Lectin-binding glycoproteins in the developing and adult snail CNS

Glycoproteins are complex molecules of the cell surface and the extracellular matrix (ECM) playing a fundamental role in the migration, guidance and synapse formation of neurons. In the present study, the glycosylated protein composition and localization were investigated in the adult and developing CNS of an aquatic (Lymnaea stagnalis) and a terrestrial (Helix pomatia) snail species, applying lectin histochemistry and blotting. Lectin probes that are specific for N-acetyl-glucosamine (GlcNAc) oligomers frequently appeared in anatomically different regions of the adult ganglia of both species, such as, the periganglionic sheath, the interperikaryonal space and the neuropil. Different GlcNAc residues were found to intensively glycosylate five, high-molecular weight proteins characteristic for the ECM of Lymnaea CNS and localized mainly in the interperikaryonal space. N-acetyl-galactosamine oligomers were less pronounced in the adult snail ganglia, they were detected only in the periganglionic sheath and the attached basement lamina. Apart from some similarities, the glycosylation pattern of proteins and the distribution of glycoproteins in the neuropil displayed significant differences in Lymnaea and Helix. All continuous and increasing level of and also transient presence of glycoproteins were detected during Lymnaea CNS development. Our results indicate a rich glycosylated pattern of specific proteins in the snail CNS, displaying remarkable species- and age-dependent changes which suggest the wide importance of protein glycosylation in the CNS of invertebrates.     

Nitric oxide in the crustacean brain: regulation of neurogenesis and morphogenesis in the developing olfactory pathway.

Nitric oxide (NO) plays major roles during development and in adult organisms. We examined the temporal and spatial patterns of nitric oxide synthase (NOS) appearance in the embryonic lobster brain to localize sources of NO activity; potential NO targets were identified by defining the distribution of NO-induced cGMP. Staining patterns are compared with NOS and cyclic 3,5 guanosine monophosphate (cGMP) distribution in adult lobster brains. Manipulation of NO levels influences olfactory glomerular formation and stabilization, as well as levels of neurogenesis among the olfactory projection neurons. In the first 2 days following ablation of the lateral antennular flagella in juvenile lobsters, a wave of increased NOS immunoreactivity and a reduction in neurogenesis occur. These studies implicate nitric oxide as a developmental architect and also support a role for this molecule in the neural response to injury in the olfactory pathway.     

Topographical relationship between neuronal nitric oxide synthase immunoreactivity and cyclic 3′,5′-guanosine monophosphate accumulation in the brain of the adult Xenopus laevis

Previous immunohistochemical staining procedures of the brain and pituitary in Xenopus laevis, using an antiserum against neuronal nitric oxide (NO) synthase (nNOS) and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry, have revealed NOS activity in neurons and fibers in a number of brain areas, as well as in fibers in the pituitary. In the present study we have localized the target structures of the NOergic system in the Xenopus brain by visualizing the sites of NO-sensitive cyclic 3′,5′-guanosine monophosphate (cGMP) accumulation, according to a method for cGMP visualization in rat brain slices. Brain slices of unfixed Xenopus are incubated in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine and the NO donor sodium nitroprusside, followed by fixation and cryosectioning. Sections were then processed for immunohistochemistry using rabbit and sheep antisera against cGMP and a sheep antiserum against nNOS. Visualization of single and double labeling of cGMP immunoreactive and/or nNOS immunoreactive structures was performed with combined CY3/fluorescein isothiocyanate fluorescence microscopy. Following this procedure, we provide immunohistochemical evidence for the distribution of cGMP-accumulating neurons in the brain of adult Xenopus. In most brain areas, the distribution of nNOS and cGMP immunoreactive structures (neuron somata and fibers) is distinct and separate, for instance in the dorsal pallium, the lateral thalamic nuclei, the optic tectum, the locus coeruleus and the reticular formation. However, nNOS and cGMP immunoreactive structures are often found in the vicinity of each other, and in the optic tectum even in adjacent neuron fibers and somata. The present observations are in line with the presence of an NO-dependent soluble guanylate cyclase in distinct brain areas of Xenopus laevis, corroborating similar data in the mammalian brain. Further, our observations may add to the understanding of the anatomical connectivity pattern and functional relevance of the NOergic system in the amphibian brain.     

