Effects of atrial natriuretic factor, nitroprusside and acetylcholine on cGMP immunostaining in the isolated perfused rat kidney.

In the present study the localization of the cGMP production in response to the vasodilators acetylcholine (ACh) and sodium nitroprusside (SNP) and to atrial natriuretic factor (ANF) was studied in the isolated perfused rat kidney using cGMP immunocytochemistry. After ACh (0.3 microM) infusion increased cGMP immunoreactivity was found in kidney interlobar and segmental arteries and in glomeruli. SNP (1 microM) and ANF (0.01 microM) elevated cGMP staining in the same elements of the kidney as ACh. In the glomeruli ACh and SNP stimulated cGMP production in mesangial cells whereas ANF stimulated cGMP production in mesangial cells whereas ANF stimulated cGMP production in epithelial cells (podocytes). However, SNP at higher doses (10 microM) stimulated cGMP production not only in glomeruli, but also in interstitial cells throughout the cortex. In addition SNP and ANF increased cGMP production in the medulla.

Immunocytochemistry of cGMP in the Cerebellum of the Immature, Adult, and Aged Rat: the Involvement of Nitric Oxide. A Micropharmacological Study.

In this study we describe the localization of formaldehyde-fixed cGMP-immunoreactivity (cGMP-IR) in rat cerebellar tissue slices incubated in vitro. In the absence of phosphodiesterase inhibition, cGMP-immunofluorescence was of low intensity in tissue slices prepared from immature cerebella. Addition of isobutylmethylxanthine (IBMX) to the incubation medium resulted in the appearance of cGMP-IR in clusters of astrocytes in the internal granular layer. Addition of N-methyl-d-aspartate (NMDA), kainic acid, atrial natriuretic factor (ANF), or sodium nitroprusside (SNP) gave an intense cGMP-IR in Bergmann fibres, Bergmann cell bodies, and astrocytes in the internal granular layer. Astrocytes in the white matter showed cGMP-IR after incubation of the slice in the presence of ANF or nitroprusside, but not after NMDA or kainic acid. In addition, after SNP stimulation of cGMP production, cGMP-IR was found in fibres which were not positive for glial fibrillary acidic protein (GFAP). In the adult cerebellar slice, intense basal cGMP-immunostaining was observed in Bergmann fibres, Bergmann cell bodies, and astrocytes in the granular layer. No cGMP-IR was observed in Purkinje cells. Stimulation of the cGMP-content in the glial structures by NMDA, ANF, or SNP, was suggested by the immunocytochemical results. However, when measured biochemically, only the effect of SNP was statistically significant, and immunocytochemistry showed that SNP clearly stimulated cGMP synthesis in neuronal cell structures. In the cerebellum of the aged rat a reduced cGMP-IR was found compared to the adult, in the same structures which showed cGMP-IR in the adult. Basal cGMP-immunostaining was reduced in the presence of haemoglobin, methylene blue, by inhibiting nitric oxide synthesis with NG-monomethyl-l-arginine (NGMAr), or by depletion of external Ca2+. Also the stimulatory effect of NMDA and of ANF (partly) on the cGMP-IR was inhibited by these compounds. cGMP-IR after stimulation of guanylate cyclase by SNP was reduced by the concomitant presence of haemoglobin or methylene blue, but not by NGMAr, or by omission of Ca2+. Our results point to an important role for cGMP in the functioning of glial tissue in the cerebellum and also suggest a role for nitric oxide as an intercellular mediator in the functioning of glutamate and ANF in the cerebellum.

cGMP immunocytochemistry in aorta, kidney, retina and brain tissues of the rat after perfusion with nitroprusside.

The distribution of cyclic guanosine 3',5' monophosphate (cGMP) producing cells in various organs of the rat were studied immunocytochemically using antibodies raised against formaldehyde-fixed cGMP. Sodium nitroprusside (SNP), a direct activator of guanylate cyclase and vasodilator, was used to enhance cGMP levels. In order to reach all organs optimally, whole body perfusion was performed using a modified Krebs-Ringer buffer at 37 degrees C, aerated with 5% CO2/95% O2, also containing isobutyl methyl xanthine (IBMX); a phosphodiesterase inhibitor. After 15-min pre-perfusion, SNP was added to the perfusate, followed by fast fixation with ice-cold 4% paraformaldehyde-phosphate buffer. After vehicle perfusion, only the retina showed cGMP immunoreactivity in the photoreceptor and ganglion layer, while other organs lacked cGMP immunoreactivity. After 15-min perfusion with SNP (10 microM), enhanced cGMP immunostaining was seen in smooth muscles of the aorta, amacrine-like cells in the retina, glomeruli of the kidney cortex, blood vessels in the dura mater, as well as cells in the pineal and in the median eminence. The results indicate that the distribution and the reactivity of cGMP producing cells, situated outside the blood brain barrier, can be studied by immunocytochemistry after pharmacological manipulations of the intact tissue with a nitrovasodilator using whole body perfusion.