| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 22, 2003 as doi:10.1096/fj.02-0359fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Biochemistry and Integrative Medical Biology;
* Department of Ophthalmology, School of Medicine, Keio University, Tokyo;
Department of Ophthalmology, Tokyo Dental College, Chiba; and
# Department of Molecular Biology, Life Science and Veterinary Science, Osaka Prefecture University, Osaka, Japan
3Correspondence: Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, Tokyo 160-8582, Japan. E-mail: msuem{at}sc.itc.keio.ac.jp
SPECIFIC AIMS
Although nitric oxide (NO) and carbon monoxide (CO) are activators of soluble guanylate cyclase (sGC), roles of the two gases to regulate sGC activities in vivo remain unknown. We therefore sought to immunohistochemically visualize the gas-mediated sGC activation, addressing synergistic and/or antagonistic effects of these monoxides on the cGMP-producing enzyme in neural tissues such as retina.
PRINCIPAL FINDINGS
1. A monoclonal antibody 3221 increases its affinity to sGC on application of NO
To examine sGC activities in vivo, we used two distinct monoclonal antibodies (mAbs) against the enzymes, mAb28131 and mAb3221. Not only did this method appear to visualize the sGC activity, it was also proved to be a good tool to provide microtopographic information on the ability of sGC for receiving gases in vivo. As seen in Fig. 1
A, both mAbs exhibited positive immunoreactivities to rat retinal lysates but with different blotting patterns; mAb28131 displayed one major band at 76 kDa, indicating reactivity to the ß subunit of sGC. mAb3221 showed two major bands at 66–68 kDa and at 76 kDa, indicating that the mAb reacts with 
and ß subunits even when protein samples were denatured. These results raised the possibility that mAb3221 recognizes a regiospecific structure determined by the two subunits of native sGC protein, consistent with our previous results. The surface plasmon resonance technology was used to examine binding interactions of native sGC with the immobilized mAb3221 (Fig. 1B
). The condition where the reaction mixture contained sufficient NO gave an elevated Bmax with a two-order reduction in the Kd compared with those in the absence of NO. The prosthetic heme-free sGC did not exhibit any increase in the binding affinity induced by NO, suggesting that the conformational change of sGC detected by mAb3221 depends on the reaction of NO with the heme. The addition of CO also enhanced the affinity of the mAb to sGC, but modestly. These results suggest that mAb3221 changes its affinity according to the activation state of the enzyme, acquiring high affinity to the activated sGC while maintaining low affinity to the quiescent one. The affinity of mAb28131 to the enzyme remained constant regardless.
|
2. Immunohistochemistry by mAb3221 visualizes local sGC activation in vivo
Not only does mAb3221 enable immunological detection of sGC activation in vitro, but it can be applied to visualize the activation on tissues undergoing controlled perfusion fixation with paraformaldehyde. We argued that immunoreactivities of mAb3221 for the fixed sGC should correlate with its activation state whereas that of mAb28131 should remain constant. Taking the intensity of chromogen formation as an index of immunoreactivities, results shown in Fig. 2
A support our argument. An i.v. injection of L-arginine, but not that of D-arginine (data not shown), increased mAb3221-associated immunoreactivities in the inner plexiform layer (INL) but did not alter those for mAb28131.
|
We attempted to determine whether endogenously generated NO and CO in retina could play a role in altering local sGC function in a site-specific manner (Fig. 2B
). In external limiting membrane (ELM), mAb3221-associated immunoreactivities were significantly enhanced by L-arginine. Treatment with N
-nitro-L-arginine methyl ester (L-NAME) which blocks endogenous NO synthesis, but not with D-NAME, significantly reduced the staining. Treatment with zinc protoporphyrin-IX (ZnPP), an inhibitor of CO-generating heme oxygenase (HO), but not with copper protoporphyrin-IX (CuPP), which does not block the enzyme, caused a substantial increase in the mAb3221 immunoreactivities, suggesting that housekeeping levels of CO limit the activity of sGC under normal conditions. To test further whether the mechanism whereby ZnPP administration increased the immunostaining depends on NO, we simultaneously administered the HO inhibitor with L-NAME. Such a concomitant inhibition of CO- and NO-producing enzymes completely attenuated the enhanced immunoreactivities induced by ZnPP alone.
Such inhibitory effects of L-NAME on the enhanced mAb3221 immunoreactivities induced by ZnPP varied among different retinal layers. ELM exhibited a significant reduction of the mAb3221 immunoreactivities when treated with ZnPP plus L-NAME vs. those with L-NAME alone. Similarly, a statistically insignificant but notable reduction of this kind was evident in optic fiber layer (OpFL). These results suggest that endogenous CO has the ability to up-regulate basal sGC activity in these layers of the retina. In contrast, immunoreactivities at INL and inner plexiform layer (IPL) under the same conditions were comparable to those in the group treated with L-NAME alone, suggesting that, in these regions, housekeeping levels of sGC activation occurs mainly through NO-dependent mechanisms but not through CO-dependent ones. The retina undergoing 24 h exposure to green light markedly increased HO-1, the inducible isozyme, in Müller’s glia cells (MGCs) and in retinal pigment epithelium (RPE). Under these circumstances, mAb3221 immunoreactivities were significantly reduced in INL and IPL but increased in ELM.
3. CO attenuates NO-elicited sGC activation in vitro
At a concentration of 10–30 µM, CO exhibited double-faced effects on the activation of purified bovine sGC induced by an NO donor S-nitroso-N-acetyl penicillamine (SNAP). In the absence or presence of SNAP at <100 nM, CO modestly activated sGC. By contrast, in the presence of SNAP at greater concentrations, application of CO modestly but significantly attenuated sGC activation. These results suggest that CO serves as a partial antagonist for sGC that limits the dynamic range of the NO-dependent activation of the enzyme.
