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Background: In phase 1 trials, the HIV-1 integrase strand transfer inhibitor cabotegravir (GSK1265744) was well tolerated, both alone, and in combination with the non-nucleoside reverse transcriptase inhibitor rilpivirine. We assessed cabotegravir plus rilpivirine, as a two-drug oral antiretroviral regimen, for the maintenance of viral suppression in antiretroviral-naive HIV-1-infected individuals., Methods: In the phase 2b Long-Acting antireTroviral Treatment Enabling (LATTE) trial, a multicentre study done in Canada and the USA, antiretroviral-naive HIV-1-infected adults (aged ≥18 years) were randomly allocated in a 1:1:1:1 ratio to oral cabotegravir 10 mg once a day, 30 mg once a day, 60 mg once a day, or oral efavirenz 600 mg once a day with dual nucleoside reverse transcriptase inhibitors (NRTIs) for 24 weeks of induction. Patients who were virologically suppressed by week 24 received a two-drug maintenance regimen consisting of their randomly allocated cabotegravir dose plus oral rilpivirine 25 mg or continued efavirenz plus NRTIs for an additional 72 weeks. Patients and investigators were masked to doses of cabotegravir received for 96 weeks, but not to the assignment of cabotegravir or efavirenz. The primary endpoint was the proportion of patients with fewer than 50 copies per mL of HIV-1 RNA (US Food and Drug Administration snapshot algorithm) at week 48. Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, NCT01641809., Findings: Of 243 patients randomly allocated and treated, 156 (86%) of 181 patients in the cabotegravir groups (52 [87%] of 60, 51 [85%] of 60, and 53 [87%] of 61 patients in the 10 mg, 30 mg, and 60 mg groups, respectively) and 46 (74%) of 62 in the efavirenz group had fewer than 50 copies per mL of HIV-1 RNA after induction therapy. After patients in the cabotegravir groups were changed over from dual NRTIs to rilpivirine at week 24, 149 (82%; 95% CI 77-88) patients in the cabotegravir groups (48 [80%; 70-90], 48 [80%; 70-90], and 53 [87%; 78-95] patients in the 10 mg, 30 mg, and 60 mg groups, respectively) versus 44 (71%; 60-82) in the efavirenz group were virologically suppressed at week 48, and 137 (76%; 69-82) receiving cabotegravir (41 [68%; 57-80], 45 [75%; 64-86], and 51 [84%; 74-93] patients in the 10 mg, 30 mg, and 60 mg groups, respectively) versus 39 (63%; 51-75) in the efavirenz group were virologically suppressed at week 96. Treatment-related adverse events were reported by 93 (51%) cabotegravir-treated patients (28 [47%], 32 [53%], and 33 [54%] patients in the 10 mg, 30 mg, and 60 mg groups, respectively) and 42 (68%) efavirenz-treated patients. Six (3%) patients in the cabotegravir groups (one [2%], one [2%], and four [7%] patients in the 10 mg, 30 mg, and 60 mg groups, respectively) withdrew because of treatment-emergent adverse events compared with nine (15%) in the efavirenz group., Interpretation: Cabotegravir plus dual NRTI therapy had potent antiviral activity during the induction phase. As a two-drug maintenance therapy, cabotegravir plus rilpivirine provided antiviral activity similar to efavirenz plus dual NRTIs until the end of week 96. Combined efficacy and safety results lend support to our selection of oral cabotegravir 30 mg once a day for further assessment. LATTE precedes studies of the assessment of longacting injectable formulations of both drugs as a two-drug regimen for the treatment of HIV-1 infection., Funding: ViiV Healthcare and Janssen Research and Development., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Renal insufficient patients suffer from a variety of complications as direct and indirect consequence of accumulation of retention solutes. Guanidinosuccinic acid (GSA) is an important probable uremic toxin, increased in plasma, urine, cerebrospinal fluid and brain of patients with uremia and supposed to play a role in the pathogenesis of some neurological symptoms. GSA, an NMDA-receptor agonist and GABA-receptor antagonist, is suggested to act as an excitotoxin and shown to be convulsive. The effect of hippocampal (i.h.) GSA injection on behavior and hippocampal volume in mice is presented here. In addition, hippocampal cGMP concentration after systemic injection of GSA was measured. The effect of co-application of NMDA-receptor antagonist CGP37849 with GSA was tested, in vivo, after hippocampal GSA injection and, in vitro, on GSA evoked currents in spinal cord neurons. A significant dose-dependent effect of i.h. injection of GSA on cognitive performance, activity and social exploratory behavior was observed. There was a protective effect of CGP37849 on GSA induced behavioral alterations. Volume of hippocampal cornu ammonis region decreased significantly and dose-dependently after GSA injection. Systemic GSA injection increased cGMP concentration in hippocampal formation. It can be concluded that GSA is an important neurotoxin. As GSA is increased in patients with uremia, it probably contributes to their neurological symptoms. Knowledge of neurotoxic effects and mechanisms of action of GSA and other uremic retention solutes could help in the development of more efficient treatment of uremic patients. Animal models like the 'GSA mouse model' are useful tools for research in this context.

