The effect of neuromuscular blockade on the efficiency of facemask ventilation in patients difficult to facemask ventilate: a prospective trial.

Facemask ventilation of the lungs can be an important rescue intervention in a 'cannot intubate' scenario. We assessed the effect of neuromuscular blockade on expiratory tidal volumes in patients with expected difficulty in mask ventilation. The lungs of patients with at least three predictors of difficulty in mask ventilation were ventilated using a facemask held with two hands, with mechanical ventilation set in a pressure-controlled mode. Tidal volumes were recorded before and after the establishment of complete neuromuscular block. In 113 patients, median (IQR [range]) tidal volume increased from 350 (260-492 [80-850]) ml initially, by 48% to 517 (373-667 [100-1250]) ml 30 s after rocuronium administration, (p < 0.001). After the onset of the complete neuromuscular block, a median tidal volume of 600 (433-750 [250-1303]) ml was observed, corresponding to an increase of 71% from baseline values (p < 0.001), and 16% from values obtained 30 s after rocuronium administration, respectively; p = 0.003). No decrease in the tidal volume during the measurements was observed. We conclude that the administration of rocuronium at a dose of 0.6 mg.kg-1 was able to improve facemask ventilation in all cases with a potentially clinically relevant increase in tidal volume. The early use of a neuromuscular blocking agent can be considered as a therapeutic option in case of difficulty with mask ventilation.

An observational study of outcome in SLE patients with biopsy-verified glomerulonephritis between 1986 and 2004 in a defined area of southern Sweden: the clinical utility of the ACR renal response criteria and predictors for renal outcome.

OBJECTIVES: To test the utility of the World Health Organization (WHO) and International Society of Nephrology/Renal Pathology Society (ISN/RPS) criteria for lupus nephritis (LN) in systemic lupus erythematosus (SLE) and the American College of Rheumatology renal response criteria (ACR-RRC) for renal follow-up in an observational cohort. METHOD: All 52 biopsy-verified cases of LN during 19 years were identified, and glomerular filtration rate (GFR), serum creatinine, proteinuria, haematuria, Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K), and complement were retrieved at diagnosis of nephritis, after 6 and 12 months, and at the latest visit. Forty-five renal biopsies were available for re-evaluation with the ISN/RPS criteria. Outcome was defined by the ACR-RRC and the final GFR. RESULTS: The mean follow-up time was 9 years; complete renal response (CRR) was achieved in 11 cases, end-stage renal disease (ESRD) in four, and nephrotic syndrome (NS) in one. The final GFR decreased with increasing age at biopsy (p < 0.01) and with interstitial manifestations added to the ISN/RPS classification (p < 0.05). The final GFR correlated with the decrease of proteinuria or casts and actual serum creatinine after 6 months of treatment (all p < 0.05). The outcome defined by ACR-RRC correlated with the nephrological components of SLEDAI-2K after 6 months of therapy (p < 0.01) and with the presence of antibodies to C1q at biopsy (p < 0.05). CONCLUSIONS: Renal outcome is correlated with the response to treatment after 6 months and with the addition of interstitial changes to the ISN/RPS classification, which might add useful information for prediction. The ACR-RRC offers a defined alternative to categorize renal response.

Severe lung lesions caused by Salmonella are prevented by inhibition of the contact system.

Vascular damage induced by trauma, inflammation, or infection results in an alteration of the endothelium from a nonactivated to a procoagulant, vasoconstrictive, and proinflammatory state, and can lead to life-threatening complications. Here we report that activation of the contact system by Salmonella leads to massive infiltration of red blood cells and fibrin deposition in the lungs of infected rats. These pulmonary lesions were prevented when the infected animals were treated with H-D-Pro-Phe-Arg-chloromethylketone, an inhibitor of coagulation factor XII and plasma kallikrein, suggesting that inhibition of contact system activation could be used therapeutically in severe infectious disease.

Topical application of dynorphin A (1-17) antibodies attenuates neuronal nitric oxide synthase up-regulation, edema formation, and cell injury following focal trauma to the rat spinal cord.

