Jan de Vente

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.

On the stimulation of soluble and particulate guanylate cyclase in the rat brain and the involvement of nitric oxide as studied by cGMP immunocytochemistry

The localization of the particulate and soluble guanylate cyclase in the rat brain was studied using cGMP-immunocytochemistry. The cGMP was fixed to tissue protein using a formaldehyde fixative, and an antibody against cGMP was used which was raised against a cGMP-formaldehyde-thyroglobulin conjugate. We used the atrial natriuretic factor (ANF) as a model compound to stimulate the particulate enzyme and sodium nitroprusside (SNP) to stimulate the soluble enzyme. Sequential immunostaining for cGMP and glial fibrillary acidic protein (GFAP) showed that the great majority of the ANF-responsive, cGMP-producing cells were astrocytes. These ANF-responsive cells were found in discrete parts of the CNS; not all astrocytes in these regions were responsive to ANF. SNP stimulated cGMP in abundantly present neuronal fibres throughout the CNS; few neuronal cell bodies showed increased cGMP production after SNP. Moreover, SNP also raised cGMP in astrocytes, however, not all astrocytes showed the response to SNP. These results suggest that cells might be present in the CNS which contain both the soluble and the particulate guanylate cyclase. It was demonstrated that in the immature cerebellum, the cGMP was raised in glial structures in response to N-methyl-D-aspartate (NMDA), ANF, SNP, and kainic acid. The response to NMDA and kainic acid was sensitive to inhibition of the nitric oxide synthesis from L-arginine by NG-methyl-L-arginine. Surprisingly the response to ANF localized in the molecular layer and the granular layer was also sensitive to inhibition by NG-methyl-L-arginine, whereas the response to ANF in the deep nuclei was not. A small depolarization induced by 10 to 20 mmol/l K+ induced an increase in cGMP in chopped hippocampus tissue which showed a biphasic temporal characteristic. The initial, fast (30 sec), peak was shown to be localized in varicose fibres throughout the hippocampus, whereas the slower response (10 min) was localized in astrocytes. These studies demonstrate that the different enzymes which synthesize cGMP are differently localized. However, there is also a time dependency in the activation of the guanylate cyclases, which becomes apparent in different structures at different times. The possible role of cGMP as a regulator of ion homeostase is discussed.

Chapter 5 Vagal efferent projections: viscerotopy, neurochemistry and effects of vagotomy

Publisher Summary This chapter focuses on the viscerotopic organization of the cells of origin of the vagus nerve with emphasis on subnuclear organization in the vagus nerve (DMV) and nucleus ambiguus (AMB). Aspects of the neurochemistry and afferent inputs of the DMV and AMB are discussed in terms of the identified viscerotopic organization. The effects of vagus nerve lesions will be assessed in terms of subnuclear differences as they relate to the connectivity connectivity and neurochemistry of the DMV and AMB. The chapter shows that the viscerotopic representation in the two nuclei comports with topographic patterns of specific neurochemicals and that vagal lesions cause differential responses in chemically and functionally distinct groups of neurons in the DMV and AMB. Neurochemical changes following vagal lesions are selective for specific viscera and are not uniform among functional subdivisions in the two nuclei. The viscerotopic organization of vagal motoneurons, thus, may also be reflected in differences in trophic support and pathophysiology.

cGMP-Producing, Atrial Natriuretic Factor-Responding Cells in the Rat Brain.

Using an in vitro incubation method, we stimulated cGMP production in rat brain slices by rat ANF‐(103–126). The localization of the cells responding to this ANF stimulation with an increase in cGMP production was studied by cGMP immunocytochemistry. ANF‐responding cells were found in specific loci throughout the central nervous system of the rat. Regions containing the highest number of these cells were: the olfactory bulb, the lateral septum, the bed nucleus of the accessory olfactory tract, the mediobasal amygdala, the central grey area, the medial vestibular nucleus, and the nucleus of the solitary tract. Scattered ANF‐responding, cGMP‐immunoreactive cells were found in the hippocampus, the cingulate cortex, the ventral pallidum, the medial preoptic area, and the endopeduncular nucleus. ANF‐responding cells in these areas had the same morphology, that is, multipolar with numerous processes. The nature of these ANF‐responding cells was studied by sequential staining with an antiserum against glial fibrillary acidic protein (GFAP). In the hippocampus it was demonstrated that all ANF‐responding cells are astroglial cells. However, not all astroglial cells in this area showed a cGMP response, demonstrating a regional heterogeneity. ANF‐responding cells, having the appearance of neuronal cell bodies, could be found in the subfornical organ, and the hypothalamic paraventricular nucleus. Fibres producing cGMP immunoreactivity in response to ANF were found in the median preoptic nucleus, the medial preoptic area, and the dorsal hypothalamus.

