A GABAergic inhibitory microcircuit controlling cholinergic outflow to the airways.

GABA is the main inhibitory neurotransmitter that participates in the regulation of cholinergic outflow to the airways. We have tested the hypothesis that a monosynaptic GABAergic circuit modulates the output of airway-related vagal preganglionic neurons (AVPNs) in the rostral nucleus ambiguus by using a dual-labeling electron microscopic method combining immunocytochemistry for glutamic acid decarboxylase (GAD) with retrograde tracing from the trachea. We also determined the effects of blockade of GABAA receptors on airway smooth muscle tone. The results showed that retrogradely labeled AVPNs received a significant GAD-immunoreactive (GAD-IR) terminal input. Out of a pooled total of 3,161 synaptic contacts with retrogradely labeled somatic and dendritic profiles, 20.2% were GAD-IR. GAD-IR terminals formed significantly more axosomatic synapses than axodendritic synapses (P < 0.02). A dense population of GABAergic synaptic contacts on AVPNs provides a morphological basis for potent physiological effects of GABA on the excitability of AVPNs. GAD-IR terminals formed exclusively symmetric synaptic specializations. GAD-IR terminals were significantly larger (P < 0.05) in both length and width than unlabeled terminals synapsing on AVPNs. Therefore, the structural characteristics of certain nerve terminals may be closely correlated with their function. Pharmacological blockade of GABAA receptors within the rostral nucleus ambiguus increased activity of putative AVPNs and airway smooth muscle tone. We conclude that a tonically active monosynaptic GABAergic circuit utilizing symmetric synapses regulates the discharge of AVPNs.     

The epithelial cholinergic system of the airways

Acetylcholine (ACh), a classical transmitter of parasympathetic nerve fibres in the airways, is also synthesized by a large number of non-neuronal cells, including airway surface epithelial cells. Strongest expression of cholinergic traits is observed in neuroendocrine and brush cells but other epithelial cell types-ciliated, basal and secretory-are cholinergic as well. There is cell type-specific expression of the molecular pathways of ACh release, including both the vesicular storage and exocytotic release known from neurons, and transmembrane release from the cytosol via organic cation transporters. The subcellular distribution of the ACh release machineries suggests luminal release from ciliated and secretory cells, and basolateral release from neuroendocrine cells. The scenario as known so far strongly suggests a local auto-/paracrine role of epithelial ACh in regulating various aspects on the innate mucosal defence mechanisms, including mucociliary clearance, regulation of macrophage function and modulation of sensory nerve fibre activity. The proliferative effects of ACh gain importance in recently identified ACh receptor disorders conferring susceptibility to lung cancer. The cell type-specific molecular diversity of the epithelial ACh synthesis and release machinery implies that it is differently regulated than neuronal ACh release and can be specifically targeted by appropriate drugs.     

Nimesulide affects antioxidant status during acute lung inflammation in rats

Antiradical activity of nimesulide, a commonly used COX-2 specific inhibitor, was estimated in vitro by 1, 1 diphenyl-2-picrylhydrazyl (DPPH) assay, nitroblue tetrazolium reduction assay and lipid peroxidation assay, respectively. The biochemistry of antioxidant functions of nimesulide was also investigated under control and inflammatory conditions, caused by intratracheal instillation of lipopolysaccharide (LPS). Pro-inflammatory conditions generally end up in oxidative insults, which have been suggested to be the cause of multiple organ failure in inflammation. A primary defense, constituted of antioxidant enzymes, against this oxidative damage has evolved in the body. In this study, male Wistar rats were orally administered with nimesulide (9 mg/kg/twice daily for 1 week), followed by intratracheal instillation with 2 microg of LPS and after 18 hr, antioxidant defense system and lipid peroxidation were measured in liver, lungs and kidneys. Nimesulide pretreatment was found to protect the tissue from enhanced levels of lipid peroxidation, and also stimulated the levels of glutathione-S-transferase (GST) in liver and glutathione reductase in kidneys. Surprisingly, nimesulide oral feeding also significantly suppressed superoxide dismutase (SOD) activity in all the three organs. Although, in our study, nimesulide proved to be an inducer of GST (a marker for chemoprevention) and a scavenger of superoxide anions at higher concentrations (> 250 microM), but the relevance of suppression of SOD enzyme activity, which may contribute to the drug's toxic effects cannot be ignored. The work suggests that further long term studies are needed to confirm nimesulide as a safe drug.     