Previous investigations from our laboratory show that up-regulation of neuronal nitric oxide synthase (NOS) following spinal cord injury (SCI) is injurious to the cord. Antiserum to dynorphin A (1-17) induces marked neuroprotection in our model of SCI, indicating an interaction between dynorphin and NOS regulation. The present investigation was undertaken to find out whether topical application of dynorphin A (1-17) antiserum has some influence on neuronal NOS up-regulation in the traumatized spinal cord. SCI was produced in anesthetized animals by making a unilateral incision into the right dorsal horn of the T10-11 segments. The antiserum to dynorphin A (1-17) was applied (1 : 20, 20 microL in 10 seconds) 5 minutes after trauma over the injured spinal cord and the rats were allowed to survive 5 hours after SCI. Topical application of dynorphin A (1-17) antiserum significantly attenuated neuronal NOS up-regulation in the adjacent T9 and T12 segments. In the antiserum-treated group, spinal cord edema and cell injury were also less marked. These observations provide new evidence that the opioid active peptide dynorphin A may be involved in the mechanisms underlying NOS regulation in the spinal cord after injury, and confirms our hypothesis that up-regulation of neuronal NOS is injurious to the cord.

Intracerebral administration of neuronal nitric oxide synthase antiserum attenuates traumatic brain injury-induced blood-brain barrier permeability, brain edema formation, and sensory motor disturbances in the rat.

The role of nitric oxide (NO) in traumatic brain injury (TBI)-induced sensory motor function and brain pathology was examined using intracerebral administration of neuronal nitric oxide synthase (nNOS) antiserum in a rat model. TBI was produced by a making a longitudinal incision into the right parietal cerebral cortex limited to the dorsal surface of the hippocampus. Focal TBI induces profound edematous swelling, extravasation of Evans blue dye, and up-regulation of nNOS in the injured cerebral cortex and the underlying subcortical areas at 5 hours. The traumatized animals exhibited pronounced sensory motor deficit, as seen using Rota-Rod and grid-walking tests. Intracerebral administration of nNOS antiserum (1 : 20) 5 minutes and 1 hour after TBI significantly attenuated brain edema formation, Evans blue leakage, and nNOS expression in the injured cortex and the underlying subcortical regions. The nNOS antiserum-treated rats showed improved sensory motor functions. However, administration of nNOS antiserum 2 hours after TBI did not influence these parameters significantly. These novel observations suggest that NO participates in blood-brain barrier disruption, edema formation, and sensory motor disturbances in the early phase of TBI, and that nNOS antiserum has some potential therapeutic value requiring additional investigation.

Immunologic proliferation marker Ki-S2 as prognostic indicator for lymph node-negative breast cancer.

BACKGROUND: Proper treatment of lymph node-negative breast cancer depends on an accurate prognosis. To improve prognostic models for this disease, we evaluated whether an immunohistochemical marker for proliferating cells, Ki-S2 (a monoclonal antibody that binds to a 100-kd nuclear protein expressed in S, G2, and M phases of the cell cycle), is an accurate indicator of prognosis. METHODS: We studied 371 Swedish women with lymph node-negative breast cancer; the median follow-up time was 95 months. The fraction of tumor cells in S phase was assessed by flow cytometry, and tumor cell proliferation was measured immunohistochemically with the monoclonal antibodies Ki-S2 and Ki-S5 (directed against the nuclear antigen Ki-67). A combined prognostic index was calculated on the basis of the S-phase fraction, progesterone receptor content, and tumor size. RESULTS: In multivariate analyses that did or did not (263 and 332 observations, respectively) include the S-phase fraction and the combined prognostic index, the Ki-S2 labeling index (percentage of antibody-stained tumor cell nuclei) emerged as the most statistically significant predictor of overall survival, disease-specific survival, and disease-free survival (all two-sided P<.0001). In the risk group defined by a Ki-S2 labeling index of 10% or less, life expectancy was not statistically significantly different from that of age-matched women without breast cancer, whereas the group with a high Ki-S2 labeling index had an increased risk of mortality of up to 20-fold. CONCLUSIONS: Cellular proliferation is a major determinant of the biologic behavior of breast cancer. Prognosis is apparently best indicated by the percentage of cells in S through M phases of the cell cycle. Measurement of the Ki-S2 labeling index of a tumor sample may improve a clinician's ability to make an accurate prognosis and to identify patients with a low risk of recurrence who may not need adjuvant therapy.

