cGMP Imaging in Brain Slices Reveals Brain Region-Specific Activity of NO-Sensitive Guanylyl Cyclases (NO-GCs) and NO-GC Stimulators.

Impaired NO-cGMP signaling has been linked to several neurological disorders. NO-sensitive guanylyl cyclase (NO-GC), of which two isoforms-NO-GC1 and NO-GC2-are known, represents a promising drug target to increase cGMP in the brain. Drug-like small molecules have been discovered that work synergistically with NO to stimulate NO-GC activity. However, the effects of NO-GC stimulators in the brain are not well understood. In the present study, we used Förster/fluorescence resonance energy transfer (FRET)-based real-time imaging of cGMP in acute brain slices and primary neurons of cGMP sensor mice to comparatively assess the activity of two structurally different NO-GC stimulators, IWP-051 and BAY 41-2272, in the cerebellum, striatum and hippocampus. BAY 41-2272 potentiated an elevation of cGMP induced by the NO donor DEA/NO in all tested brain regions. Interestingly, IWP-051 potentiated DEA/NO-induced cGMP increases in the cerebellum and striatum, but not in the hippocampal CA1 area or primary hippocampal neurons. The brain-region-selective activity of IWP-051 suggested that it might act in a NO-GC isoform-selective manner. Results of mRNA in situ hybridization indicated that the cerebellum and striatum express NO-GC1 and NO-GC2, while the hippocampal CA1 area expresses mainly NO-GC2. IWP-051-potentiated DEA/NO-induced cGMP signals in the striatum of NO-GC2 knockout mice but was ineffective in the striatum of NO-GC1 knockout mice. These results indicate that IWP-051 preferentially stimulates NO-GC1 signaling in brain slices. Interestingly, no evidence for an isoform-specific effect of IWP-051 was observed when the cGMP-forming activity of whole brain homogenates was measured. This apparent discrepancy suggests that the method and conditions of cGMP measurement can influence results with NO-GC stimulators. Nevertheless, it is clear that NO-GC stimulators enhance cGMP signaling in the brain and should be further developed for the treatment of neurological diseases.

Real-Time Imaging Reveals Augmentation of Glutamate-Induced Ca2+ Transients by the NO-cGMP Pathway in Cerebellar Granule Neurons.

Dysfunctions of NO-cGMP signaling have been implicated in various neurological disorders. We have studied the potential crosstalk of cGMP and Ca2+ signaling in cerebellar granule neurons (CGNs) by simultaneous real-time imaging of these second messengers in living cells. The NO donor DEA/NO evoked cGMP signals in the granule cell layer of acute cerebellar slices from transgenic mice expressing a cGMP sensor protein. cGMP and Ca2+ dynamics were visualized in individual CGNs in primary cultures prepared from 7-day-old cGMP sensor mice. DEA/NO increased the intracellular cGMP concentration and augmented glutamate-induced Ca2+ transients. These effects of DEA/NO were absent in CGNs isolated from knockout mice lacking NO-sensitive guanylyl cyclase. Furthermore, application of the cGMP analogues 8-Br-cGMP and 8-pCPT-cGMP, which activate cGMP effector proteins such as cyclic nucleotide-gated cation channels and cGMP-dependent protein kinases (cGKs), also potentiated glutamate-induced Ca2+ transients. Western blot analysis failed to detect cGK type I or II in our primary CGNs. The addition of phosphodiesterase (PDE) inhibitors during cGMP imaging showed that CGNs degrade cGMP mainly via Zaprinast-sensitive PDEs, most likely PDE5 and/or PDE10, but not via PDE1, 2, or 3. In sum, these data delineate a cGK-independent NO-cGMP signaling cascade that increases glutamate-induced Ca2+ signaling in CGNs. This cGMP⁻Ca2+ crosstalk likely affects neurotransmitter-stimulated functions of CGNs.

Npom-Protected NONOate Enables Light-Triggered NO/cGMP Signalling in Primary Vascular Smooth Muscle Cells.

Diazeniumdiolates (NONOates) are a class of nitric-oxide-releasing substances widely used in studies of NO/cGMP signalling. Because spatiotemporal control is highly desirable for such purposes, we have synthesised a new Npom-caged pyrrolidine NONOate. A kinetic analysis together with a Griess assay showed the photodependent release of NO with high quantum yield (UV light). In primary vascular smooth muscle cells (VSMCs), our compound was reliably able to induce fast increases in cGMP, as measured with a genetically encoded FRET-based cGMP sensor and further validated by the phosphorylation of the downstream target vasodilator-stimulated phosphoprotein (VASP). Thanks to their facile synthesis, good decaging kinetics and capability to activate cGMP signalling in a fast and efficient manner, Npom-protected NONOates allow for improved spatiotemporal control of NO/cGMP signalling.