The present study investigates the role of cGMP and cAMP on the memory performance in the object recognition task in rats. The analogue 8-Br-GMP or 8-Br-cAMP was administered bilaterally into the hippocampus (0, 1, 3 and 10 microg in 0.5 microl saline/site) immediately after the exposure to two identical objects. After 24 h, saline-treated animals spent equal times exploring a new and the familiar object demonstrating that they did not recognize the familiar one. However, a dose-dependent improvement in object recognition was found after injection of 8-Br-cGMP. In contrast, 8-Br-cAMP did not improve the memory performance at the doses tested. These results indicate that hippocampal cGMP but not cAMP is involved in early stages of consolidation of object memory.

The aim of our study was to investigate the effect of perinatal asphyxia on developmental apoptosis in the cervical and lumbar spinal cord in the neonatal rat. Perinatal asphyxia was induced by keeping pups at term in utero in a water bath at 37 degrees C for 20 min, followed by resuscitation. Effects of this treatment on developmental apoptosis were studied on postnatal days 2, 5 and 8 using terminal deoxynucleotidyl transferase (TdT)-dUTP-biotin nick end labelling (TUNEL) and caspase-3 staining. TUNEL positive cells were identified using double immunostaining. On postnatal day 2 an increase of 215% in TUNEL positive cells was detected (P=0.005) in laminae IV-VII of the lumbar spinal cord of rats which underwent perinatal asphyxia compared to controls. An increase of 55% compared to controls (P=0.03) was seen in laminae I-III of the lumbar spinal cord at postnatal day 8. TUNEL positive cells could be partly identified as microglia cells (ED1 positive) and oligodendrocytes (O4 positive). The effect of perinatal asphyxia on programmed cell death in the neonatal rat spinal cord was mainly observed in the intermediate zone and dorsal horn of the lumbar spinal cord. We conclude that perinatal asphyxia has a pronounced effect on the survival of cells in a specific region of the spinal cord and thus may have a profound effect on the development of motor networks.