The retinoblastoma gene in breast cancer: allele loss is not correlated with loss of gene protein expression.

The significance of the retinoblastoma gene (RB) in the development of human breast cancer remains unclear. In the present study, loss of heterozygosity (LOH) in RB was found in 26% of 90 informative primary breast tumors and was correlated to DNA nondiploidy, a high S-phase fraction, and LOH at chromosome 17p13.3. However, allele loss was not associated with loss of RB protein (pRB) expression. Low to absent levels of pRB were found in 15% of 73 immunoblot analyzed tumors, most of which manifested retained heterozygosity in RB. Conversely, tumors exhibiting LOH showed often high pRB expression. Our data suggest that RB may be involved in the pathogenesis of some breast tumors, as evidenced by the absence of pRB, but that this alteration is acquired by mechanisms other than the unmasking of a recessive mutation by allele loss. LOH in RB may be merely a stochastic event in the unstable genome of aneuploid, rapidly proliferating cells or, alternatively, reflect the presence of an adjacent tumor suppressor gene.

Heme oxygenase and carbon monoxide: regulatory roles in islet hormone release: a biochemical, immunohistochemical, and confocal microscopic study.

Carbon monoxide (CO) has been suggested as a novel messenger molecule in the brain. We now report on the cellular localization and hormone secretory function of a CO-producing constitutive heme oxygenase (HO-2) in mouse islets. Islet homogenates produced large amounts of CO which were suppressed dose-dependently by the HO inhibitor zincprotoporphyrin-IX (ZnPP-IX). We also show, for the first time, that glucose markedly stimulates the HO activity (CO production) in intact islets. A further potentiation was induced by the HO substrate hemin. Western blot showed that islet tissue expressed HO-2, and confocal microscopy revealed that HO-2 resided in insulin, glucagon, somatostatin, and pancreatic polypeptide cells. ZnPP-IX dose-dependently inhibited, whereas hemin enhanced, both insulin and glucagon secretion from glucose-stimulated islets. Stimulation or inhibition of CO production was accompanied by corresponding changes in islet cGMP levels. Exogenously applied CO stimulated insulin and glucagon release from isolated islets, whereas exogenous nitric oxide (NO) inhibited insulin and stimulated glucagon release. Islets stimulated by glucose or L-arginine displayed a marked increase in their NO-synthase (NOS) activity. Such an increase was suppressed by hemin, conceivably because NOS activity was inhibited by hemin-derived CO. Consequently, hemin enhanced L-arginine-induced insulin secretion. Insulin release stimulated by either hemin-derived CO or exogenous CO was strongly inhibited by the guanylate cyclase inhibitor ODQ, but it was unaffected by ZnPP-IX. Glucagon release induced by CO (but not by hemin) was inhibited by ODQ and partly inhibited by ZnPP-IX. We propose that the islets of Langerhans are equipped with a heme oxygenase-carbon monoxide pathway, which constitutes a novel regulatory system of physiological importance for the stimulation of insulin and glucagon release. This pathway is stimulated by glucose, is at least partly dependent on the cGMP system, and displays interaction with islet NOS activity.

Reduced level of uterine norepinephrine transmitter during hCG-induced pseudopregnancy in the rabbit.

The bulk of rabbit uterine norepinephrine is present in short adrenergic neurons which have unique functional properties. Pseudopregnancy was induced in adult nulliparous rabbits by a single injection of 1,500 IU of human chorionic gonadotrophin (hCG). By the 6th day of pseudopregnancy, the total uterine content of norepinephrine was reduced to a level only 40% of that found in untreated controls. At 12 days after the injection the amine level was greater than at 6 days but still significantly less than in controls. By 18 days, when the progestational proliferation of the endometrium had subsided, the levels in uteri of control and injected animals were similar. The findings support previous findings indicating the presence of a peripheral neuro-endocrine mechanism influencing uterine motor function.