Localisation of neuronal nitric oxide synthase-immunoreactivity in rat and rabbit retinas

The distribution of neuronal nitric oxide synthase (NOS) immunoreactivity was examined in rat and rabbit retinas and was compared with the distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase reactivity and vasoactive intestinal peptide (VIP) immunoreactivity. An antibody raised against a C-terminal fragment of a cloned rat cerebellar NOS was used to localise NOS immunoreactivity. NOS immunoreactive cells were not detected in rat retinas at postnatal day 1 or 4, but were seen from postnatal day 7 onwards. NOS immunolabelling was seen in a small population of cells in the proximal inner nuclear layer. Most of the labelled cells had the position of amacrine cells and were seen to send processes into the inner plexiform layer. A few labelled cells were at times also seen in the ganglion cell layer, which are likely to correspond to displaced amacrine cells. The same NOS-labelling pattern was seen in rat and rabbit retinas.NADPH-diaphorase staining was observed in both species, in photoreceptor inner segments, in cells with the position of horizontal cells, in a subset of amacrine and displaced amacrine cells, in large cell bodies in the ganglion cell layer, in both plexiform layers, and in endothelium. Colocalisation of NOS immunoreactivity and NADPH-diaphorase staining was only observed among amacrine cells. However, not all NADPH-diaphorase-reactive amacrine cells were found to be NOS immunoreactive. VIP immunoreactivity was also localised in rat retinas in a subpopulation of amacrine cells, but no colocalisation of NOS and VIP immunoreactivity was observed.Our observations indicate that only amacrine cells contain the NOS form recognisable by the antibody used, and suggest that different isoforms of neuronal NOS may be present in retinal cells. Further, the onset of NOS expression in rat amacrine cells appears to occur independently of neuronal activity.Paper in honour of Professor Rolf Elofsson on the occasion of his retirement from the chair of Zoology at the University of Lund     

Neuronal nitric oxide synthase (bNOS) messenger RNA and protein expression in developing rat brainstem nuclei

Neuronal nitric oxide synthase (bNOS) messenger RNA expression and immunoreactivity were mapped in series of cryosections through the developing rat brainstem nuclei. Between embryonic day E16 and postnatal day P16, brainstem nuclei expressed both bNOS messenger RNA (mRNA) in situ hybridization signals and protein immunoreactivity. However, NOS mRNA signals were absent from the Edinger Westphal, facial or motory trigeminal nucleus. Strong patterns of mRNA signals and immunoreactivity occurred in neurons located in the substantia nigra pars compacta and the laterodorsal tegmental nuclei. Between E24 and P16, altered patterns of bNOS mRNA positive and immunoreactive neurons, e.g. superior and inferior colliculi, raphe nuclei, solitary tract or pontine nucleus were documented. Altered NOS expression patterns thus may reflect developmental processes within distinct neuronal populations such as cell phenotype discrimination or synaptogenesis within efferent or afferent brainstem pathways. The NOS/NO system therefore appears to be a modulator for intra-/intercellular adjustment processes in normal development.     

Nitric Oxide Synthase in Human Parathyroid Glands and Parathyroid Adenomas

Nitric oxide (NO) is a novel gaseous intercellular transmitter thought to play important physiological roles in the regulation of blood flow and hormone secretion in, for example, the pituitary, the thyroid, and the endocrine pancreas. Whether nitric oxide synthase (NOS) is present in the human parathyroid glands has not yet been demonstrated. In the present study, histologically normal, but functionally suppressed human parathyroid glands and parathyroid adenomas from patients with primary hyperparathyroidism were investigated by immunocytochemistry with antibodies against neuronal NOS and by reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. We also used H&E to identify the NOS-immunoreactive cells. Immunocytochemistry demonstrated the presence of neuronal-type NOS in a subpopulation of glandular cells, identified as oxyphilic cells, in both normal parathyroid glands and adenomas. NADPH-diaphorase staining visualized NOS in the endothelium of blood vessels and in glandular cells, corresponding to those containing immunoreactive NOS. In addition, we found NADPH-diaphorase staining in many chief cells. Our results indicate that both glandular cells and vascular endothelium in human parathyroid glands and adenomas express NOS. There is thus a morphological substrate for locally produced NO that may be involved in the regulation of parathyroid blood flow and hormone secretion.     