The present study investigated the effects of two cyclic GMP-specific phosphodiesterase enzyme type 5 inhibitors, sildenafil and vardenafil, on the memory performance in the object recognition task. Both compounds were given per orally (1, 3 and 10 mg/kg sildenafil; 0.1, 0.3, 1 and 3 mg/kg vardenafil) immediately after the exposure to two identical objects. The memory for the objects was tested 24 h later. Vehicle-treated rats spent equal times exploring a new and the familiar object demonstrating that they did not remember the familiar one. However, sildenafil improved the object discrimination performance of the rats with a high discrimination performance at a dose of 3 mg/kg. Rats treated with vardenafil also showed an improved object discrimination performance. Compared with sildenafil, vardenafil appeared to be even more potent in this respect since it already produced a high discrimination performance at a dose of 0.3 mg/kg. The effects of both compounds on cyclic GMP and cyclic AMP accumulation were studied in rat hippocampal slices incubated in vitro. Cyclic GMP levels were increased after incubation with the highest concentration of 100 microM vardenafil (together with 0.1 mM sodium nitroprusside), although no changes in cyclic GMP levels were detected after incubation with different concentrations of sildenafil. Both compounds had no effect on cyclic AMP levels. Additional cyclic GMP immunocytochemistry showed that incubation with vardenafil (in the presence of sodium nitroprusside) resulted in a concentration-dependent staining of cyclic GMP. Staining was predominantly found in neuronal fibres in the hippocampal CA2/CA3 region. It was already detected at a concentration of 0.1 microM vardenafil. Also positive fibres were detected after incubation with sildenafil but at a higher concentration of 10 microM. Taken together, these results suggest that inhibition of phosphodiesterase enzyme type 5 improves object recognition memory. This effect might be explained by increased levels of central cyclic GMP., (Copyright 2002 IBRO)

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.

In brain, signaling pathways initiated by atrial natriuretic peptide, or transmitters which stimulate nitric oxide synthesis, increase cGMP as their second messenger. One important class of target molecules for cGMP is cGMP-dependent protein kinases, and in the present study, biochemical and immunocytochemical analyses demonstrate the widespread distribution of type II cGMP-dependent protein kinase in rat brain, from the cerebral cortex to the brainstem and cerebellum. Also, colocalization of cGMP-dependent protein kinase type II with its activator, cGMP, was found in several brain regions examined after in vitro stimulation of brain slices with sodium nitroprusside. In western blots, cGMP-dependent protein kinase type II was observed in all brain regions examined, although cerebellar cortex and pituitary contained comparatively less of the kinase. Immunocytochemistry revealed cGMP-dependent protein kinase type II in certain neurons, and occasionally in putative oligodendrocytes and astrocytes, however, its most striking and predominant localization was in neuropil. Electron microscopy examination of neuropil in the medial habenula showed localization of the kinase in both axon terminals and dendrites. As a membrane-associated protein, cGMP-dependent protein kinase type II often appeared to be transported to cell processes to a greater extent than being retained in the cell body. Thus, immunocytochemical labeling of cGMP-dependent protein kinase type II often did not coincide with the localization of kinase mRNA previously observed by others using in situ hybridization. We conclude that in contrast to cGMP-dependent protein kinase type I, which has a very restricted localization to cerebellar Purkinje cells and a few other sites, cGMP-dependent protein kinase type II is a very ubiquitous brain protein kinase and thus a more likely candidate for relaying myriad cGMP effects in brain requiring protein phosphorylation.

The 3-nitropropionic acid animal model is a model where excitotoxicity, mitochondrial dysfunction and oxidative stress, mechanisms common to various neurodegenerative diseases, are involved. The present study investigated whether behavioural alterations in this model were related to striatal damage. Wistar and Lewis rats were exposed to 3-nitropropionic acid and their behavioural performance (open field, walking pattern and Morris Water Maze task) was tested after the injections and after a recovery period of 3 weeks. No changes in activity were found in the open field test. Altered walking pattern was observed in the footprint analysis, although a different response was observed in the Wistar rats compared to the Lewis rats. Initially increased latency times were observed during visual discrimination learning in the Morris Water Maze task in 3-nitropropionic acid-treated Wistar rats compared to Wistar controls. During spatial discrimination learning (invisible platform) in the Morris Water Maze task the swimming velocity was decreased in both rat strains as a result of 3-nitropropionic acid treatment. Increased striatal glial fibrillary acidic protein concentration in Wistar rats correlated with several parameters of the footprint analysis and with the latency and distance in visual as well as spatial discrimination learning in the Morris Water Maze. It is concluded that measurement of walking pattern and spatial orientation performance are sensitive indicators to monitor behavioural changes in relation to striatal degeneration in the 3-nitropropionic acid animal model. In addition, Lewis rats are less sensitive towards 3-nitropropionic acid treatment than Wistar rats.