Distribution, origin and projections of nitric oxide synthase-containing neurons in gut and pancreas.

Nitric oxide has been put forward as an important inhibitory neurotransmitter in the gut. Nitric oxide synthase-containing neurons were visualized by immunocytochemistry using antibodies against neuronal nitric oxide synthase or by beta-nicotinamide adenine dinucleotide phosphate diaphorase staining in whole mounts and cryostat sections from the gastrointestinal tract and pancreas of several mammals (mouse, rat, hamster, guinea-pig, cat and man). Nitric oxide synthase-containing neuronal cell bodies were numerous in the myenteric but fewer in the submucous ganglia all along the gut of all species. Varicose nerve terminals formed extensive networks in the circular smooth muscle and the myenteric ganglia. Nitric oxide synthase-containing nerve terminals were frequently found around the Brunner glands in the duodenum; scattered nerve terminals were also found in the gastric and colonic mucosa and around blood vessels in the submucosa all along the gut. In the rat small and large intestine nitric oxide synthase-containing submucous neurons terminated within the mucosa/submucosa and nitric oxide synthase-containing myenteric neurons issued short descending projections, approximately 3 mm, to the smooth muscle and other myenteric ganglia. In the pancreas of all species nitric oxide synthase-containing nerve cell bodies were regularly seen in intrapancreatic ganglia. Positive nerve fibers were mainly found within nerve trunks in interlobular spaces and as delicate fibers within the islets. Double staining for nitric oxide synthase and neuropeptides in intestine and pancreas of rat, guinea-pig and man revealed that only occasionally the nitric oxide synthase-containing nerve cell bodies stored in addition vasoactive intestinal peptide and neuropeptide Y, or enkephalin. However, nitric oxide synthase-containing nerve terminals, particularly those in the circular muscle of the gut, frequently contained vasoactive intestinal peptide/neuropeptide Y (rat and man) or vasoactive intestinal peptide/enkephalin (guinea-pig). In intrapancreatic ganglia few nitric oxide synthase-containing nerve cell bodies were also vasoactive intestinal peptide-immunoreactive. Coexistence of nitric oxide synthase and vasoactive intestinal peptide in nerve terminals could here be detected around blood vessels and interlobular ducts. The distribution of nitric oxide synthase indicates a major role of nitric oxide in the regulation of gut motility; a role in the regulation of blood flow and secretion in both gut and pancreas is also likely.

Distribution and effects of pituitary adenylate cyclase-activating peptide in the rabbit eye.

Pituitary adenylate cyclase-activating peptide (PACAP)-like immunoreactivity was demonstrated by immunocytochemistry together with calcitonin gene-related peptide (CGRP)-like immunoreactivity in small to medium-sized neurons in the trigeminal ganglion and in nerve fibers in the iris, ciliary body, cornea, choroid and sclera of the rabbit eye. The regional distribution of PACAP-27- and PACAP-38-like immunoreactivity in the eye was studied by radioimmunoassay: the highest concentrations were found in the iris sphincter and ciliary body. The distribution pattern resembled that of CGRP-like immunoreactivity, which is a well-known constituent of sensory C-fibre neurons. Intravitreal injection of PACAP-27 or PACAP-38 induced conjunctival hyperemia, swelling of the anterior segment of the eye, miosis and breakdown of the blood-aqueous barrier, manifested as a marked aqueous flare response. Tetrodotoxin pretreatment inhibited the conjunctival hyperemia, the swelling of the anterior segment of the eye, and the miosis but not the aqueous flare response. The concentration of PACAP-like immunoreactivity in the aqueous humor was increased greatly following infrared irradiation of the iris, topical application of formaldehyde to the cornea, or intravitreal injection of endotoxin or bovine serum albumin. Also the concentration of CGRP-like immunoreactivity in the aqueous humor was increased greatly. Both in vivo and in vitro studies showed that capsaicin caused a parallel release of PACAP-like immunoreactivity and CGRP-like immunoreactivity from the uvea. Injection of PACAP-27 and PACAP-38 resulted in the release of CGRP-like immunoreactivity (and PACAP-like immunoreactivity) into the aqueous humor and PACAP-27 and PACAP-38 were also found to evoke tachykinin-mediated contractions of the isolated iris sphincter muscle, indicating that PACAP induces positive feedback on C-fibres. Thus, PACAP is a sensory neuropeptide in the eye. Since the PACAP-induced ocular responses mimicked the symptoms of inflammation, and since the PACAP-like immunoreactivity concentration in the aqueous humor was greatly increased following noxious stimulation, we suggest that it takes part in the inflammatory responses of the rabbit eye.