Atriopeptins and Escherichia coli enterotoxin STa have different sites of action in mammalian intestine

Studies with Escherichia coli-induced heat-stable enterotoxin (STa), an activator of intestinal particulate guanylate cyclase, have established an independent role for cyclic guanosine monophosphate (cGMP) as an intracellular mediator of intestinal salt and water secretion. The present study addressed whether atriopeptins (APs), known activators of particulate guanylate cyclase in other tissues, function as physiological agonists for cGMP-linked Cl- secretion in intestine. APs, in contrast to STa, caused no or only minor changes in cGMP levels in freshly isolated rat intestinal villus and crypt cells and in cultured human colon carcinoma cell lines (HT29glc-, CaCo-2, and T84). Conversely, APs, but not STa, induced a large increase in intracellular cGMP levels in the undifferentiated small intestinal cell lines IEC-6, IEC-18, and INT407. Addition of AP II (atrial natriuretic peptide fragment 5-27) to stripped mucosa of rat proximal colon in Ussing chambers caused a transient increase in the transepithelial potential difference (PD), which most likely represents an increase in Cl- secretion. In contrast, a sustained increase in PD was observed in response to STa or 8Br-cGMP. The AP II-provoked increase in PD was blocked by the neurotoxin tetrodotoxin. Immunohistochemical detection of cGMP in this tissue provided evidence for a different localization pattern of cells responding with an increase in cGMP levels to STa (colonocytes and goblet cells) or AP (specific cells in the submucosa) in rat proximal colon. This indicates that APs, unlike STa, do not directly stimulate the colonic epithelial cells but possibly provoke Cl- secretion by release of a neurotransmitter in the submucosa.

The NO-cGMP Pathway in Neonatal Rat Dorsal Horn

Incubation of slices of neonatal rat spinal cord with nitric oxide donor compounds produced marked elevations in cyclic guanosine 3′,5’monophosphate (cGMP) levels. The excitatory amino acid receptor agonists N‐methyl‐d‐aspartate (NMDA) and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA) produced smaller increases, which were blocked by the nitric oxide synthase (NOS) inhibitor Ml‐NG‐nitroarginine (NOArg), indicating that these cGMP responses were mediated by nitric oxide. Immunocytochemistry revealed that, in response to NMDA, cGMP accumulated in a population of small cells and neuropil in laminae II and III of the dorsal horn. This area was also shown, by reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry, to contain NOS. These observations suggest that, in the rat spinal cord, NMDA receptor activation is linked to the formation of NO and, hence, of cGMP. This pathway is located selectively in the superficial dorsal horn, consistent with a role in the processing of nociceptive signals.

Levels of basic fibroblast growth factor, glutamine synthetase, and interleukin-6 in subretinal fluid from patients with retinal detachment.

PURPOSE To investigate the presence of basic fibroblast growth factor, glutamine synthetase activity, and interleukin-6 in subretinal fluid from patients with retinal detachment. METHODS In a prospective study we measured basic fibroblast growth factor, glutamine synthetase activity, interleukin-6, and total protein in subretinal fluid samples from 96 eyes from 94 consecutive patients with a retinal detachment corrected by a conventional scleral buckling operation in our clinical practice. As controls, vitreous fluid samples from eyes with a macular hole (n = 6) or pucker (n = 11) were used. Laboratory data of the patient group were compared with the control group and correlated with various clinical data. RESULTS Levels (median, range) of basic fibroblast growth factor, glutamine synthetase activity, interleukin-6, and total protein were significantly higher in patients than in controls (P <.0001). An increased level of glutamine synthetase and total protein correlated with a longer duration of the retinal detachment (r =.4, P =.002, and r =.34, P =.001, respectively). Interleukin-6 and basic fibroblast growth factor levels did not correlate with the duration of the detachment. After multivariate logistic regression analysis, no significant relation was found between any of the tested subretinal proteins and a low visual outcome or redetachment. CONCLUSIONS We found increased levels of basic fibroblast growth factor and glutamine synthetase in subretinal fluid from patients with retinal detachment. Basic fibroblast growth factor and glutamine synthetase may play a role in the pathogenesis and recovery after retinal detachment. The questions of whether the increased levels of basic fibroblast growth factor and glutamine synthetase result from leakage of dying glia cells (including Müller cells) and neurons and if basic fibroblast growth factor is actively produced to protect the photoreceptor cells need further research.