Lymnea stagnalis as experimental intermediate host for the protostrongylid nematodeElaphostrongylus rangiferi

The aquatic snail species,Lymnea stagnalis was examined as experimental intermediate host for the nematodeElaphostrongylus rangiferi, a neurotrophic parasite of reindeer,Rangifer tarandus. All juvenile snails exposed to first-stage larvae ofE. rangiferi became infected, but the mean number of larvae per snail ( ) was higher when snails were exposed in water ( =14.3) than when exposed on filterpaper moistened with a suspension of larvae ( =8.1), an efficient method of infecting terrestrial snails. Juvenile snails had a significant higher mean number of larvae ( =17.2) than adults ( =1.4). The development of the parasite, in both juvenile and adultL. stagnailis, was comparable to the most susceptible terrestrial snails. At 21 days postinfection about 40% of the larvae had reached the infective L3 stage, whilst 80% were in the third stage at 29 days postinfection. When kept at 12°C, larvae remained infective toL. stagnalis for about 100 days, but the period of infectivity was reduced to about 17 days when larvae were kept at 20° C.L. stagnalis is a good experimental host forE. rangiferi, and may be of epidemiological significance in transmitting the parasite to reindeer.     

Relative significance of the nitric oxide (NO)/cGMP pathway and K+ channel activation in endothelium‐dependent vasodilation in the femoral artery of developing piglets

Mechanisms mediating endothelium‐dependent vasodilation were investigated in femoral artery rings from <2‐day‐old (newborn) and 2‐week‐old piglets. Based on previous results we hypothesized an age difference in the relative contribution of nitric oxide(NO)‐cyclic 3′,5′‐guanosine monophosphate (cGMP) and K⁺ channel‐activation to acetylcholine (ACh)‐induced vasodilation. Changes in vascular tone were studied in organ baths in the absence or presence of NO synthase(NOS) inhibition or K⁺ channel blockade and the intra‐arterial accumulation of cGMP in response to ACh was measured with radioimmunoassay (RIA). In control experiments, relaxant responses to ACh were equal in the two age groups. In the presence of the NOS‐inhibitors N ^G‐monomethyl‐L ‐arginine acetate (L ‐NMMA; 100 μM ) or N^G‐nitro‐L ‐arginine (L ‐NOARG; 1–100 μM ), however, relaxation was significantly more reduced in femoral artery rings from 2‐week‐old than from newborn, with lower pD₂ values in the older age group. Inhibition of large (BK_Ca) conductance calcium‐sensitive K⁺ channels with tetraethylammonium chloride (TEA; 1 mM ), gave a significant rightward shift in the concentration‐response curves to ACh which was of the same magnitude in both age groups. The ACh‐induced vasodilation was abolished in both age groups by high K⁺ (20 mM ) in combination with L ‐NOARG (100 μM ). The relative increase in cGMP levels after addition of ACh (10 nM ) was significantly larger in rings from newborn compared with 2‐week‐old piglets (12‐ vs. four‐fold). In summary, sensitivity to NOS inhibition increased with age while the effect of K⁺ channel blockade with TEA was the same in femoral artery rings from newborn to 2‐week‐old piglets. Lower sensitivity to NOS inhibition and a larger increase in cGMP in response to ACh could indicate a higher efficacy of the NO/cGMP pathway in this vessel in the newborn piglet.     