The structures capable of synthesizing cyclic GMP in response to nitric oxide in the rat brain were compared relative to the anatomical localization of neuronal nitric oxide synthase. In order to do this, we used brain slices incubated in vitro, where cyclic GMP-synthesis was stimulated using sodium nitroprusside as a nitric oxide-donor compound, in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Nitric oxide-stimulated cyclic GMP synthesis was found in cells and fibers, but was especially prominent in varicose fibers throughout the rat brain. Fibers containing the nitric oxide-stimulated cyclic GMP production were present in virtually every area of the rat brain although there were large regional variations in the density of the fiber networks. When compared with the localization of nitric oxide synthase, it was observed that although nitric oxide-responsive and the nitric oxide-producing structures were found in similar locations in general this distribution was complementary. Only occasionally was nitric oxide-mediated cyclic GMP synthesis observed in structures which also contained nitric oxide synthase. We conclude that the nitric oxide-responsive soluble guanylyl cyclase and nitric oxide synthase are usually juxtaposed at very short distances in the rat brain. These findings very strongly support the proposed role of nitric oxide as an endogenous activator of the soluble guanylyl cyclase in the central nervous system and convincingly demonstrate the presence of the nitric oxide-cyclic GMP signal transduction pathway in virtually every area of the rat brain.

In the present study we evaluated the consequences of interference with nitric oxide synthesis during development on brain function and behaviour in later life. Rat pups received a daily injection of the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME, 25 mg/kg, s.c.) from postnatal day 0 to 24. At postnatal day 8 L-NAME-treated rats had enlarged and heavier stomachs, while body weights appeared to be reduced. The stomachs were not affected in size and weight anymore at postnatal day 24, whereas the body weights were still reduced by the L-NAME treatment, although they soon recovered after termination of the treatment. At four months-of-age, rats were tested in non-cognitive (open field) and cognitive (Morris water escape, two-way active avoidance) tasks. Open field behaviour of adult rats postnatally treated with L-NAME was not affected. In the water escape task there were no differences between the saline and L-NAME-treated rats in spatial discrimination learning and spatial reversal learning. Furthermore, postnatal L-NAME treatment did not have an effect on the acquisition of the two-way active avoidance task. Subsequently, we tested rat pups during the L-NAME treatment at postnatal day 19 through 24 in the open field and the two-way active avoidance task. L-NAME treatment appeared to increase the behavioural activity in the open field. There was no difference in behaviour in the active avoidance task between saline and L-NAME-treated rats. Biochemical and immunocytochemical studies showed that at postnatal day 8 the basal cyclic GMP level was reduced, while the cyclic GMP formation due to incubation with the nitric oxide donor sodium nitroprusside appeared to be increased in the hippocampus, striatum and frontal cortex of L-NAME-treated rats. Hence, nitric oxide synthase was inhibited whereas the soluble guanylyl cyclase activity may be increased in sensitivity. At postnatal day 24 basal cyclic GMP levels and nitric oxide-mediated cyclic GMP formation in the brain structures of L-NAME-treated rats had normal values again. Taken together, the findings of this study suggest that postnatal inhibition of nitric oxide synthase has profound neurochemical effects during development and may have short-lasting effects on non-cognitive behaviour, but it does not affect behaviour and brain function in later life.

