A mechanism of retinal protection from light-induced degeneration by hydrogen sulfide

Abstract

Since our initial demonstrations that hydrogen sulfide (H₂S) may function as a neuromodulator in the brain and a smooth muscle relaxant in the vascular system, accumulating evidence shows that H₂S may function as a signaling molecule. We and others also found that H₂S has a cytoprotective effect. Because H₂S is well-known toxic gas, a cytoprotective role has been overlooked. H₂S protects neurons from oxidative stress. It also protects cardiac muscle from ischemia-reperfusion injury. The finding led to the application of H₂S to the bypass surgery patients in Phase II clinical trial. Cystathionine β–synthase (CBS) and cystathionine γ–lyase (CSE) are well known as H₂S-producing enzymes. We recently demonstrated that the other H₂S-producing enzyme, 3-mercaptopyruvate sulfurtransferase (3MST) along with cysteine aminotransferase (CAT) is localized to neurons in the brain and to the vascular endothelium. However, the regulation of H₂S production by 3MST/CAT pathway had not been well understood. The present study shows that H₂S production by 3MST/CAT pathway is regulated by Ca²⁺ and that H₂S protects retinal photoreceptor cells from light induced degeneration by suppressing excessive Ca²⁺ influx caused by intense light.

Keywords: :

• 3MST
• Ca²⁺
• CAT
• cytoprotective
• H₂S
• retina

This article refers to:

We demonstrated that CBS is expressed in the brain and can produce H₂S, which facilitates the induction of hippocampal long-term potentiation (LTP), a synaptic model of memory, by enhancing the activity of NMDA receptors.¹ H₂S also induces Ca²⁺ influx and Ca²⁺ waves in astrocytes.^2,3 Another H₂S-producing enzyme, CSE, was found in the thoracic aorta, the ileum and the portal vein and H₂S relaxes these tissues.⁴ Based on these observations we proposed that H₂S may function as a neuromodulator and a smooth muscle relaxant. Subsequently, H₂S was found to activate ATP-dependent K⁺ channels to relax vascular smooth muscle.⁵ In addition to the function as a signaling molecule, H₂S also has a role as a cytoprotectant.^6-10 It protects neurons from oxidative stress by reinstating the levels of glutathione, an intracellular major antioxidant.^6-8 It also protects cardiac muscle from ischemia-reperfusion injury by preserving the mitochondrial function.¹⁰

In the brain CBS is localized to astrocytes,^11,12 a type of glia, while 3MST is localized to neurons.¹³ 3MST and CAT localized to vascular endothelium also produce H₂S that may regulate vascular tone.¹⁴ 3MST produces H₂S from 3-mercaptopyruvate, which is produced by CAT from cysteine and α–ketoglutarate. H₂S production by 3MST/CAT pathway requires a reducing substance, such as dithiothreitol (DTT). However, the corresponding endogenous reducing substance has not been identified. We recently demonstrated that thioredoxin and dihydrolipoic acid (DHLA) are endogenous reducing substances for 3MST to produce H₂S.¹⁵

3MST along with CAT is also localized to retinal neurons, and H₂S production by the enzymes is regulated by Ca²⁺.¹⁶ In the absence of Ca²⁺ the production is the maximum and is decreased by Ca²⁺ in a concentration-dependent manner. There is no change in the activity of 3MST/CAT pathway to produce H₂S in the presence or absence of calmodulin or a calmodulin inhibitor, W-7, suggesting that calmodulin is not involved in the regulation on the pathway by Ca²⁺ (Fig. 1).¹⁶

The center-surround organization is one of the most important characteristics in the retinal neurons. The negative feedback from horizontal cells to photoreceptor cells plays a key role in the center-surround organization. When retinal photoreceptor cells are exposed to light, the intracellular concentrations of Ca²⁺ are decreased to 10 nM that activates 3MST/CAT pathway to produce H₂S. In darkness Ca²⁺ concentrations are increased to 600 nM that cause the cessation of H₂S production. H₂S, in turn, suppresses voltage-gated L-type Ca²⁺ channels (VGCC) in photoreceptor cells by activating vacuolar-type H⁺-ATPase (V-ATPase) in horizontal cells, leading to maintaining intracellular Ca²⁺ in photoreceptor cells in low levels (Fig. 1).¹⁶

The retina is susceptible to oxidative stress because of its high consumption of oxygen and daily exposure to light. Excessive light exposure leads to photoreceptor degeneration whose death is an irreversible injury caused by reactive oxygen species and elevated intracellular concentrations of Ca²⁺. The regulation of Ca²⁺ by endogenous H₂S may fail by the excessive levels of light, and the photoreceptor cell degeneration occurs. Even under such conditions the administration of a donor of H₂S suppresses photoreceptor degeneration. The increased number of TUNEL- and 8-hydroxy-2’-deoxyguanosine positive cells by intense light was decreased by administration of H₂S.¹⁶ The enhancement of 3MST/CAT pathway or the administration of H₂S may have clinical benefit for diseases with retinal cell degeneration.

Figure 1. When retinal photoreceptor cells are exposed to light, cGMP-gated channels are closed and the cell membrane is hyperpolarized. The intracellular concentrations of Ca²⁺ in photoreceptor cells are decreased to approximately 10 nM, which activates 3MST/CAT to produce H₂S. H₂S activates vacuolar-type H⁺-ATPase in horizontal cells to released H⁺ that suppresses the activity of voltage gated Ca²⁺ channels in photoreceptor cells. By this mechanism H₂S maintains intracellular Ca²⁺ in low levels. Excessive light exposure leads to photoreceptor degeneration caused by reactive oxygen species and elevated intracellular concentrations of Ca²⁺. The regulation of Ca²⁺ by endogenous H₂S may fail by the excessive levels of light, and the photoreceptor cell degeneration occurs. Even under such conditions the enhancement of 3MST/CAT pathway or the administration of H₂S may have clinical benefit for diseases with retinal cell degeneration.

Abbreviations:
H2S	=	hydrogen sulfide
CBS	=	cystathionine β–synthase
CSE	=	cystathionine γ–lyase
3MST	=	3-mercaptopyruvate sulfurtransferase
CAT	=	cysteine aminotransferase
LTP	=	long-term potentiation
DTT	=	dithiothreitol
DHLA	=	dihydrolipoic acid
VGCC	=	voltage-gated Ca2+
V-ATPase	=	vacuolar-type H+-ATPase

Acknowledgments

This work was supported by a grant from National Institute of Neuroscience and by KAKENHI (23659089) from Grant-in-Aid for Challenging Exploratory Research to H.K., by KAKENHI (23790316) from Grant-in-Aid for Young Scientists (B) to Y.M.