一氧化氮合酶阳性结构在大鼠三叉神经感觉核簇内的分布及其与面部伤害性刺激诱发的FOS蛋白表达的关系

用还原型尼克酰胺腺嘌呤二核苷酸脱氢酶组织化学技术与ＦＯＳ免疫组化（ＡＢＣ法）相结合的方法，观察了大鼠三叉神经感觉核簇内一氧化氮合酶阳性神经元和纤维的分布及其与面部伤害性刺激诱发的ｃ－ｆｏｓ原癌基因蛋白表达的关系．证明，浓密的一氧化氮合酶阳性纤维和终末分布于三叉神经脊束核尾侧亚核的Ⅰ和Ⅱ层，阳性成分呈蓝黑色的带状分布，而在三叉神经感觉核簇的其余核区内很稀疏．一氧化氮合酶阳性神经元胞体与面部伤害性刺激诱发的ｃ－ｆｏｓ原癌基因蛋白表达的阳性神经元在此核簇内的分布节段非常相似，都主要存在于与伤害性传入传递和调控有关的三叉神经脊束核尾侧亚核，并且二者都主要分布于与痛觉调控有关的浅层．约有１０～１５％的一氧化氮合酶阳性神经元同时呈ｃ－ｆｏｓ原癌基因蛋白表达．在三叉脊束核尾侧亚核的Ⅲ～Ⅴ层仅出现散在的阳性细胞，除在吻侧亚核的背内缘与孤束核交界处有少量一氧化氮合酶同性神经元外，在三叉神经感觉核簇的其余核区两者均几无表达．本文及文献结果提示，一氧化氮可能对面部伤害性信息的传递和调控起重要作用．     

Atypical guanylyl cyclase from the pond snail Lymnaea stagnalis: cloning,sequence analysis and characterization of expression

Soluble guanylyl cyclases (sGCs) are traditionally recognized as the main molecular receptor for nitric oxide (NO), a gaseous transmitter involved in many functions of the nervous system. Some sGCs are however insensitive to NO and therefore are known as atypical. Although atypical sGCs have been shown to exist in both vertebrate and invertebrate nervous systems, our understanding of their functional role is incomplete. Here we report on the cloning, sequencing and localization of an atypical sGC named Lym-sGCβ3 from the snail Lymnaea stagnalis. We found that Lym-sGCβ3 shares a number of structural characteristics with some previously characterized atypical sGCs including the presence of Tyr140 in the regulatory domain. This residue is thought to be of a critical importance in determining sensitivity of atypical sGCs to oxygen. These findings raise the possibility that Lym-sGCβ3 is an oxygen receptor. The results of our in situ hybridization and RT-PCR experiments support this idea further by showing that Lym-sGCβ3 is expressed in the osphradium, a peripheral sense organ in which oxygen-sensing neurons are located. Also of interest are our observations that many neurons in Lymnaea CNS co-express conventional and atypical sGC subunits. These data are consistent with a possible dominant negative regulatory role of atypical sGC subunits through the formation of heterodimers exhibiting low enzymatic activity.     

Signalling mechanisms involved in the induction of inducible nitric oxide synthase by Lactobacillus rhamnosus GG,endotoxin, and lipoteichoic acid

Background and Aims: Probiotic Lactobacillus rhamnosus GG (Lactobacillus GG) has been found beneficial in the treatment of viral and antibiotic-associated diarrhea. Recently, it has also been shown to induce nitric oxide (NO) production, and have some other immunostimulatory effects. The aim of the present study was to investigate the mechanisms involved in the induction of inducible nitric oxide synthase (iNOS) and NO production by Lactobacillus GG. Methods and Results: iNOS expression and NO production induced by Lactobacillus GG, lipopolysaccharide (LPS), and lipoteichoic acid (LTA) was abrogated by NOS inhibitors L-NMMA and 1400W, by a protein synthesis inhibitor cycloheximide, by a tyrosine kinase inhibitor genistein and by a NF-B inhibitor pyrrolidinedithiocarbamate (PDTC) in J774 macrophages. Polymyxin B inhibited NO production induced by LPS, but did not inhibit Lactobacillus GG induced NO production. _P42/44 MAP-kinase inhibitor PD98059, dexamethasone and cyclosporine A inhibited partially iNOS protein expression and NO formation in LactobacillusGG, LPS and LTA treated cells. Ro 31-8220 (protein kinase C inhibitor) and SB203580 (p38 MAP-kinase inhibitor) had only a minor effect on NO production. Conclusions: Lactobacillus GG induced NO production through iNOS pathway and the mechanisms mediating that process were very similar with those involved in LPS and LTA induced NO synthesis.     