The effects of 7-nitroindazole, a putative selective inhibitor of neuronal nitric oxide (NO) synthase and zaprinast, a cGMP-selective phosphodiesterase inhibitor, were evaluated on recognition memory of rats in the object recognition test. This test is based on the differential exploration of a new and a familiar object. Two doses of 7-nitroindazole (10 and 30 mg/kg) and zaprinast (3 and 10 mg/kg) were used. The substances were administered i.p. immediately after the exposure to two identical objects, i.e., at the start of the delay interval. After a delay interval of 1 h, control rats spent more time exploring the new object which demonstrates that they recognized the familiar one. Both doses of 7-nitroindazole impaired the discrimination between the two objects after the 1 h interval. After a 4 h interval, control rats did not discriminate between the objects. The highest dose of zaprinast facilitated object recognition after the 4 h interval. In addition, this dose of zaprinast (10 mg/kg) reversed the recognition memory deficit induced by 7-nitroindazole (10 mg/kg) at the 1 h interval. The highest dose of 7-nitroindazole slightly increased mean arterial blood pressure 1 h after its administration. 4 h after administration of zaprinast (10 mg/kg), mean arterial blood pressure was also slightly increased, but not after 1 h after zaprinast administration. However, these effects on blood pressure do not explain the differential effects on object recognition memory. These results therefore suggest that NO-cGMP signal transduction is involved in object recognition memory independently of its cardiovascular role. Finally, since 7-nitroindazole affected mean arterial blood pressure it can not be regarded as a selective inhibitor of neuronal NO synthase.

In the rat central nervous system, cyclic GMP can be produced by two isoforms of guanylyl cyclase: a cytosolic isoform, which is activated by nitric oxide, and a membrane-bound isoform, activated by atrial natriuretic factor. We studied the development of guanylyl cyclase activity upon maturation of the rat forebrain from postnatal days 4 to 24, using a combined immunocytochemical and biochemical approach. Atrial natriuretic factor-activated particulate guanylyl cyclase activity was found to decrease in the frontal cortex, in the lateral septum and in the piriform cortex upon maturation. A transient expression of atrial natriuretic factor-sensitive guanylyl cyclase activity was observed at postnatal day 8 in the caudate putamen complex, whereas an increase was observed in the lateral olfactory tract from postnatal days 8 to 24. Biochemical and immunocytochemical studies using the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester, or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinaloxin-1-one, indicated high levels of endogenous nitric oxide release at postnatal days 4 and 8. This activity decreased strongly in all brain areas examined. From postnatal day 8 onwards, atrial natriuretic factor-responsive cyclic GMP-immunoreactive cells could be characterized as astrocytes, with the exception of those in the the lateral olfactory tract, where the myelinated fibers became cyclic GMP producing. Furthermore, our results on activation of both guanylyl cyclases at postnatal day 8 leads to the suggestion that both isoforms might be found in the same cells. This study shows that there are pronounced differences between various frontal brain areas in the development of the responsiveness of both the particulate and soluble isoforms of guanylyl cyclase, and lends further support to the hypothesis that natriuretic peptides have a role in neuronal growth and plasticity of the rat brain.

Using a novel technique combining immunohistochemistry and in vitro quantitative autoradiography, we were able simultaneously to localize and quantitate cyclic guanosine 3',5'-monophosphate (cGMP)-immunoreactive binding in adult rat cerebellum. The cGMP-immunoreactive binding was predominantly detected in the molecular layer of the cerebellum under both basal and N-methyl-D-aspartate-stimulated conditions. N-Methyl-D-aspartate significantly increased the cGMP binding density in the molecular layer. This increased cGMP level was dose-dependently and significantly inhibited by the inhalational anesthetics halothane and isoflurane. This increased cGMP level was also significantly inhibited by L-NG-nitroarginine methyl ester, an inhibitor of nitric oxide synthases. L-Arginine, the substrate of nitric oxide synthase, reversed the inhibition by L-NG-nitroarginine methyl ester on the cGMP increase. This novel combination of immunohistochemistry and quantitative autoradiography may be used to localize and quantitate simultaneously cGMP or other substances in animal tissues. Our data also confirm that nitric oxide is involved in the stimulation of cGMP formation by N-methyl-D-aspartate. Halothane and isoflurane inhibit the nitric oxide-guanylyl cyclase signaling pathway activated by the excitatory amino acid N-methyl-D-aspartate in the brain, which may be a component of the mechanisms by which these two inhalational anesthetics produce their anesthetic effects.

