Bo Holmqvist

NITRIC OXIDE SYNTHASE-CONTAINING NEURONS IN RAT PARASYMPATHETIC, SYMPATHETIC AND SENSORY GANGLIA : A COMPARATIVE STUDY

SummaryIn rats, the distribution of nerve structures staining for NADPH-diaphorase, and showing immunoreactivities for nitric oxide synthase (NOS), tyrosine hydroxylase and various neuropeptides was studied in sensory ganglia (dorsal root, nodose and trigeminal ganglia), in sympathetic ganglia (superior cervical, stellate, coeliac-superior and inferior mesenteric ganglia), parasympathetic ganglia (sphenopalatine, submandibular, sublingual and otic ganglia), and in the mixed parasympathetic/ sympathetic ganglia (major pelvic ganglia). The coincidence of neuronal cell bodies with strong NOS-immunoreactivity and strong NADPH diaphorase reactivity was almost total. The relative proportions of NOS-immunoreactive nerve cell bodies were largest in parasympathetic ganglia and major pelvic ganglia followed by sensory ganglia. In sympathetic ganglia no NOS-immunoreactive neuronal cell bodies could be detected. In parasympathetic and major pelvic ganglia, there was a very significant neuronal co-localization of immunoreactivities for NOS and vasoactive intestinal polypeptide (VIP). This was almost total in major pelvic ganglia, in which NOS-/VIP-immunoreactive nerve cell bodies were separate from sympathetic (tyrosine hydroxylase-/neuropeptide Y-immunoreactive), suggesting that NOS-/VIP-immuno-reactive neurons might also be parasympathetic.

Nitric oxide synthase in the brain of a teleost

The presence and distribution of the nitric oxide (NO) converting enzyme, NO synthase (NOS), was investigated in the brain of a teleost, the Atlantic salmon. Both NOS immunoreactive and NADPH diaphorase positive, non-neuronal and neuronal cell bodies, fibers and putative nerve terminals were identified throughout the brain. Even so, the staining was not identical in all regions. NO, synthesized by NOS-like enzymes, may play an important role in a diversity of cellular mechanisms in the brain of the salmon, including in neural systems related to olfactory, visual, hypophysiotrophic, viscero-sensoric and motor functions.

Binding and Uptake of A beta 1-42 by Primary Human Astrocytes In Vitro

Clearance of the amyloid‐β peptide (Aβ) as a remedy for Alzheimers disease (AD) is a major target in on‐going clinical trials. In vitro studies confirmed that Aβ is taken up by rodent astrocytes, but knowledge on human astrocyte‐mediated Aβ clearance is sparse. Therefore, by means of flow cytometry and confocal laser scanning microscopy (CLSM), we evaluated the binding and internalization of Aβ1‐42 by primary human fetal astrocytes and adult astrocytes, isolated from nondemented subjects (n = 8) and AD subjects (n = 6). Furthermore, we analyzed whether α1‐antichymotrypsin (ACT), which is found in amyloid plaques and can influence Aβ fibrillogenesis, affects the Aβ uptake by human astrocytes. Upon over night exposure of astrocytes to FAM‐labeled Aβ1‐42 (10 μM) preparations, (80.7 ± 17.7)% fetal and (52.9 ± 20.9)% adult Aβ‐positive astrocytes (P = 0.018) were observed. No significant difference was found in Aβ1‐42 uptake between AD and non‐AD astrocytes, and no influence of ApoE genotype on Aβ1‐42 uptake was observed in any group. There was no difference in the percentage of Aβ‐positive cells upon exposure to Aβ1‐42 (10 μM) combined with ACT (1,000:1, 100:1, and 10:1 molar ratio), versus Aβ1‐42 alone. CLSM revealed binding of Aβ1‐42 to the cellular surfaces and cellular internalization of smaller Aβ1‐42 fragments. Under these conditions, there was no increase in cellular release of the proinflammatory chemokine monocyte‐chemoattractant protein 1, as compared with nontreated control astrocytes. Thus, primary human astrocytes derived from different sources can bind and internalize Aβ1‐42, and fetal astrocytes were more efficient in Aβ1‐42 uptake than adult astrocytes.

