43. A similar synergy was also observed between SNAP andthe PDE4 inhibitor rolipram. These observations indicated apossibility that the potentiating action of SNAP on Cx43 expressionis due to inhibition of PDE3 activity.
Cx43 Expression Was not Altered by Cyclooxygenase or NO Synthase Inhibitor
cAMP signaling pathway and NO have extensive cross-talk. Besidesinhibition of PDE3, NO is reported to be able to induce andactivate the cyclooxygenase (32,33). The increased formationof prostaglandin E2, a known activator of the adenyl cyclase,may contribute to the action of NO on Cx43 expression. In addition,cAMP has been reported to trigger the expression of inducibleNO synthase in MC (34). The positive reciprocal interactionsbetween endogenous NO and cAMP may also be implicated in thecontrol of Cx43 expression. To examine these possibilities,we pretreated MC with the specific cyclooxygenase inhibitorindomethacin or with the NO synthase inhibitor NG-nitro-l-argininemethyl ester (L-NAME) before exposing the cells to NO, 8-Br-cAMPalone, or both in combination. Figure 4 shows that neither indomethacinnor L-NAME affected the expression of Cx43 as induced by thestimuli. Therefore, these possibilities were excluded.
Figure 4. Influences of cyclooxygenase or NO synthase inhibitor on the synergistic action of SNAP and 8-bromo-cAMP. MC were either left untreated (control) or treated with 50 µM indomethacin or 100 µM NG-nitro-l-arginine methyl ester 15 min before exposure to SNAP (100 µM) and 8-bromo-cAMP (500 µM) alone or in combination for 24 h. Expression of Cx43 was examined by Western blot analysis. Similar results were obtained by two additional experiments.
NO Increases Cx43 mRNA Expression and Membrane Location of Cx43 Protein
To examine whether NO affects Cx43 expression at the transcriptionallevel, we performed Northern blot analysis. Consistent withthe increase in the protein level, SNAP significantly inducedmRNA expression of Cx43 under both basal and cAMP-stimulatedconditions. This effect of NO could also be mimicked by 8-bromo-cGMP(Figure 5, A and B).
Figure 5. Effects of NO on mRNA expression and protein distribution of Cx43. (A) Northern blot analysis of Cx43 mRNA expression in MC that were treated with 100 µM SNAP and 500 µM 8-bromo-cGMP alone or in combination with 250 µM 8-bromo-cAMP for 12 h (top). Staining of ribosomal RNA 18S and 28S was shown as a loading control (bottom). One representative result from three independent experiments is shown. (B) Densitometric analysis of data shown in A. Data are expressed as relative percentages against the value of untreated control (means ± SEM; n = 3). #P < 0.01 versus basal control; *P < 0.01 versus single stimulus. (C) MC were treated with 100 µM SNAP or 500 µM 8-bromo-cGMP alone (Figure 4C, top) or in combination with 250 µM 8-bromo-cAMP (Figure 4, bottom) for 24 h and subjected to immunofluorescent staining of Cx43. Magnification, x600.
Cx43 is known to be located at cell-to-cell contact portionsand perinuclear regions in cultured MC (23), and only membrane-associatedCx43 molecules are active for GJIC. We looked at effects ofSNAP and 8-bromo-cGMP on Cx43 protein distribution by usingimmunofluorescent staining. As shown in Figure 5C, Cx43 wasmainly located at the perinuclear regions in untreated controls.After exposure to SNAP (100 µM) or 8-bromo-cGMP (500 µM)for 24 h, a modest but clear enhancement of Cx43 staining wasobserved along the region of cell-to-cell contact. When SNAPor 8-bromo-cGMP was added in combination with 8-bromo-cAMP,a dramatic increase of Cx43 was observed at both the cell-to-cellcontact region and the perinuclear region. 8-Bromo-cAMP alonehad only a marginal effect on Cx43 staining at the concentrationof 250 µM.
NO Augments Functional GJIC
The increased expression and membrane distribution of Cx43 maylead to enhancement of GJIC. To examine this possibility, weanalyzed the GJIC in MC using the Lucifer Yellow dye-transferassay (23,24,26). As shown in Figure 6, incubation of MC with100 µM SNAP or 500 µM cGMP was found to be sufficientfor promotion of GJIC (number of dye-coupled cells: control,2.83 ± 1.75; SNAP, 6.28 ± 1.54; 8-bromo-cGMP,6.26 ± 1.59; means ± SEM; n = 10 to 15). In thepresence of 250 µM 8-bromo-cAMP, the effects of SNAP and8-bromo-cGMP were more pronounced (8-bromo-cAMP alone, 4.09± 2.40; cAMP + SNAP, 11.35 ± 3.61; cAMP + 8-bromo-cGMP,10.73 ± 4.52; means ± SEM; n = 10 to 15).