The distribution of nerves with the potential to synthesize nitric oxide was examined within the urinary bladder and proximal urethra of humans and guinea-pigs, using an antibody to nitric oxide synthase. Further experiments identified cells in which cGMP-immunoreactivity was induced following exposure to the nitric oxide donor, sodium nitroprusside. These cells represent the potential physiological targets of neuronally released nitric oxide, since activation of soluble guanylate cyclase, and a consequent rise in intracellular cGMP, mediate many of the effects of this transmitter. Nitric oxide synthase-immunoreactivity was widely distributed in the lower urinary tract. In guinea-pigs, 50-68% of all intrinsic vesical neurons expressed nitric oxide synthase-immunoreactivity, while in humans 72-96% of neurons in the wall of the bladder contained nitric oxide synthase. In both humans and guinea-pigs, varicose nitric oxide synthase-immunoreactive nerve terminals provided a moderate innervation to the detrusor muscle of the bladder body, and a denser innervation to the urethral muscle. Immunoreactive nerves also projected to the subepithelium and around blood vessels, but were rarely observed encircling intramural vesical ganglia. Following stimulation with sodium nitroprusside, smooth muscle cells of the urethra expressed strong cGMP-immunoreactivity, but detrusor muscle cells remained uniformly negative. Although the detrusor muscle fibres did not express cGMP, numerous interstitial cells throughout the bladder body demonstrated an intense induction of cGMP-immunoreactivity by sodium nitroprusside. These cells had long dendritic processes extending parallel to the smooth muscle fibres, and contained vimentin, an intermediate filament expressed by cells of mesenchymal origin. Other cell types in which sodium nitroprusside exposure induced cGMP-immunoreactivity were the uroepithelial cells, vascular smooth muscle cells and pericytes, and a small number of varicose nerve terminals. In the guinea-pig, a minor proportion (less than 10%) of intrinsic neurons in the wall of the bladder also expressed cGMP. No intrinsic neurons were observed in specimens of human bladder processed for cGMP immunohistochemistry. The results provide anatomical evidence that nitric oxide may function as a neurotransmitter in the lower urinary tract. Although nerves with the capacity to produce nitric oxide supply both the detrusor muscle and the urethra, distinct regional differences exist in the effects of nitric oxide on the induction of cGMP. If the nitric oxide-mediated induction of cGMP is a reliable indicator of the physiological responsiveness of a cell to nitric oxide, then smooth muscle cells appear to be the predominant targets of nitric oxide in the urethra, while in the bladder body, interstitial cells may serve this role. These findings support previous studies which have implicated nitric oxide as an inhibitory transmitter involved in the relaxation of the bladder neck. Our experiments further indicate that a number of cell types within the lower urinary tract could potentially mediate the effects of endogenously released nitric oxide.

In the mammalian retina there are two populations of nitric oxide synthase-containing amacrine cells that stain with the nicotinamide adenine dinucleotide phosphate-diaphorase reaction. To determine the response of these neurons to light, immunoreactivity to Fos proteins was used as a marker of synaptic activation. Fos immunoreactivity is absent in dark-adapted retinas, but 70% of large, Type I nicotinamide adenine dinucleotide phosphate-diaphorase-reactive amacrine cells and 5-10% of the smaller but more numerous Type II nicotinamide adenine dinucleotide phosphate-diaphorase-reactive amacrine cells contain Fos proteins after light stimulation. To localize putative cellular targets of nitric oxide in the retina, retinas were stained immunocytochemically for cyclic GMP after the local administration of the nitric oxide donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine. Both compounds induce strong cyclic GMP immunoreactivity in ON cone bipolar cells. The data suggest that the light-induced inward current in ON cone bipolar cells is enhanced by a nitric oxide-cyclic GMP pathway and that the major source of nitric oxide is the nicotinamide adenine dinucleotide phosphate-diaphorase-reactive amacrine cells in the rabbit retina.