Nitric oxide synthase and background adaptation in Xenopus laevis

Adaptation of the skin colour to the background light condition in the amphibian Xenopus laevis is achieved by migration of pigment granules in the skin melanophores, a process regulated by α-MSH secretion from melanotrope cells in the pituitary pars intermedia (PI). α-MSH secretion in turn, is regulated by various stimulatory and inhibitory messengers synthesized in brain nuclei, especially the hypothalamic suprachiasmatic and magnocellular nuclei and the locus coeruleus in the hindbrain.In the present study, the roles in background adaptation of nitric oxide (NO) and NO synthase (NOS) enzyme activity were evaluated. In situ, using both immunohistochemistry with anti-human brain NOS (bNOS) serum in paraffin-embedded material and using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry in cryo-sections, we showed NOS in neurons in the optic tectum and in the locus coeruleus. NADPH-d reactivity was also found in neurons in the lateral amygdala, the ventral hypothalamic nucleus and in fibers in the median eminence. Using a Western blot stained with an anti-human bNOS serum, we demonstrated a 150 kDa band in Xenopus hindbrain lysates, which is similar to the NOS protein present in the rat anterior pituitary, but which was not detectable in the lysates from both the neurointermediate and distal lobes in Xenopus. No differences in histochemical staining pattern or on Western blotting were observed between animals adapted to a black or a white background.Paraffin sections of the endocrine PI and pars distalis did not reveal bNOS-like immunoreactivity. NADPH-d reactivity was observed in the endothelia of this gland. However, using a new procedure of thin cryo-sections of pituitary neurointermediate lobes, we observed bNOS-immunoreactive fibers as well as cyclic 3′,5′ guanosine monophosphate (cGMP)-accumulating fibers in the PI.The PI may be regulated by NOergic neurons from higher brain centers. The possibility that NOergic neurons in the locus coeruleus are involved in the innervation of the PI needs further investigation. The latter neurons are probably not noradrenergic because double labeling studies show no co-localization of NADPH-d reactivity and tyrosine hydroxylase immunoreactivity in locus coeruleus neurons.     

Nitric oxide‐induced signalling in rat lacrimal acinar cells

The aim of the present study was to investigate the physiological role of nitric oxide (NO) in mediating secretory processes in rat lacrimal acinar cells. In addition, we wanted to determine whether the acinar cells possess endogenous nitric oxide synthase (NOS) activity by measuring NO production using the fluorescent NO indicator 4,5‐diaminofluorescein (DAF‐2). We initiated investigations by adding NO from an external source by means of the NO‐donor, S‐nitroso‐N‐acetyl‐penicillamine (SNAP). Cellular concentrations of cyclic guanosine 5′‐phosphate (cGMP) ([cGMP]) were measured by radioimmunoassay (RIA), and we found that SNAP induced a fast increase in the [cGMP], amounting to 350% of the [cGMP] in resting cells. Moreover, addition of SNAP and elevating [cGMP] in fura‐2 loaded lacrimal acinar cells, resulted in a cGMP‐dependent protein kinase‐mediated release of Ca²⁺ from intracellular stores, leading to a rise in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i). The Mn²⁺ quenching studies revealed that the Ca²⁺ release was not accompanied by Ca²⁺ influx. Finally, we demonstrate that lacrimal acinar cells possess endogenous NOS activity, which is activated by β‐adrenergic stimulation and not by a rise in [Ca²⁺]_i alone. We show that in rat lacrimal acinar cells, NO and cGMP induce Ca²⁺ release from intracellular stores via G kinase activation. However, the changes in [Ca²⁺]_i are relatively small, suggesting that this pathway plays a modulatory role in Ca²⁺ signalling, thus not by itself causing fast transient increases in [Ca²⁺]_i. In addition, we suggest that endogenously produced NO activated by β‐adrenergic receptor stimulation, plays an important role in signalling to the surrounding tissue.     

Blood cell types and blood cell formation in gastropod molluscs

The results of previous and recent investigations on the structure, function and formation of blood cells of the freshwater snail $\text{Lymnaea}$$\text{stagnalis}$ are described and discussed. Two hypotheses on blood cell formation and blood cell types are presented.     