Nitric oxide synthase in the gill of Atlantic salmon : colocalization with and inhibition of Na+,K+-ATPase

SUMMARY We investigated the relationship between nitric oxide (NO) and Na+,K+-ATPase (NKA) in the gill of anadromous Atlantic salmon. Cells containing NO-producing enzymes were revealed by means of nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, which can be used as an indicator of NOS activity, i.e. NO production. Antibodies against the two constitutive NOS isoforms, neuronal and endothelial NOS, both produced immunoreactivity restricted to large cells at the base and along the secondary lamellae. NADPHd-positive cells showed a corresponding distribution. Antibodies against the inducible NOS isoform only labeled small cells located deep in the filament. Using in situ hybridization and NKA immunoreactivity, cells expressing Na+,K+-ATPaseα -subunit mRNA were found to have a similar distribution to the NOS cells. Double labeling for NOS immunoreactivity and NKA α-subunit mRNA revealed cellular colocalization of NKA α-subunit mRNA and nNOS protein in putative chloride cells at the base of the lamellae and interlamellar space. Along the lamellae, some NOS- or NKA-immunoreactive cells possessed a relatively lower expression of NKA α-subunit mRNA in smolts. A clear increase in NADPHd staining in the gill was demonstrated from parr to smolt. The regulatory role of NO on gill NKA activity was studied in vitro using sodium nitroprusside (SNP; 1 mmol l-1) and PAPA-NONOate (NOC-15; 0.5 mmol l-1) as NO donors. Both SNP and NOC-15 inhibited gill NKA activity by 30% when compared to controls. The study shows that NO systems are abundant in the gill of Atlantic salmon, that NO may be produced preferentially by a constitutive NOS isoform, and suggests that NO influence on gill functions is mediated via intracellular, possibly both auto/paracrine, inhibition of Na+,K+-ATPase activity in chloride cells. Furthermore, the increase in NADPHd in the gill during smoltification suggests a regulatory role of NO in the attenuation of the smoltification-related increase in Na+,K+-ATPase activity prior to entering seawater.

Nitric oxide synthase in the CNS of the atlantic salmon

This study describes for the first time the presence and distribution of the nitric oxide (NO) synthesizing enzyme, NO synthase (NOS), in the retina of a teleost. NADPH diaphorase (NADPHd) histochemistry and NOS immunohistochemistry revealed both NOS immunoreactive and NADPHd positive structures in photoreceptor outer segments, amacrine cells, horizontal cells and ganglion cells. Since NO is known to stimulate the synthesis of cGMP, our results implicate an important role for NO in retinal function, especially in cGMP related events in the photoreceptors.

Identification and distribution of nitric oxide synthase in the brain of adult zebrafish.

Nitric oxide (NO) is proposed to be involved in developmental and plastic processes. We investigated the presence and distribution of nitric oxide synthase (NOS) in the zebrafish (Danio rerio) using molecular and histochemical techniques. A partial gene sequence corresponding to the neuronal NOS isoform (nNOS) was identified, and in situ hybridization revealed cellular nNOS mRNA expression throughout the brain of adult zebrafish, distributed in distinct central nuclei and in proliferation zones. NOS immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase activity partly coincided with the nNOS mRNA expression, however was present also in additional neuronal and non-neuronal cell types. The results indicate the occurrence of different NOS isoforms in the adult brain, of which nNOS may participate in neurotransmission, and in mechanisms related to the continuous growth and neuronal plasticity of the teleost brain.

Partial cloning of constitutive and inducible nitric oxide synthases and detailed neuronal expression of NOS mRNA in the cerebellum and optic tectum of adult Atlantic salmon (Salmo salar)

Studies of different species have implicated nitric oxide (NO) synthase (NOS) in various physiological and pathological events. Three major NOS isoforms are present in the brain of mammals; endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). Little is known about the significance of the presence of these proteins in the brain. We report the first investigation into the presence of nNOS and iNOS isoforms in a teleost, adult Atlantic salmon (Salmo salar). Complementary DNA was synthesized from cerebellum and thymus mRNA using RT-PCR techniques with primers against conserved regions of NOS. Cloning and sequencing revealed a partial gene sequence of 560 bp corresponding to mammalian nNOS from cerebellum cDNA. The predicted protein sequence of identified salmon nNOS possessed 85% identity to that of mammalian nNOS. Northern blot analysis of different tissues revealed expression in brain and heart, and indicated expression of three different nNOS mRNAs in the brain. In addition, a 389 bp sequence corresponding to iNOS was identified in thymus cDNA. Salmon iNOS is almost identical to rainbow trout iNOS (95%), but shows much less amino acid identity to goldfish (65%) and mammalian (52%) iNOS. Phylogenetically, all vertebrate nNOS and iNOS homologues are clustered separately. Expression studies by means of in situ hybridization revealed abundant nNOS mRNA transcripts in distinct neuronal populations throughout the Purkinje cell layer of the corpus cerebellum and the periventricular layer of the optic tectum. Our data show that adult Atlantic salmon possess a gene encoding an nNOS isoform and putative alternatively spliced forms, which are expressed in distinct neuronal populations in the cerebellum and optic tectum, and in yet unidentified cell types in the heart. The data suggest that the arising of different vertebrate NOS isoforms is an evolutionary old event. The well conserved sequences present in salmon and mammalian nNOS may reflect their importance in protein function, whereas interspecies distributional differences in cellular expression of nNOS and sequence differences of iNOS may reflect variations and specializations in routes of NO action in the vertebrate phylogeny.