There is growing evidence that nitric oxide serves as a neurotransmitter released from enteric inhibitory nerves in the gastrointestinal tract. The distribution of nitric oxide synthase suggests that nitric oxide may also be a neurotransmitter within enteric ganglia. Since many actions of nitric oxide are mediated by stimulation of soluble guanylate cyclase and a subsequent increase in 3',5'-cyclic guanosine monophosphate (cGMP) concentration, targets for nitric oxide in the canine proximal colon were investigated by immunohistochemical localization of cGMP. In the presence of phosphodiesterase inhibitors (M&B 22948, 100 microM and 3-isobutyl-1-methyl-xanthine, 1 mM), exogenous nitric oxide and electrical field stimulation caused an accumulation of cGMP-like immunoreactivity in several cell-types including colonic smooth muscle cells. cGMP-like immunoreactivity was also observed in a subpopulation of neurons in both myenteric and submucosal ganglia. Sequential labeling with the NADPH diaphorase technique showed that 94% of neurons that responded to exogenous nitric oxide with an increase in cGMP-like immunoreactivity were NADPH diaphorase negative. None of the myenteric neurons that responded to electrical field stimulation with an increase in cGMP-like immunoreactivity were NADPH diaphorase positive, and only one submucosal neuron with cGMP-like immunoreactivity was also NADPH diaphorase positive. The electrical field-stimulated increase in cGMP-like immunoreactivity was blocked by nitroarginine (100 microM). An increase in cGMP-like immunoreactivity also occurred in interstitial cells located at the submucosal surface of the circular muscle layer. These cells are interposed between nerve varicosities and smooth muscle cells and may partially mediate neuromuscular transmission. Sodium nitroprusside and nitric oxide also caused an accumulation of cGMP-like immunoreactivity in smooth muscle cells of intramural arterioles and venules. The results of this study further support the role of nitric oxide as a neurotransmitter in colonic muscles, and provide support for the hypothesis that interstitial cells are functionally innervated by enteric inhibitory neurons. The data also suggest that nitric oxide may serve as a neurotransmitter in enteric ganglia.

The immunohistochemical localization of cyclic GMP was used to determine potential physiological sites of action of nitric oxide in the guinea-pig small intestine and colon. In control tissue, cyclic GMP-immunoreactivity was observed only in macrophages, whose identity was confirmed by double-label experiments using either F4/80, a macrophage-specific antibody, or fluorescein isothiocyanate-labelled dextran injected intravenously. Following exposure to the nitric oxide donor, sodium nitroprusside, cyclic GMP-immunoreactivity was induced in subpopulations of neurons in the myenteric and submucosal plexuses of the ileum and colon. In the colon, cyclic GMP-immunoreactivity was induced in 5-10% of myenteric neurons. The cyclic GMP-immunoreactive neurons did not contain nitric oxide synthase. In the ileum, cyclic GMP-immunoreactive neurons comprised about 2% of myenteric neurons and 40% of submucosal neurons; these cyclic GMP-immunoreactive neurons were also immunoreactive for vasoactive intestinal peptide, but they did not contain nitric oxide synthase. Interstitial cells between the mesothelium and the longitudinal muscle layer, vascular smooth muscle and vascular pericytes also showed sodium nitroprusside-induced cyclic GMP-immunoreactivity. The interstitial cells of Cajal at the inner surface of the circular muscle layer and the smooth muscle cells of the circular and longitudinal muscle layers showed increases in cyclic GMP-immunoreactivity that varied in extent from animal to animal. The results suggest that nitric oxide could act at several sites in the intestine through the stimulation of guanylyl cyclase.