Reassessment of inflammation of airways in chronic bronchitis.

The term chronic bronchitis has been criticised because it is associated with hypersecretion of mucus rather than bronchial inflammation. This study was designed to establish the presence or absence of clinical chronic bronchitis and measure pulmonary function in 45 patients about to undergo resection of the lung. The condition in the cartilaginous and small airways and the severity of the emphysema were then measured in the resected specimen. The results from 20 patients who had clinical chronic bronchitis were compared with those in 25 patients who did not. The data show that patients with chronic bronchitis had greater inflammation (a) on mucosal surfaces (p less than 0.05) of all bronchi larger than 2 mm luminal diameter and (b) around glands (p less than 0.005) and gland ducts (p less than 0.05) in bronchi larger than 4 mm diameter. A variable degree of inflammation was present in the submucosa of smaller bronchi. The groups had equivalent proportions of mucous glands and Reid''s indices in central airways, and no differences were noted in measurements of pulmonary function, condition of small airways, or emphysema. These data show that the term chronic bronchitis is justified by inflammation of cartilaginous airways and suggest that this abnormality may be the cause of the chronic productive cough.     

The role of the cholinergic system in the central control of thermoregulation in rats

Systemic and central administration of methacholine (a synthetic choline derivative) both produced dose-dependent decreases in rectal temperature in rats at all the ambient temperatures studied. Both at room temperature (22 degrees C) and in the cold (8 degrees C), the hypothermia in response to methacholine application was brought about by both a decrease in metabolic heat production and an increase in cutaneous circulation. In the heat (29 degrees C), the hypothermia was due solely to an increase in respiratory evaporative heat loss. Furthermore, the methacholine-induced hypothermia was antagonized by central pretreatment of atropine (a selective blocker of cholinergic receptors), but not by the central administration of either 6-hydroxydopamine (a relative depletor of catecholaminergic nerve fibers) or 5,6-dihydroxytryptamine (predominately a serotonin depletor). The data indicate that activation of the cholinergic receptors within brain with methacholine decreases heat production and (or) increases heat loss which leads to hypothermia in rats.     

Resistance to outflow of cerebrospinal fluid after central infusions of angiotensin.

Infusions of artificial cerebrospinal fluid (CSF) into the cerebroventricles of conscious rats can raise CSF pressure (CSFp). This response can be modified by some neuropeptides. One of these, angiotensin, facilitates the rise in CSFp. We measured CSFp in conscious rats with a computerized system and evaluated resistance to CSF outflow during infusion of artificial CSF, with or without angiotensin, from the decay kinetics of superimposed bolus injections. Angiotensin (10 ng/min) raised CSFp (P less than 0.05) compared with solvent, but the resistance to CSF outflow of the two groups was similar (P greater than 0.05). Because CSFp was increased by angiotensin without an increase in the outflow resistance, a change in some volume compartment is likely. Angiotensin may raise CSFp by increasing CSF synthesis; this possibility is supported, since the choroid plexuses contain an intrinsic isorenin-angiotensin system. Alternatively, angiotensin may dilate pial arteries, leading to an increased intracranial blood volume.     

Chemical profile of vagal preganglionic motor cells innervating the airways in ferrets: the absence of noncholinergic neurons.