Histochemical demonstration of a concomitant reduction in neural vasoactive intestinal polypeptide, acetylcholinesterase, and noradrenaline of cat uterus during pregnancy.

Noradrenaline, acetylcholinesterase, and vasoactive intestinal polypeptide (VIP) were visualized in uterine nerves of cats by histochemical techniques. Alterations were followed in different regions of the organs at various stages of pregnancy and compared with the situation in non-pregnant controls. Positively stained nerve fibres, the adrenergic type being particularly well developed, were found along the muscle bundles and around blood vessels in the smooth muscle layers, as well as in the mucosa, of both uterine horns and cervix. The nerve supply was especially prominent in the upper part of the cervix. The distribution of VIP-immunoreactive and acetylcholinesterase-positive nerve fibres resembled each other, but they were less numerous than the adrenergic fibres. In the course of pregnancy there was a marked reduction in the number of all positively reacting nerves, so that almost no fibres were visible in the uterine horns near term. A small number of positive nerve fibres was found to remain, however, in the wall of the sterile (empty) horn during unilateral pregnancy. The reduction was less prominent in the cervix, particularly its lower part. Distinct changes were encountered already during early and mid pregnancy in those parts of the uterine wall distended by the growing conceptus, where almost no fibres were seen. The nerve supply was more intact in the non-distended portions located between the fetuses, and especially in the empty horn of unilateral pregnancy. No overt reduction in the number of positively stained nerve fibres was found in the cervix at these pregnancy stages. The results show that marked alterations take place in the uterine autonomic innervation during such an entirely physiological event as pregnancy. There is reason to assume that the histochemical observations reflect both structural and functional alterations in the innervation related both to the type of nerves involved and to the localization of the conceptus.

Morphological and functional evidence against a sensory and sympathetic origin of nitric oxide synthase-containing nerves in the rat lower urinary tract.

To establish which type of nerves (parasympathetic, sympathetic or sensory) produce nitric oxide in the rat lower urinary tract, chemical denervation of primary afferents and sympathetic nerves was carried out by systemic treatment with capsaicin and 6-hydroxydopamine, respectively, followed by identification of neuronal nitric oxide synthase immunoreactivity. Functional in vitro studies were also performed to examine whether the synthesis and release of nitric oxide was affected following treatment with the respective neurotoxins. Nerve fibres immunoreactive for substance P and calcitonin gene-related peptide were found in control tissue, but could not be detected following capsaicin treatment. In comparison, nitric oxide synthase-immunoreactive fibres appeared to be unaffected by capsaicin treatment. Administration of 6-hydroxydopamine resulted in a complete disappearance of tyrosine hydroxylase-immunoreactive nerves, whereas nitric oxide synthase-containing nerve fibres did not appear to be affected by the treatment. In ultrastructural studies, nitric oxide synthase immunoreactivity, as studied by colloidal gold particles, was found in the axoplasm and not in association with intraneuronal structures or synaptic vesicles. Gold particles representing substance P immunoreactivity were seen as clusters associated with large granular vesicles. In consecutive sections of nerve fibres, substance P and nitric oxide synthase were not found in the same axon profile. In functional studies on urethral tissue, application of capsaicin (1 microM) produced a long-lasting relaxation. The nitric oxide synthase inhibitor NG-nitro-L-arginine (0.1 mM) had no effect on this response. Systemic treatment with capsaicin or 6-hydroxydopamine had no effect on nerve-evoked, nitric oxide-mediated relaxations. The data suggest that nitric oxide synthase-containing nerves in the rat lower urinary tract do not belong to nerve populations sensitive to either the sympathetic neurotoxin, 6-hydroxydopamine, or the sensory neurotoxin, capsaicin.

