Distribution of cGMP-dependent protein kinase type I and its isoforms in the mouse brain and retina

doi:10.1016/j.neuroscience.2005.06.051

Neuroscience

Volume 135, Issue 3, 2005, Pages 863-868

https://doi.org/10.1016/j.neuroscience.2005.06.051 Get rights and content

Abstract

Nitric oxide (NO) modulates a variety of processes in the mammalian brain, but the mechanisms of neuronal NO signaling are poorly understood. In the periphery, many effects of NO are mediated via the generation of the second messenger cyclic guanosine-3′,5′-monophosphate (cGMP) and activation of the cGMP-dependent protein kinase type I (cGKI). However, previous studies suggested that the expression of cGKI in the nervous system is rather restricted, thus, questioning the functional significance of the cGMP/cGKI pathway as a mediator of NO signaling in the brain. Here we have performed a detailed immunohistochemical study to elucidate the distribution of cGKI in the CNS and eye of the mouse. Expression of cGKI protein was detected not only in the previously described areas (cerebellum, hippocampus, dorsomedial hypothalamus) but also in a number of additional regions, such as medulla, subcommissural organ, cerebral cortex, amygdala, habenulae, various hypothalamic regions, olfactory bulb, pituitary gland, and retina. Immunoblotting with isoform-specific antibodies indicated that the cGKIα isoform is prominent in the cerebellum and medulla, whereas the cGKIβ isoform is predominant in the cortex, hippocampus, hypothalamus, and olfactory bulb. Similar levels of the isoforms were detected in the pituitary gland and eye. Thus, it appears that distinct brain regions express distinct cGKI isoforms that signal via distinct pathways. Together, these results improve our understanding of the cellular and molecular mechanisms of NO/cGMP/cGKI signaling and indicate that the distribution and functional relevance of this pathway in the mammalian brain is broader than previously thought.

Section snippets

Experimental animals

For experiments seven male and five female wild-type mice and two male and two female cGKI knockout mice (Wegener et al., 2002) were used. Animals were one- to three-month-old and had a 129/Sv, C57BL/6 or mixed 129Sv/C57BL6 genetic background. Results were independent of gender, age or genetic background of the analyzed mice. This study conformed to the German animal protection law and had been approved by the committee on animal care and welfare of the local government.

Western blot analysis

Mouse tissues were

Immunohistochemical detection of cGKI in the mouse CNS and retina

For immunohistochemistry, a rabbit polyclonal antiserum detecting both cGKI isoforms, cGKIα and cGKIβ, was used (cGKI common antibody). The specificity of the cGKI common antibody was validated by using recombinant proteins as well as tissues from wild-type and cGKI knockout mice (Wegener et al 2002, Feil et al 2003, Kleppisch et al 2003, Geiselhoringer et al 2004; see also below). Importantly, immunohistochemical staining of sections revealed signals in wild-type but not cGKI knockout tissues (

Discussion

Based on immunohistochemistry and immunoblot analysis with cGKI-specific antibodies, this study shows that the cGKI is more widely distributed in the CNS than reported previously. In addition, this is the first analysis of the distribution and relative expression level of the cGKIα and cGKIβ proteins in the brain. The specificity of the cGKI antibodies was demonstrated by immunostaining of tissues from cGKI knockout mice. This strategy is presumably superior to conventional controls for

Conclusion

This study suggests that the distribution and functional role of cGKI in the mammalian CNS is broader than previously thought. Together with the localization of NO synthase and cGMP (De Vente et al., 1998), the new expression data for cGKI should be useful to draw an anatomical map of the NO/cGMP/cGKI pathway in the brain and to correlate it with functional data.

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft and the VolkswagenStiftung.

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¹: Present address: Interfakultäres Institut für Biochemie der Universität Tübingen, Hoppe-Seyler-Straβe 4, 72076 Tübingen, Germany.

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Molecular neuroscienceDistribution of cGMP-dependent protein kinase type I and its isoforms in the mouse brain and retina

Abstract

Section snippets

Experimental animals

Western blot analysis

Immunohistochemical detection of cGKI in the mouse CNS and retina

Discussion

Conclusion

Acknowledgments

Physiol Behav

Neuroscience

J Chem Neuroanat

FEBS Lett

J Biol Chem

Neurosci Lett

J Biol Chem

Presynaptic role of cGMP-dependent protein kinase during long-lasting potentiation

J Neurosci

Functional reconstitution of vascular smooth muscle cells with cGMP-dependent protein kinase I isoforms

Circ Res

Impairment of LTD and cerebellar learning by Purkinje cell-specific ablation of cGMP-dependent protein kinase I

J Cell Biol

Molecular neuroscience
Distribution of cGMP-dependent protein kinase type I and its isoforms in the mouse brain and retina