In ferrets, we investigated the presence of choline acetyltransferase (ChAT), vasoactive intestinal peptide (VIP), and markers for nitric oxide synthase (NOS) in preganglionic parasympathetic neurons innervating extrathoracic trachea and intrapulmonary airways. Cholera toxin beta-subunit, a retrograde axonal transganglionic tracer, was used to identify airway-related vagal preganglionic neurons. Double-labeling immunohistochemistry and confocal microscopy were employed to characterize the chemical nature of identified airway-related vagal preganglionic neurons at a single cell level. Physiological experiments were performed to determine whether activation of the VIP and ChAT coexpressing vagal preganglionic neurons plays a role in relaxation of precontracted airway smooth muscle tone after muscarinic receptor blockade. The results showed that 1) all identified vagal preganglionic neurons innervating extrathoracic and intrapulmonary airways are acetylcholine-producing cells, 2) cholinergic neurons innervating the airways coexpress ChAT and VIP but do not contain NOS, and 3) chemical stimulation of the rostral nucleus ambiguus had no significant effect on precontracted airway smooth muscle tone after muscarinic receptor blockade. These studies indicate that vagal preganglionic neurons are cholinergic in nature and coexpress VIP but do not contain NOS; their stimulation increases cholinergic outflow, without activation of inhibitory nonadrenergic, noncholinergic ganglionic neurons, stimulation of which induces airway smooth muscle relaxation. Furthermore, these studies do not support the possibility of direct inhibitory innervation of airway smooth muscle by vagal preganglionic fibers that contain VIP.     

Inhibition of allergic inflammation in the airways using aerosolized antisense to Syk kinase

Activation of the protein tyrosine kinase Syk is an early event that follows cross-linking of Fc gamma R and Fc epsilon R, leading to the release of biologically active molecules in inflammation. We reported previously that aerosolized Syk antisense oligodeoxynucleotides (ASO) depresses Syk expression in inflammatory cells, the release of mediators from alveolar macrophages, and pulmonary inflammation. To study the effect of Syk ASO in allergic inflammation and airway hyperresponsiveness, we used the Brown Norway rat model of OVA-induced allergic asthma. Syk ASO, delivered in a liposome, carrier/lipid complex by aerosol to rats, significantly inhibited the Ag-induced inflammatory cell infiltrate in the bronchoalveolar space, decreasing both neutrophilia and eosinophilia. The number of eosinophils in the lung parenchyma was also diminished. Syk ASO also depressed up-regulation of the expression of beta(2) integrins, alpha(4) integrin, and ICAM-1 in bronchoalveolar lavage leukocytes and reversed the Ag-induced decrease in CD62L expression on neutrophils. Furthermore, the increase in TNF levels in bronchoalveolar lavage following Ag challenge was significantly inhibited. Syk ASO also suppressed Ag-mediated contraction of the trachea in a complementary model. Thus, aerosolized Syk ASO suppresses many of the central components of allergic asthma and inflammation and may provide a new therapeutic approach.     

Control of the maturation and the survival of central noradrenergic neurons in culture.

Central noradrenergic neurons from the locus coeruleus express unique plastic properties. The aim of this study was to identify factors that specifically regulate the development and the survival of the noradrenergic cells. Primary dissociated cultures of embryonic locus coeruleus (LC) neurons were established. Norepinephrine (NE) uptake was used as an index of maturation of the noradrenergic neurons. The noradrenergic cells were identified and quantified following immunocytochemical staining for tyrosine hydroxylase antibody. We have examined the effect of hippocampal target tissue and of cyclic-AMP (cAMP) on the development of these cells. Coculturing LC cells with a low density of hippocampal target cells, resulted in a significant increase in NE uptake. However, when the amount of hippocampal target cells was doubled an enormous decrease in NE uptake occurred. The target stimulatory effect was mediated by both neurons and glia, whereas the inhibitory effect was mediated by direct contact between target glia and LC neurons and detected only in the presence of serum. In addition to target effect, we also tested the effect of elevated intracellular cAMP level on NE uptake versus GABA uptake. GABA uptake served as a developmental index of the non noradrenergic cells. Increasing the intracellular cAMP level, by application of the membrane permeable analog dibutyryl cyclic AMP (DbcAMP), resulted in a selective stimulation of NE uptake, due to enhanced survival of noradrenergic neurons. GABA uptake and the number of non-noradrenergic cells were not changed in the presence of DbcAMP. DbcAMP could maintain the survival of LC neurons in the absence of glial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Area postrema: cholinergic and noradrenergic regulation of emesis. A new concept