Facilitatory effect of pituitary adenylate cyclase activating polypeptide on micturition in normal, conscious rats.

In unanaesthetized, normal rats, continuous cystometry revealed that pituitary adenylate cyclase activating peptide (PACAP-27), administered intrathecally or intra-arterially near the bladder, stimulated micturition. The localization of PACAP-27 in the rat lower urinary tract was studied by immunohistochemistry, and the direct effects on the smooth muscles of the rat detrusor and urethra were investigated in vitro. In the intact rat, 1.0 nmol of PACAP-27 administered intrathecally as well as intra-arterially close to the bladder, but not intravenously, increased micturition pressure, decreased micturition volume and bladder capacity, and facilitated spontaneous bladder contractions. PACAP-27 immunoreactive structures were extremely scarce in the lower urinary tract, and the peptide had negligible effects on isolated detrusor muscle contracted by carbachol or stimulated electrically, or on urethral preparations contracted by noradrenaline. These results suggest that PACAP-27 has facilitatory actions on micturition both at the spinal cord and peripheral ganglionic levels.

Heme oxygenase-1, heme oxygenase-2 and biliverdin reductase in peripheral ganglia from rat, expression and plasticity.

The expression of inducible and constitutive heme oxygenase and biliverdin reductase was studied in normal and cultured peripheral ganglia from adult rats, using immunocytochemistry and in situ hybridization. Dramatic changes were induced by one to two days' culturing of dorsal root ganglia, nodose ganglia, otic ganglia, sphenopalatine ganglia and superior cervical ganglia. An up-regulation of inducible heme oxygenase was found in satellite cells of the cultured nodose ganglia, dorsal root ganglia, sphenopalatine ganglia and otic ganglia, whereas only a few satellite cells in the superior cervical ganglia responded with an increase in inducible heme oxygenase immunoreactivity. In the superior cervical ganglia inducible heme oxygenase also appeared in a subpopulation of macrophages. During culturing, expression of inducible heme oxygenase immunoreactivity also increased in axons and in nerve cell bodies. In situ hybridization corroborated the immunocytochemical findings, revealing a strong up-regulation of inducible heme oxygenase messenger RNA in satellite cells, and less pronounced up-regulation in nerve cell bodies. Constitutive heme oxygenase immunoreactivity was found in most neurons in all of the ganglia studied. No significant changes in constitutive heme oxygenase immunoreactivity could be observed in cultured ganglia. Biliverdin reductase immunoreactivity was barely detectable in any of the normal ganglia; however, after culturing it appeared in axons, single nerve cell bodies and nerve cell nuclei. The results show that inducible heme oxygenase is up-regulated in peripheral ganglia after axonal injury, and suggest a role for carbon monoxide in cellular signaling and a requirement for the antioxidant (bilirubin) during the regeneration process.

The contribution of nitric oxide to endotoxin-induced ocular inflammation: interaction with sensory nerve fibres.