Figure 6. Promotion of gap junctional intercellular communication (GJIC) by NO. (A) Microphotographic analysis of dye-coupled cells. Confluent culture of MC was treated with SNAP (100 µM) or 8-bromo-cGMP (500 µM) together with (+) or without (–) 8-bromo-cAMP (250 µM) for 24 h, and Lucifer Yellow (LY) was pressure-injected into a single cell. LY diffusion into adjacent cells was recorded by video camera. (B) Quantitative analysis of dye-coupled cells. The number of cells into which LY was transferred from the injected cells within 3 min is shown. Data are presented as the means ± SEM (n = 10 to 15). #P < 0.01 versus basal control; *P < 0.01 versus single stimulus. Magnification, x320 in A.
Amplification of the cAMP Signaling by NO
To provide direct evidence showing that NO is able to amplifythe cAMP signaling, the level of intracellular cAMP was measured.Significant elevation of the cAMP level was detected in cellsthat were treated with 100 µM SNAP for 30 min (cAMP concentration[fmol/L per µg protein]: basal, 6.29 ± 1.47 versus100 µM SNAP, 10.84 ± 1.98; mean ± SEM; n= 3; P < 0.05).
The increase of cAMP causes PKA activation. It subsequentlyactivates cAMP response element (CRE), leading to expressionof genes that have CRE in their regulatory regions. Becausethe promoter region of the Cx43 gene contains CRE (35) and thatCRE activity serves as a good indicator of PKA activation, weexamined activity of CRE in MC that were treated with SNAP.For this purpose, MC were stably transfected with pCRE-SEAP,and reporter cells were established. The cells were then exposedto various stimuli, and activity of SEAP in conditioned mediawas evaluated. As shown in Figure 7A, SNAP alone induced a modestincrease of CRE activity in a dose-dependent manner. The activationof CRE by SNAP was dramatically enhanced in the presence of500 µM 8-bromo-cAMP. Figure 7B shows enhancement of 8-bromo-cAMP–inducedactivation of CRE by SNAP. This effect was observed at a widerange of concentrations of 8-bromo-cAMP (50 to 500 µM).Similar to our previous data on Cx43 expression, the potentiatingeffect of SNAP on CRE activity was mimicked by other NO donorsNONOate and SNP and by cGMP analogue 8-bromo-cGMP but not by8-pCPT-cGMP (Figure 7C). It is interesting, however, that 8-pCPT-cGMPalone induced a higher basal level of CRE activity, as comparedwith SNAP or 8-bromo-cGMP. Consistent with the results shownin Figure 2A, the effect of SNAP on CRE activation was preventedby ODQ but not by PKG inhibitor Rp-8-Br-PET-cGMP (Figure 7D).The synergistic effect of SNAP or 8-bromo-cGMP on cAMP-elicitedactivation of CRE was also confirmed by the mRNA level of thereporter gene SEAP (Figure 7, E and F).
Figure 7. Potentiating action of NO on cAMP response element (CRE) activity. (A) Dose-dependent potentiation of 8-bromo-cAMP–elicited CRE activation by SNAP. MC that were stably transfected with pCRE–secreted alkaline phosphatase (pCRE-SEAP) were exposed to the indicated concentrations of SNAP in the presence or absence of 500 µM 8-bromo-cAMP for 24 h. The conditioned media were harvested and assayed for SEAP activity. (B) Enhancement of the action of 8-bromo-cAMP by SNAP. MC were treated with the indicated concentrations of 8-bromo-cAMP alone or together with 100 µM SNAP for 24 h. (C) Various NO donors and cGMP analogues on CRE activity. MC were treated with 100 µM SNAP, 100 µM NONOate, 200 µM SNP, 500 µM 8-bromo-cGMP, or 500 µM 8-pCPT-cGMP alone or in combination with 250 µM 8-bromo-cAMP for 24 h. (D) Effects of soluble guanylate cyclase (sGC) inhibitor ODQ and PKG inhibitor Rp-8-Br-PET-cGMP on the action of NO. MC were preincubated with 25 µM ODQ or 50 µM PKG inhibitor Rp-8-Br-PET-cGMP for 15 min before exposing to the mixture of SNAP (100 µM) and 8-bromo-cAMP (250 µM) for 24 h in the presence of these agents. Data are presented as fold induction of SEAP activity against untreated control (means ± SD). Assays were performed in triplicate. #P < 0.05 versus basal control; *P < 0.05 versus single stimulus. Similar results were obtained from an additional experiment. (E) Northern blot analysis of SEAP mRNA expression in reporter cells that were treated with SNAP or 8-bromo-cGMP alone or in combination with 8-bromo-cAMP for 12 h (top). The staining of ribosomal RNA 18S and 28S is shown in the bottom panel as a loading control. Representative results from three independent experiments are shown. (F) Densitometric analysis of data shown in E. The results are expressed as fold induction against the untreated control (means ± SEM; n = 3). #P < 0.01 versus basal control; *P < 0.01 versus single stimulus.