In unanaesthetized cats the biochemical mechanisms and the functional characteristics of the emetic action of injection of noradrenaline and McN-A-343, a ganglionic muscarinic stimulant into the cerebral ventricle (i.c.v.) through chronically implanted cannulae were investigated. Both produced dose-dependent and shortlasting emetic response. The emesis evoked by noradrenaline was abolished, whereas the emesis induced by McN-A-343 was not completely blocked after ablation of the area postrema. Further, the emetic response to noradrenaline as well as to McN-A-343 was attenuated or blocked in cats pretreated with 6-hydroxydopamine (i.c.v.) and hemicholinium (i.c.v.); it was abolished in cats pretreated with reserpine (i.c.v.). On the other hand, the emetic response to i.c.v. noradrenaline and to i.c.v. McN-A-343 was not virtually altered in cats pretreated with bretylium (i.c.v.), alpha-methyl-p-tyrosine (i.c.v.) and 5,6-dihydroxytryptamine (i.c.v.). It is postulated that noradrenergic neurones as well as cholinergic axon terminals within the area postrema are necessary for the emetic action of noradrenaline, whereas cholinergic axon terminals within the area postrema subserve the emetic response to McN-A-343. A functional link between cholinergic terminals and noradrenergic neurones as well as a modulatory role of noradrenergic afferents on cholinergic afferents mediating emesis within the area postrema is further proposed. Thus, noradrenergic neurones might represent a common site of confluence of different inputs subserving the emesis in the area postrema. Finally, cholinergic terminals sometimes bypass this area and synapse in the emetic regions of the brainstem regulating emesis.     

Involvement of cholinergic mechanisms in the central control of respiration in the cane toad, Bufo marinus

Chemical substrates, central sites and central mechanisms underlying the regulation of breathing in lower vertebrates have not been well characterized. The present study was undertaken to determine the effect of pH changes and cholinergic agents on the central control of respiration in the cane toad, Bufo marinus. Adult toads were anesthetized, catheterized and unidirectionally ventilated before exposing the brainstem. An airtight buccal cannula was also inserted through the tympanum to record buccal pressure. The animal was decerebrated, anesthetic removed and the responses to pH changes of solutions bathing the ventral surface of the medulla (VSM) were tested by superfusing the VSM with mock cerebrospinal fluid (mCSF) of pH 7.8-normal, 7.2-acidic and 8.4-basic. The acidic solution increased respiratory activity, the basic solution decreased activity and the normal solution had no effect. In addition, cholinergeric agents (acetylcholine-ACh, physostigmine-Phy, nicotine-Nic, and atropine-Atr) dissolved in mCSF were applied bilaterally onto the VSM using filter paper pledgets. ACh, Phy and Nic increased episodic breathing frequency by 14.3+/-9.7, 9.4+/-5.4 and 29.1+/-11.8 %, respectively, whereas, Atr caused a decrease (-26.6+/-16.6%). These agents had no effect on blood pressure. It is therefore, concluded that the VSM is pH sensitive and a cholinergic mechanism is involved in the central modulation of respiration in Bufo.     

Pharmacological analysis of reactivity changes in airways due to acute inflammation in cats

Our recent in vitro studies on airways smooth muscles of the cat with turpentine oil inflammation showed the occurrence of a contractile response of tracheal preparations and a significant increase in the isometric tension of lung strips to histamine application. This study was aimed to establish whether histamine H2-receptors participated in the changed in vitro reactivity of the airways smooth muscles of cats suffering from experimentally induced airway inflammation. Pretreatment of control tracheal preparations, control and experimental groups of the lung strips by cimetidine did not change the character of the histamine response. Similarly, the amplitude of histamine relaxation, of the tracheal preparations partially contracted by carbachol was unchanged by experimental inflammation. Clemastine significantly shifted the histamine dose-response curves to the right in both groups of lung strips. However, significant differences in lung strip reactivity between control and experimental groups of cats were not eliminated. Our results do not support the role of histamine H2-receptors in the pathologically increased airway reactivity to histamine in vitro.     

Relationships between eupnoeic pattern of breathing and ventilatory control in man: I. Response to airways occlusion during active lung inflation.