1. The actions of nitric oxide (NO) have been investigated in an endotoxin-evoked ocular inflammatory model in the rabbit, with particular emphasis on the relationship between NO, sensory nerves (C-fibres) and the C-fibre neuropeptides, calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase activating peptide (PACAP). 2. Endotoxin, injected intravitreally, evoked inflammatory responses, i.e. conjunctival hyperaemia, miosis and protein extravasation, reflected by the aqueous flare response (AFR). In control rabbits, the maximum AFR was 66.5 +/- 9.5 (arbitrary units). Pretreatment with the NO synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NAME, 200 mg kg-1) given by intravenous injection, inhibited the endotoxin-evoked responses; the AFR was 16.5 +/- 1.9 (n = 8, P < 0.001) and the conjunctival hyperaemia was abolished. 3. Endotoxin-evoked ocular inflammation is associated with the release of CGRP and PACAP from C-fibres. In the eyes challenged with endotoxin, the concentrations of PACAP-27, -38 and CGRP in the aqueous humour were 58.2 +/- 10.9, 54.4 +/- 12.4 and 5526 +/- 519 (pmoll'), respectively. L-NAME inhibited the release of PACAP-27, -38 and CGRP; the concentrations were 14.3 +/- 2.5, 13.5 +/- 2.5 and 510 +/- 67 (pmoll-1), respectively (n = 8, P < 0.01 or 0.001). 4. Intravitreal injection of 0.3 nmol CGRP induced conjunctival hyperaemia and AFR; the maximum AFR was 140.2 +/- 11.4. L-NAME suppressed the response induced by CGRP; the AFR was 23.4 +/- 5.5 (n = 8, P < 0.001). L-NAME abolished the conjunctival hyperaemia induced by PACAP-27 and -38 (0.3 nmol) and reduced the AFR. 5. The inflammatory cells that infiltrated the uvea, cornea and aqueous humour in large numbers in response to intravitreal injection of endotoxin were found to express inducible NOS. L-NAME prevented the appearance of such cells. 6. Our findings suggest that NO plays an important role in the endotoxin-evoked ocular inflammation in the rabbit: NO activates C-fibres causing release of C-fibre neuropeptides into the aqueous humour. In addition, NO mediates scme of the ocular effects of CGRP and PACAP, since L-NAME suppressed the AFR induced by these peptides.

NO synthase in cholinergic nerves and NO-induced relaxation in the rat isolated corpus cavernosum.

1. In the rat corpus cavernosum (CC), the distribution of immunoreactivity for neuronal and endothelial NO synthase (nNOS and eNOS), and the pattern of NOS-immunoreactive (-IR) nerves in relation to some other nerve populations, were investigated. Cholinergic nerves were specifically immunolabelled with antibodies to the vesicular acetylcholine transporter protein (VAChT). 2. In the smooth muscle septa surrounding the cavernous spaces, and around the central and helicine arteries, the numbers of PGP- and tyrosine hydroxylase (TH)-IR terminals were large, whereas neuropeptide Y (NPY)-, VAChT-, nNOS-, and vasoactive intestinal polypeptide (VIP)-IR terminals were found in few to moderate numbers. 3. Double immunolabelling revealed that VAChT- and nNOS-IR terminals, VAChT- and VIP-IR terminals, nNOS-IR and VIP-IR terminals, and TH- and NPY-IR terminals showed coinciding profiles, and co-existence was verified by confocal laser scanning microscopy. TH immunoreactivity was not found in VAChT-, nNOS-, or VIP-IR nerve fibres or terminals. 4. An isolated strip preparation of the rat CC was developed, and characterized. In this preparation, cumulative addition of NO to noradrenaline (NA)-contracted strips, produced concentration-dependent, rapid, and almost complete relaxations. Electrical field stimulation of endothelin-1-contracted preparations produced frequency-dependent responses: a contractile twitch followed by a fast relaxant response. After cessation of stimulation, there was a slow relaxant phase. Inhibition of NO synthesis, or blockade of guanylate cyclase, abolished the first relaxant phase, whereas the second relaxation was unaffected. 5. The results suggest that in the rat CC, nNOS, VAChT- and VIP-immunoreactivities can be found in the same parasympathetic cholinergic neurons. Inhibitory neurotransmission involves activation of the NO-system, and the release of other, as yet unknown, transmitters.

Morphological and functional in vitro and in vivo characterization of the mouse corpus cavernosum.