The mammalian carotid body is a peripheral arterial chemoreceptor organ involved in the regulation of respiration, and in the modulation of blood pressure through reflex control of peripheral vascular resistance and cardiac output. In addition to its responsiveness to blood gases, the organ is also sensitive to hyperosmotic solutions, and we have recently shown that a systemic hormonal regulator of natriuresis and diuresis, atrial natriuretic peptide, is a potent inhibitor of chemoreceptor activity evoked by hypoxia in the cat carotid body. The present study demonstrates atrial natriuretic peptide immunoreactivity in type I cells of the carotid body, and shows further that a biologically active atrial natriuretic peptide fragment, atriopeptin III, increases cyclic guanosine monophosphate immunoreactivity in type I cells in a dose-dependent manner. Moreover, double-labeling techniques demonstrate co-existence of atrial natriuretic peptide immunoreactivity with the atriopeptin III-enhanced cyclic guanosine monophosphate reaction product. These findings indicate the probable existence of atrial natriuretic peptide receptors coupled to membrane-bound guanylate cyclase on the parenchymal type I cells. Our findings support the view that cyclic guanosine monophosphate functions as a second messenger in this organ, and may serve as a functional activity marker in identifying type I cells which respond to atrial natriuretic peptide.

The development of a new 3',5'-cyclic guanosine monophosphate (cGMP) antiserum was initiated starting from the following considerations: (a) adequate fixation of cGMP is a prerequisite for a reliable demonstration of soluble cGMP, and (b) fixation might influence the specificity of the immunocytochemical demonstration of cGMP. Therefore, cGMP-protein conjugate was prepared in a way which equals tissue fixation. cGMP was coupled to bovine thyroglobulin using formaldehyde. Antibodies against this conjugate were raised in rabbits. The specificity of the antisera was evaluated in a gelatin model system. No immunoreactivity was observed with nucleotides other than cGMP or with rabbit preimmune sera. Immunoinhibition experiments showed that only the cGMP-formaldehyde-thyroglobulin conjugate and, to a lesser extent free cGMP, absorbed onto the antiserum. In rat brain an extensive localization of cGMP-immunostaining was found. Examples are hippocampus CAI and CAII, and cortical layers II and V. No cGMP-immunostaining was found in the cerebellum. In vitro incubated superior cervical ganglia showed cGMP-immunostaining in the large postganglionic neuronal cell bodies; this cGMP-immunostaining increased upon incubation of the ganglia in iso-osmolar 100 mM K+. In conclusion, we prepared a new-type highly specific antiserum against cGMP, suitable to demonstrate cGMP-immunoreactivity in tissue material.

The nature of the rat adipocyte beta-adrenoceptor was studied in further detail using a selected series of tolamol-type beta-adrenoceptor antagonists. Isoprenaline antagonism by these compounds was evaluated on adipocytes, right atrium, left atrium and left hemidiaphragm of the rat. Adipocyte pA2 values were corrected for binding to bovine serum albumin determined separately for each antagonist. A strong correlation between adipocyte and diaphragm pA2 values was found with those antagonists which had an identical N-substituent but a different substitution pattern in the phenoxypropanolamine ring. This relationship was absent with those compounds which had the same 2-methylphenoxypropanolamine moiety but a different N-substituent. With the latter antagonists, however, adipocyte pA2 values correlated significantly with the two cardiac pA2 values. These results support the idea that the interaction site of the rat adipocyte beta-adrenoceptor for the aromatic moiety of arylethanolamines and aryloxypropanolamines has beta 2-characteristics whereas the alkanolamine side-chain interaction site has a beta 1-nature. Finally, it was ascertained that the apparently dualistic (beta 1 and beta 2) character of the rat adipocyte adrenoceptor is not due to the presence of both a beta 1- and a beta 2-receptor population on the fat cell plasma membrane.