Modulation of Penile Erection in Rabbits by Mondia Whitei: Possible Mechanism of Action

Mondia whitei root was evaluated to validate its anecdotal use and determine its possible mode of action in the management of erectile dysfunction. Rabbits were administered with daily oral doses of 100–400 mg kg⁻¹ crude ethanolic extract of M. whitei and sildenafil (50 mg kg⁻¹) as positive control for 6 weeks. Cavernosal tissue NOS activity and levels of NO and cGMP, and NOS and PDE protein expressions were investigated. The effect of the crude extract, chloroform and petroleum ether fractions in vitro on cavernosal tissue NOS activity and levels of NO and cGMP at 0.01 and 0.10 mg g⁻¹ tissue were also investigated. Results indicate that the crude extract increased NOS activity by 7% at 200 mg kg⁻¹ with corresponding increases in NO (88%) and cGMP (480%) levels. No significant changes in these measurements were observed with the 100 and 400 mg kg⁻¹ doses whilst sildenafil slightly reduced them (15.9–37.5%). NOS and PDE protein expressions in test animals were not different from controls. Pre-incubation of cavernosal tissue in vitro with the crude extract of M. whitei and its chloroform fraction markedly increased NOS activity (26–132%) and levels of NO (25%) and cGMP (50–400%) at 0.01 mg g⁻¹ tissue but these were reduced to near control levels when their concentrations were increased to 0.10 mg g⁻¹ tissue whilst the petroleum ether fraction had no effect. These findings suggest that M. whitei may influence erectile function through activation/stimulation of NOS with corresponding increases in tissue NO and cGMP levels and that certain chemical constituents present in the chloroform fraction may be responsible for biological activity.     

Expression and distribution of GABA and GABAB‐receptor in the rat adrenal gland

The inhibitory effects of gamma‐aminobutyric acid (GABA) in the central and peripheral nervous systems and the endocrine system are mediated by two different GABA receptors: GABA_A‐receptor (GABA_A‐R) and GABA_B‐receptor (GABA_B‐R). GABA_A‐R, but not GABA_B‐R, has been observed in the rat adrenal gland, where GABA is known to be released. This study sought to determine whether both GABA and GABA_B‐R are present in the endocrine and neuronal elements of the rat adrenal gland, and to investigate whether GABA_B‐R may play a role in mediating the effects of GABA in secretory activity of these cells. GABA‐immunoreactive nerve fibers were observed in the superficial cortex. Some GABA‐immunoreactive nerve fibers were found to be associated with blood vessels. Double‐immunostaining revealed GABA‐immunoreactive nerve fibers in the cortex were choline acetyltransferase (ChAT)‐immunonegative. Some GABA‐immunoreactive nerve fibers ran through the cortex toward the medulla. In the medulla, GABA‐immunoreactivity was seen in some large ganglion cells, but not in the chromaffin cells. Double‐immunostaining also showed GABA‐immunoreactive ganglion cells were nitric oxide synthase (NOS)‐immunopositive. However, neither immunohistochemistry combined with fluorescent microscopy nor double‐immunostaining revealed GABA‐immunoreactivity in the noradrenaline cells with blue‐white fluorescence or in the adrenaline cells with phenylethanolamine N‐methyltransferase (PNMT)‐immunoreactivity. Furthermore, GABA‐immunoreactive nerve fibers were observed in close contact with ganglion cells, but not chromaffin cells. Double‐immunostaining also showed that the GABA‐immunoreactive nerve fibers were in close contact with NOS‐ or neuropeptide tyrosine (NPY)‐immunoreactive ganglion cells. A few of the GABA‐immunoreactive nerve fibers were ChAT‐immunopositive, while most of the GABA‐immunoreactive nerve fibers were ChAT‐immunonegative. Numerous ChAT‐immunoreactive nerve fibers were observed in close contact with the ganglion cells and chromaffin cells in the medulla. The GABA_B‐R‐immunoreactivity was found only in ganglion cells in the medulla and not at all in the cortex. Immunohistochemistry combined with fluorescent microscopy and double‐immunostaining showed no GABA_B‐R‐immunoreactivity in noradrenaline cells with blue‐white fluorescence or in adrenaline cells with PNMT‐immunoreactivity. These immunoreactive ganglion cells were NOS‐ or NPY‐immunopositive on double‐immunostaining. These findings suggest that GABA from the intra‐adrenal nerve fibers may have an inhibitory effect on the secretory activity of ganglion cells and cortical cells, and on the motility of blood vessels in the rat adrenal gland, mediated by GABA‐Rs.     