1. In normal mice, the distribution of adrenergic, cholinergic, some peptidergic, and neuronal nitric oxide synthase (nNOS)-containing nerves were investigated. Functional in vitro correlates were obtained. An in vivo model was developed in which erectile haemodynamics in response to drugs or nerve-stimulation were studied. 2. Immunoreactivities for vesicular acetylcholine transporter protein (VAChT), nNOS-, and vasoactive intestinal polypeptide (VIP), co-existed in nerve fibres and terminal varicosities. Immunoreactivities for neuropeptide Y (NPY) and tyrosine hydroxylase (TH) were found in the same nerve structures. 3. Chemical sympathectomy abolished TH- and NPY-IR nerve structures in cavernous smooth muscle bundles. The distribution of calcitonin gene-related peptide (CGRP)-, nNOS-, VAChT- and VIP-IR nerve structures was unchanged. 4. In endothelial cells of the central and helicine arteries, veins and venules, intense immunoreactivity for endothelial NOS (eNOS) was observed. No distinct eNOS-IR cells were found lining the cavernous sinusoids. 5. In vitro, nerve-induced relaxations were verified, and endothelial NO/cyclic GMP-mediated relaxant responses were established. VIP and CGRP had small relaxant effects. A functioning adenylate cyclase/cyclic AMP pathway was confirmed. 6. Neuronal excitatory responses were abolished by prazosin, or forskolin. VIP and CGRP counteracted contractions, whereas NPY and scopolamine enhanced excitatory responses. 7. In vivo, erectile responses were significantly attenuated by L-NAME (50 mg kg(-1)) and facilitated by sildenafil (200 microg kg(-1)). 8. It is concluded that the mouse is a suitable model for studies of erectile mechanisms in vitro and in vivo.

Nitric oxide synthase in pig lower urinary tract: immunohistochemistry, NADPH diaphorase histochemistry and functional effects.

1. The distribution and colocalization of nitric oxide synthase (NOS)-like immunoreactivity and NADPH diaphorase activity in the pig lower urinary tract were investigated by immunohistochemical and histochemical staining techniques. Functional in vitro studies were performed to correlate the presence of NOS-immunoreactivity/NADPH diaphorase staining with smooth muscle responses involving the L-arginine/nitric oxide (NO) pathway. 2. NOS-immunoreactivity and NADPH diaphorase activity were expressed in nerve trunks and fine nerve fibres in and/or around muscular bundles in the detrusor, trigone and urethra. Thin nerve fibres that dispersed within the muscle bundles were mainly found in the urethral/trigonal area, whereas such fibres were less common in the detrusor. 3. Almost all neuronal structures that were NOS-immunolabeled were also stained for NADPH diaphorase. In contrast, the urothelium, which was intensively stained by the NADPH diaphorase technique, remained unstained by immunohistochemistry. 4. Electrical field stimulation of pig isolated trigonal and urethral preparations induced relaxations, which were inhibited by tetrodotoxin (1 microM) and NG-nitro-L-arginine (L-NOARG, 10 microM). 5. L-Arginine (1 mM), but not D-arginine, inhibited (25-30%) electrically evoked detrusor contractions. This inhibition was reversed by L-NOARG (0.1 mM). L-Arginine did not inhibit detrusor contractions in the presence of scopolamine (1 microM) and had no direct smooth muscle effects per se. 6. Acetylcholine (1 nM-10 microM) caused concentration-dependent relaxations of noradrenaline-induced contractions in pig vesical arteries. Removal of the endothelium practically abolished the acetylcholine-induced relaxation. Pretreatment with L-NOARG (0.1 mM and 0.3 mM) caused a rightward shift of the concentration-response curves to acetylcholine, but the maximal relaxation obtained was significantly reduced (to 65 +/- 12%; n = 6; P < 0.05) only at 0.3 mM L-NOARG. 7. In vessel segments contracted with K+ (60 mM), acetylcholine induced concentration-dependent relaxations. When the vessels were incubated with 0.3 mM L-NOARG and then contracted with K+ (60 mM) all relaxant responses to acetylcholine were abolished. 8. The presence of NO synthesizing enzyme in nerve fibres and the pharmacological evidence for NO-mediated relaxation of the trigone and urethra suggest that NO or a NO-related substance may have a role in inhibitory neurotransmission in these regions. In the detrusor, the presence of NO-synthesizing enzyme in nerves can be demonstrated, but its functional importance is unclear. NO, as well as other endothelium-derived factors seem to be involved in the endothelium-dependent acetylcholine-induced relaxation of pig vesical arteries.