Sustained pharmacological inhibition of nitric oxide synthase does not affect the survival of intrastriatal rat fetal mesencephalic transplants

The objective of the present study was to investigate the potential role of the free radical nitric oxide (NO) in the development of fetal rat mesencephalic neurons grafted in a 6-hydroxydopamine (6-OHDA) lesioned rat model of Parkinsons disease. First, using nitric oxide synthase (NOS)-immunocytochemistry and reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, we investigated the presence of the neuronal isoform of NOS (nNOS) in intrastriatal mesencephalic grafts. During the course of the experiment (16 weeks) an increase in the staining intensity and the number of nNOS/NADPH-d positive cells within the grafts was observed, as well as a gradual maturation of dopaminergic neurons. In addition, within both the host striatal and grafted mesencephalic tissue, a NO-dependent accumulation of cyclic guanosine monophosphate (cGMP) was detected, indicating the presence of guanylate cyclase, i.e., the target-enzyme for NO. Secondly, to determine the impact of NO on the survival of grafted dopaminergic neurons, 6-OHDA lesioned rats received mesencephalic grafts and were subsequently treated with the competitive NOS-inhibitor Nomega-nitro-l-arginine methylester (l-NAME). After chronic treatment for 4 weeks, tyrosine hydroxylase immunocytochemistry revealed no apparent differences between the survival of grafted dopaminergic neurons in control- or l-NAME treated animals, respectively. As the maturation of grafted dopaminergic neurons coincides with a gradual increase in the expression of nNOS within the graft and since dopaminergic cell numbers are not changed upon administration of l-NAME, it is concluded that endogenously produced and potentially toxic NO does not affect the survival of grafted fetal dopaminergic neurons.

Development and Application of Antibodies to Primary (DA, L-DOPA) and Secondary (cGMP) Messengers: A technical report

In the last decade antibodies have been raised against a large number of amine- and amino-acid neurotransmitters and candidates. These antibodies have been used in tracing neuronal connections in the central nervous system and locating the cell bodies and varicose fibers synthesizing these molecules. There are a number of specific difficulties inherent to the use of antibodies against these small, water soluble, molecules. To obtain these antibodies, the small haptens have to be coupled to a larger protein molecule to acquire an immunogenic conjugate. Two fixatives, formaldehyde and glutaraldehyde, have been used in most cases, and highly specific antibodies have been raised against conjugates prepared with both fixatives. In visualizing a molecule cross-linked to tissue constituents the fixative applied is probably the most important factor in combination with the antibody used. Ideally, the procedure for cross-linking small molecules to tissue constituents should be strictly identical with the procedure to prepare the immunogen against which the antibody is raised. The fixative might alter the structure of the hapten and the antibody against this molecule might not recognize the free, unmodified hapten in solution. Furthermore, in the cases of formaldehyde and glutaraldehyde, the fixatives are part of the antigenic determinant, together with an unknown part of the protein molecule to which the hapten is coupled. One more problem is the lack of knowledge about the amount of hapten actually retained by the tissue after fixation. The specificity of an antibody raised against an antigen is the most important factor in determining the usefulness in all its applications. A commonly applied testing criterion is the sensitivity and specificity of a particular antibody in a radioimmunoassay. However, in immunocytochemistry the situation is more complex because one has to deal with an antigen that might be modified in a decisive way by some chemical agent (the fixative) in a surrounding which is not defined (the cellular matrix).

No colocalization of immunoreactivities for VIP and neuronal NOS, and a differential relation to cGMP-immunoreactivity in bovine penile smooth muscle.

The distribution of immunoreactivity (IR) for the neuropeptide vasoactive intestinal polypeptide (VIP) and neuronal nitric oxide synthase (nNOS) in the bovine retractor penis muscle (RP) and penile artery (PA) was studied by using two different methods. The distribution of these immunoreactivities was also compared with that of the immunoreactivity for cyclic guanosine monophosphate (cGMP). In both tissues the nerve fibers and terminals immunoreactive for VIP had a distribution that was completely different from that of the nerve fibers and terminals immunoreactive for nNOS. This contrasts with the previous observations in penile smooth muscle of other species. In the RP, as well as in the PA, many of the VIP-IR fibers were also immunoreactive for neurofilaments (NF), whereas the nNOS-IR fibers were consistently devoid of NF-IR. Stimulation with sodium nitroprusside, a nitric oxide donor, considerably increased cGMP-IR in the smooth muscle cells in both RP and PA, and in several nerve fibers in PA. Many of these cGMP-IR nerve fibers exhibited nNOS-IR, whereas none of them was immunoreactive for VIP. Our results suggest that the degree of coexistence of VIP-IR and nNOS-IR in the nerve fibers and terminals innervating penile smooth muscle show wide species differences. They also suggest that the mechanisms by which VIP could be involved in neurogenic penile erection may vary between species.