4. Microtopographic relationship between gaseous monoxide-generating systems and sGC in the retina
We then specified cell types responsible for expression of NO synthases, HO, and the cyclase. Neuronal NO synthase (nNOS) was sparsely localized in a small population of amacrine cells whereas endothelial NO synthase (eNOS) was found on the endothelia from relatively large microvessels near the vitreous side of retina. HO-2 was expressed abundantly in MGCs. Sustained exposure to visible light not only resulted in a further elevation of CO by stress-inducible HO-1 in MGCs but caused a notable induction of HO-1 in RPE. sGC, the sensor protein for NO and CO, was found in MGCs and in on-type bipolar cells. Anatomical and functional relationship between the gas-producing systems and sGC are summarized in Fig. 3
. CO generated in MGC appears to modulate the sensitivity of sGC function. The ways in which CO affects the sGC function appear to be autocrine and paracrine in nature, inhibiting activities of sGC located in MGC and those in the on-type bipolar cells. These data could imply housekeeping roles for CO in maintaining neuroglial homeostasis in the retina.
|
CONCLUSIONS AND SIGNIFICANCE
Our findings provide the first direct evidence that the sGC activity is controlled by locally produced NO and CO in vivo. One striking implication of our results is that mechanisms for sGC regulation by these gases appear to be executed not uniformly, but site-specifically. Under conditions where housekeeping levels of CO were suppressed by ZnPP, all retinal cell layers homogeneously exhibited enhancement of NO-dependent activation of sGC. On the other hand, under NO-suppressing conditions, eliminating endogenous CO abrogated the basal sGC activation in a layer-specific manner at the ELM and OpFL, suggesting roles for the gas in local maintenance of housekeeping levels of cGMP in these layers.
Direct detection of the sGC function with mAb3221 led us to propose a novel hypothesis to explain the long-standing controversy concerning synergistic or antagonistic interactions of the two diatomic gases to regulate sGC activities in vivo. The effect of CO on modulating sGC activity is not static but dynamic in that low tissue NO makes CO a stimulatory modulator of sGC whereas high tissue NO makes CO an inhibitory one. As seen in Fig. 1
, coadministration of ZnPP and L-NAME resulted in heterogeneous responses of the mAb3221 immunoreactivities. Basal sGC activation by endogenous CO was notable in ELM and OpFL, but not in INL and IPL. One possible explanation for such heterogeneous responses is the difference in local NO availability (Fig. 3)
. Although OpFL is abundant in eNOS expression, the local NO availability should not be so high. This is because the high oxygen tension (PO2) in this layer makes the half-life of NO short. ELM, the layer distal from the NO-generating system, and OpFL could share a similar environment in which free NO is limited. On the other hand, NO could be ample in INL and IPL, since these layers are proximal to NO-generating enzymes (see model in Fig. 3
). Our results in vitro that the effect of CO on the NO-mediated activation of purified sGC is also double-faced support this(see Finding 3).
Although physiologic roles of CO in neural tissues are unknown, retina could benefit from this nonradical sGC agonist to maintain housekeeping cGMP without causing potential degradation of retinoids through the radical agonist such as NO. Such a way to use CO appears to be the case in stress-induced spermatogenic control or in relaxation of hepatic stellate cells for increasing sinusoidal blood flow where NO-breakable DNA or vitamin A is heavily stored, respectively. The current findings enable a new understanding of the link between the two gases and the sGC function in vivo.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0359fje; to cite this article, use FASEB J. (January 22, 2003) 10.1096/fj.02-0359fje 
2 The first and second authors equally contributed to the present study.
This article has been cited by other articles:
|
|
 |
|
  N. K. Idriss, A. D. Blann, and G. Y.H. Lip Hemoxygenase-1 in Cardiovascular Disease J. Am. Coll. Cardiol., September 16, 2008; 52(12): 971 - 978. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Huang, F. Shi, T. Lei, Y. Song, C. L. Hughes, and G. Liu Effect of the Isoflavone Genistein Against Galactose-Induced Cataracts in Rats Experimental Biology and Medicine, January 1, 2007; 232(1): 118 - 125. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ishikawa, M. Kajimura, T. Adachi, K. Maruyama, N. Makino, N. Goda, T. Yamaguchi, E. Sekizuka, and M. Suematsu Carbon Monoxide From Heme Oxygenase-2 Is a Tonic Regulator Against NO-Dependent Vasodilatation in the Adult Rat Cerebral Microcirculation Circ. Res., December 9, 2005; 97(12): e104 - e114. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Wu and R. Wang Carbon Monoxide: Endogenous Production, Physiological Functions, and Pharmacological Applications Pharmacol. Rev., December 1, 2005; 57(4): 585 - 630. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Morita Heme Oxygenase and Atherosclerosis Arterioscler. Thromb. Vasc. Biol., September 1, 2005; 25(9): 1786 - 1795. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Fujimoto, M. Ohno, S. Ayabe, H. Kobayashi, N. Ishizaka, H. Kimura, K.-i. Yoshida, and R. Nagai Carbon Monoxide Protects Against Cardiac Ischemia--Reperfusion Injury In Vivo via MAPK and Akt--eNOS Pathways Arterioscler. Thromb. Vasc. Biol., October 1, 2004; 24(10): 1848 - 1853. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
= a decrease compared with the vehicle-treated control, # = a decrease compared with L-NAME alone. The location of each domain is indicated at the left. ELM, external limiting membrane; INL, inner nuclear layer; IPL, inner plexiform layer; OpFL, optic fiber layer. Values are mean ± SD.