The apnoeic response following interruption of the air flow at different levels of the inspiratory capacity (deltaVL) was studied in conscious children and adults. Changes in mouth pressure were used to measured the duration of the apnoe. The total duration of the interrupted breath (T1) was compared to mean value of the ventilatory period of the five preceding breaths (T0). A monoexponential regression could be fitted to the relationship between T1/T0 ratio and change in lung volume (deltaVL) measured at the onset of interruption: T1/T0=k-exp (S-deltaVL), S begin the sensitivity of the response to lung inflation. When T1/T0=1, the intrathoracic lung volume was called threshold volume (VTh.L.). The parameters S and VTh.L. were used for characterization of the individual importance of the Breuer-Hering inspiratory-inhibitory reflex (B.H. reflex). The high reproducibility of the T1/T0 vs. deltaVL relationship in many subjects showed the light influence of voluntary control on apnoea's duration. In each subject, S and VTh.L. were compared with ventilatory variables measured during eupnoea. A fast pattern of breathing (i.e. small inspired volume and short inspiratory duration) was associated with high value of S and low VTh.L. Moreover VTh.L. was near the tidal volume range in subjects where the B.H. reflex was the more potent. Thus, vagal afferents relating to this reflex could modulate the eupnoeic pattern of some subjects.     

Desensitization of central cholinergic mechanisms and neuroadaptation to nicotine

This review focuses on neuroadaptation to nicotine. The first part of the paper delineates some possible general mechanisms subserving neuroadaptation to commonly abused drugs. The postulated role of the mesocorticolimbic neuroanatomical pathway and drug-receptor desensitization mechanisms in the establishment of tolerance to, dependence on, and withdrawal from psychoactive drugs are discussed. The second part of the review deals with the pharmacological effects of nicotine at both pre- and postsynaptic locations within the central nervous system, and the still-perplexing upregulation of brain nicotine-binding sites seen after chronic nicotine administration. A special emphasis has been put on desensitization of presynaptic cholinergic mechanisms, and postsynaptic neuronal nicotinic-receptor function and its modulation by endogenous substances. A comparison with the inactivation process occuring at peripheral nicotinic receptors is also included. Finally, a hypothesis on the possible connections between desensitization of central cholinergic mechanisms and neuroadaptation to nicotine is advanced. A brief comment on the necessity of fully understanding the effects of nicotine on the developing nervous system closes this work.     

Participation of the central noradrenergic system in the mechanisms of latent inhibition

Male Wistar rats were learnt to conditioned reaction of passive avoidance 10 days after intraperitoneal administration of 50 mg/kg of specific neurotoxin DSP4. The character of conditioned reaction reproduction, duration of its conservation and its stability against amnestic processing were analyzed. It has been found that reduction of activity of noradrenergic coerulo-cortical system does not influence the conditioned reaction reproduction but inhibits its spontaneous extinction and prevents amnesia development. The obtained data are discussed in aspect of central noradrenaline participation in latent inhibition mechanisms.     

Bombesin affects the central nervous system to produce hyperglycemia in rats

Bombesin, a peptide isolated from frog skin, acts through the central nervous system to produce hyperglycemia in rats. Bombesin induced hyperglycemia may be mediated via adrenomedullary-catecholamine induced changes in insulin and glucagon secretion resulting in enhanced hepatic glucose output.     

Role of nitric oxide in central sympathetic outflow

The gaseous molecule nitric oxide (NO) plays an important role in cardiovascular homeostasis. It plays this role by its action on both the central and peripheral autonomic nervous systems. In this review, the central role of NO in the regulation of sympathetic outflow and subsequent cardiovascular control is examined. After a brief introduction concerning the location of NO synthase (NOS) containing neurons in the central nervous system (CNS), studies that demonstrate the central effect of NO by systemic administration of NO modulators will be presented. The central effects of NO as assessed by intracerebroventricular, intracisternal, or direct injection within the specific central areas is also discussed. Our studies demonstrating specific medullary and hypothalamic sites involved in sympathetic outflow are summarized. The review will be concluded with a discussion of the role of central NO mechanisms in the altered sympathetic outflow in disease states such as hypertension and heart failure.