HAMLET Treatment Delays Bladder Cancer Development

PURPOSE HAMLET is a protein-lipid complex that kills different types of cancer cells. Recently we observed a rapid reduction in human bladder cancer size after intravesical HAMLET treatment. In this study we evaluated the therapeutic effect of HAMLET in the mouse MB49 bladder carcinoma model. MATERIALS AND METHODS Bladder tumors were established by intravesical injection of MB49 cells into poly L-lysine treated bladders of C57BL/6 mice. Treatment groups received repeat intravesical HAMLET instillations and controls received alpha-lactalbumin or phosphate buffer. Effects of HAMLET on tumor size and putative apoptotic effects were analyzed in bladder tissue sections. Whole body imaging was used to study HAMLET distribution in tumor bearing mice compared to healthy bladder tissue. RESULTS HAMLET caused a dose dependent decrease in MB49 cell viability in vitro. Five intravesical HAMLET instillations significantly decreased tumor size and delayed development in vivo compared to controls. TUNEL staining revealed selective apoptotic effects in tumor areas but not in adjacent healthy bladder tissue. On in vivo imaging Alexa-HAMLET was retained for more than 24 hours in the bladder of tumor bearing mice but not in tumor-free bladders or in tumor bearing mice that received Alexa-alpha-lactalbumin. CONCLUSIONS Results show that HAMLET is active as a tumoricidal agent and suggest that topical HAMLET administration may delay bladder cancer development.

Ontogeny of vasotocin-expressing cells in zebrafish: Selective requirement for the transcriptional regulators orthopedia and single-minded 1 in the preoptic area

The neurohypophysial peptide arginine vasotocin, and its mammalian ortholog arginine vasopressin, influence a wide range of physiological and behavioral responses, including aspects of sexual and social behaviors, osmoregulation, stress response, metabolism, blood pressure, and circadian rhythms. Here, we demonstrate that, in zebrafish (Danio rerio), the vasotocin precursor gene arginine vasotocin‐neurophysin (avt) is expressed in two domains in the developing embryo: the dorsal preoptic area and the ventral hypothalamus. In the dorsal preoptic area, avt‐expressing cells are intermingled with isotocin‐neurophysin (ist) ‐expressing cells, and these neurons project to the neurohypophysis (posterior pituitary). In the dorsal preoptic area, the transcriptional regulators orthopedia b (otpb) and simple‐minded 1 (sim1) are required for expression of both avt and ist. In contrast, otp and sim1 are not required for avt expression in the ventral hypothalamus. Thus, the development of these two avt expression domains is influenced by separate gene regulatory networks. Developmental Dynamics 237:995–1005, 2008.

The early ontogeny of neuronal nitric oxide synthase systems in the zebrafish.

SUMMARY To examine a putative role for neuronal nitric oxide synthase (nNOS) in early vertebrate development we investigated nNOS mRNA expression and cGMP production during development of the zebrafish Danio rerio. The nNOS mRNA expression in the central nervous system (CNS) and periphery showed a distinct spatio–temporal pattern in developing zebrafish embryo and young larvae. nNOS mRNA expression was first detected at 19 h postfertilisation (h.p.f.), in a bilateral subpopulation of the embryonic ventrorostral cell cluster in the forebrain. The number of nNOS mRNA-expressing cells in the brain slowly increased, also appearing in the ventrocaudal cell cluster from about 26 h.p.f., and in the dorsorostral and hindbrain cell cluster and in the medulla at 30 h.p.f. A major increase in nNOS mRNA expression started at about 40 h.p.f., and by 55 h.p.f. the expression constituted cell populations in differentiated central nuclei and in association with the proliferation zones of the brain, and in the medulla and retina. In parts of the skin, nNOS mRNA expression started at 20 h.p.f. and ended at 55 h.p.f. Between 40 and 55 h.p.f., nNOS mRNA expression started in peripheral organs, forming distinct populations after hatching within or in the vicinity of the presumptive swim bladder, enteric ganglia, and along the alimentary tract and nephritic ducts. Expression of nNOS mRNA correlated with the neuronal differentiation pattern and with the timing and degree of cGMP production. These studies indicate spatio–temporal actions by NO during embryogenesis in the formation of the central and peripheral nervous system, with possible involvement in processes such as neurogenesis, organogenesis and early physiology.