Consistent with the critical role of PDE3 inhibition in theaction of SNAP, PDE3 inhibitor cilostamide was found to mimicthe potentiating action of SNAP on the cAMP-elicited activationof CRE. Furthermore, in the presence of cilostamide, SNAP failedto exhibit any additional effects on CRE activity (Figure 8A),suggesting that both agents may function through the same mechanism.Consistent with the previous data showing the synergistic effectbetween PDE3 and PDE4 inhibitors on Cx43 expression, a similar,additive effect between these agents on CRE activation was observed(Figure 8B). The role of PDE3 inhibitor cilostamide in thiscooperative action was completely reproduced by SNAP (Figure 8B).In other words, SNAP acted like the PDE3 inhibitor cilostamidein modulating cAMP signaling.
Figure 8. Reproduction of the action of SNAP on CRE activity by PDE3 inhibitor. The CRE reporter MC were treated with 8-bromo-cAMP (250 µM), SNAP (100 µM), cilostamide (20 µM), or rolipram (20 µM) alone or in various combinations for 24 h. Conditioned media were collected and used for SEAP assay. Data are presented as fold induction (means ± SD) of SEAP activity against untreated control. Assays were performed in triplicate. Similar results were also obtained from at least one additional experiment. *P < 0.05 versus single stimulus.
In this study, we investigated the effects of NO on Cx43 expressionand GJIC in cultured MC. Using NO donor SNAP as an exogenoussource of NO, we demonstrated that NO markedly increased basaland cAMP-stimulated levels of Cx43 expression, leading to enhancedGJIC. The mechanism underlying this effect involved activationof sGC, elevation of cGMP, and amplification of the cAMP signalingvia the cGMP-mediated inhibition of PDE3. Our current hypothesisis illustrated in Figure 9.
Figure 9. Schematic diagram illustrating potential mechanisms involved in the NO-mediated regulation of Cx43 expression and GJIC. NO activates sGC, causing the generation and action of cGMP. The cGMP-dependent inhibition of PDE3 activity results in the increase of the cAMP level and subsequent activation of PKA, leading to the enhancement of Cx43 expression and GJIC.
In this report, we showed, for the first time, that NO is ableto enhance both basal and cAMP-elicited Cx43 expression andGJIC. The effect of NO donor on Cx43 expression was detectedat several different levels; i.e., the protein level detectedby immunofluorescent staining and Western blot, the transcriptionallevel detected by Northern blot, and the functional level examinedby the dye-transfer assay. The regulation of GJ by NO has beenreported previously in some cell types. Sladek et al. (36) showedthat endogenous NO attenuated myometrial Cx43 expression duringrat pregnancy. Another report, by Roh et al. (37), also demonstratedan inhibitory effect of exogenous NO on Cx43 levels in cultureduterine myocytes. The mechanisms involved are currently unknown.The discrepancy of the results from uterine myocytes and glomerularMC may be due to the different properties of the cells usedfor study. Contrary to most of the previous observations (18,19),cAMP has been shown to inhibit Cx43 expression in myocytes (37).In a recent study using human umbilical vein endothelial cells,NO was shown to enhance endothelial cell coupling and selectivelyincrease the expression of Cx40 but not Cx43 (12). However,information is very limited regarding how NO affects Cx43 expressionand cell coupling in vascular effector cells. Because Cx43 isthe predominant GJ protein in SMC and plays a critical rolein transmission of vasomotor signals as well as in coordinationof vascular responses (1,4), regulation of Cx43 expression andGJIC by the major endothelium-derived vasorelaxing factor NOshould have significant pathophysiologic relevance.