New aspects of renal potassium transport

The positive contractile effect of nitric oxide (NO) donors was studied on isolated rat ventricular cardiomyocytes within a range of a positive force/frequency relationship. We determined whether the observed effect depended on cGMP. The NO donors S-nitroso-acetyl-d,l-penicillamine (SNAP) and N-{4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl}-1,3-propanediamine (spermine-NONO) increased contractile responsiveness transiently in a concentration- and frequency-dependent manner. The influence of NO donors on cGMP levels was enhanced under beating conditions. The positive contractile effect of NO donors was inhibited by adenosine 3,5-cyclic monophosphothioate Rp diastereomer (Rp-cAMPS), but not by bisindolylmaleimide. Inhibition of the soluble guanylyl cyclase (sGC) by 1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ) inhibited the positive contractile effect of NO donors. Direct activation of sGC by 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole (YC1) or addition of 8-bromo cGMP increased cell contractility comparably to NO donors. Inhibition of G_s proteins by NF441 inhibited the positive contractile effect of NO donors. In contrast, NO donors did not potentiate the positive contractile effect of forskolin. These results demonstrate that the positive contractile effect of NO donors on rat ventricular cardiomyocytes working in a range of a positive force/frequency relationships is enhanced. It is mediated by NO-dependent stimulation of the sGC interacting with G_s proteins.     

Constitutive nitric oxide synthases in rat pancreatic islets: direct imaging of glucose-induced nitric oxide production in β-cells

The production of nitric oxide (NO) by constitutive nitric oxide synthase (NOS) was investigated in isolated rat pancreatic islets and dispersed -cells. Double-immunocytochemical analyses with a confocal microscope demonstrated the presence of NOS1 in -, -, -, and PP-cells and that of NOS3 in -, -, and PP-cells, but not -cells, in the isolated rat islets. Image analyses with the NO-reactive fluorescence dye DAF-2 clearly showed that an elevation in glucose concentrations from 0 to 11.1 mM increased intracellular NO in most cells of the isolated islets. The glucose-induced elevation of intracellular NO in the islet cells was abolished in the presence of the Ca²⁺ channel blocker nicardipine and after treatment with the NOS inhibitor N ^G-nitro-l-arginine. Similarly, the ATP-sensitive K⁺ channel blocker tolbutamide, which elevates intracellular Ca²⁺ concentrations, increased DAF-2 fluorescence in most cells of the isolated islets. In isolated -cells, 11.1 mM glucose increased DAF-2 fluorescence, which was suppressed by N ^G-nitro-l-arginine and by reducing the glucose concentration to 0 mM. DAF-2 fluorescence in -cells was also increased by 50 mM K⁺, which was suppressed by N ^G-nitro-l-arginine. These results suggest that NOS1 and NOS3 are present in rat pancreatic -cells, and that glucose produces NO by Ca²⁺-dependent activation of the constitutive NOS isoforms.     

Brain Blood Flow Modulates the Neurotoxic Action of Hyperbaric Oxygen via Neuronal and Endothelial Nitric Oxide

Studies on conscious rats with inhibition of NO synthase were used to assess the dynamics of brain blood flow and EEG traces during hyperbaric oxygenation at 4 or 5 atm. Oxygen at a pressure of 4 atm induced cerebral vasoconstriction in intact animals and decreased blood flow by 11–18% (p < 0.05) during 60-min exposure to hyperbaric oxygenation. Paroxysmal EEG activity and oxygen convulsions did not occur in rats at 4 atm of O₂. At 5 atm, convulsive activity appeared on the EEG at 41 ± 1.9 min, and blood flow decreased significantly during the first 20 min; blood flow increased by 23 ± 9%, as compared with controls, (p < 0.01) before the appearance of convulsions on the EEG. Prior inhibition of NO synthase I (NOS I) and NO synthase III (NOS III) with N-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg) or inhibition only of NOS I with 7-nitroindazole (7-NI, 50 mg/kg) prevented the development of hyperoxic hyperemia and paroxysmal spikes on the EEG during hyperbaric oxygenation at 5 atm. These results show that hyperbaric oxygen induces changes in cerebral blood flow which modulate its neurotoxic action via nitric oxide synthesized both in neurons and in cerebral vessels.