We demonstrated that the effects of the NO donors were significantlyattenuated by sGC inhibitor ODQ. This inhibitory effect pointsto a possible role of cGMP in the NO-mediated regulation ofCx43 expression and GJIC. Indeed, a stable analogue of cGMP,8-bromo-cGMP, completely mimicked the ability of NO to potentiateCx43 expression and GJIC. cGMP-dependent actions of NO are usuallymediated by PKG (16). However, in our experimental setting,blockade of PKG with specific PKG inhibitor Rp-8-bromo-PET-cGMPdid not influence the promoting effect of NO on Cx43 expression.In addition, the selective PKG agonist 8-pCPT-cGMP did not mimicthe action of NO. The PKG-independent induction of Cx43 by NOled us to explore alternative responsible pathways. BecausecAMP is the well-characterized second messenger that promotesGJIC and Cx43 expression (18,19), we investigated the relationshipbetween NO and cAMP signaling. Our data demonstrated that theaction of NO was totally prevented by PKA inhibitor H89. ThecGMP-mediated activation of PKA is caused by either a directeffect of cGMP on PKA (17) or an indirect action of cGMP viainhibition of cGMP-inhibited cAMP-hydrolyzing PDE3 that increasesthe level of cAMP and activates PKA (15). Our findings supportthe later possibility, because (1) PDE3 inhibitor mimicked theaction of NO in potentiating cAMP-elicited Cx43 expression;(2) increased levels of cAMP were detected after exposure ofthe cells to SNAP; (3) cGMP analogue 8-pCPT-cGMP, which selectivelyactivates PKG but does not interact with PDE3, had no obviouseffect; and (4) in the presence of PDE3 inhibitor, the potentiatingaction of SNAP on CRE activation could not be observed. Takentogether, our current data support the idea that the PDE3 inhibition–mediatedincrease of cAMP and subsequent activation of PKA is the majormechanism for SNAP-mediated potentiation of Cx43 expressionand GJIC (Figure 9).
The findings described in this report may have significant pathophysiologicimplications as follows: (1) because GJ channels are known tobe important in many cellular functions, modulation of GJ mightbe one of the mechanisms by which NO regulates cell behavior;and (2) besides NO, endothelium-derived vasorelaxing factors,including prostacyclin and EDHF, exert their biologic effectsvia the cAMP pathway (5). NO may facilitate their vasodilativefunction via amplification of the cAMP signaling. This synergisticaction of NO may be especially important for EDHF, because itsfunction is presumably mediated by GJIC (5,6). Furthermore,a positive reciprocal interaction between intracellular cAMPand EDHF vasorelaxing activity has been documented recently(3,5). The co-treatment of cells with PDE3 and PDE4 inhibitorsinduced dramatic activation of CRE and remarkable upregulationof Cx43. This finding may lead to the development of a novelstrategy for therapeutic intervention in diseases in which impairedGJIC is involved. Activation of CRE and upregulation of Cx43expression may be the important underlying pharmacologic mechanismsof their actions under a variety of conditions (30,31).
In summary, our study demonstrated that NO is a potent modulatorof Cx43 expression and GJIC. This finding may open a new windowtoward our further understanding of the vascular role of NOunder various pathophysiologic situations.
Acknowledgments
This study was supported in part by Grants-in-Aid for scientificresearch (15590845 to J.Y.; 14657257 to M.K.) from the Ministryof Education, Science, Sports and Culture, Japan.
We thank Dr. Shuichiro Maeda (Department of Biochemistry, Universityof Yamanashi, Japan) for generous, continuous support of ourstudy.
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Received for publication June 9, 2004.
Accepted for publication September 15, 2004.
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Top
Abstract
Introduction
]quinoxalin-1–1. Guanosine 3',5'-cyclic monophosphate (cGMP) analogue 8-bromo-cGMP exerted an effect similar to NO, whereas another cGMP analogue, 8-pCPT-cGMP, which selectively activates cGMP-dependent kinase without affecting cGMP-inhibited phosphodiesterase (PDE3), had no effect. Moreover, the synergistic action of NO on Cx43 expression was completely prevented by protein kinase A inhibitor H89 but not by cGMP-dependent kinase inhibitor Rp-8-Br-PET-cGMP. These results suggested a possible involvement of NO-cAMP interaction via cGMP-mediated inhibition of PDE3. Indeed, PDE3 inhibitor cilostamide caused potentiation of 8-bromo-cAMP–elicited elevations of Cx43 expression that is similar to the effect of SNAP, and an elevation of intracellular cAMP was detected in SNAP-treated cells. With the use of genetically engineered reporter MC that express secreted alkaline phosphatase under the control of the cAMP response element, significant potentiation of cAMP-elicited activation of cAMP response element by SNAP was found. This effect was abrogated in the presence of PDE3 inhibitor cilostamide. Taken together, the results suggest that NO is involved in the control of GJIC and Cx43 expression. This effect of NO is due to activation of protein kinase A via cGMP-dependent inhibition of PDE3 activity. 
-mercaptoethanol in 62.5 mM Tris-HCl (pH 6.8) for 30 min at 60°C and reprobed for 
