Sympathetic activity and early mobilization in patients in intensive and intermediate care with severe brain injuries: a preliminary prospective randomized study.

BACKGROUND: Patients who experience severe brain injuries are at risk of secondary brain damage, because of delayed vasospasm and edema. Traditionally, many of these patients are kept on prolonged bed rest in order to maintain adequate cerebral blood flow, especially in the case of subarachnoid hemorrhage. On the other hand, prolonged bed rest carries important morbidity. There may be a clinical benefit in early mobilization and our hypothesis is that early gradual mobilization is safe in these patients. The aim of this study was to observe and quantify the changes in sympathetic activity, mainly related to stress, and blood pressure in gradual postural changes by the verticalization robot (Erigo®) and after training by a lower body ergometer (MOTOmed-letto®), after prolonged bed rest of minimum 7 days. METHODS: Thirty patients with severe neurological injuries were randomized into 3 groups with different protocols of mobilization: Standard, MOTOmed-letto® or Erigo® protocol. We measured plasma catecholamines, metanephrines and blood pressure before, during and after mobilization. RESULTS: Blood pressure does not show any significant difference between the 3 groups. The analysis of the catecholamines suggests a significant increase in catecholamine production during Standard mobilization with physiotherapists and with MOTOmed-letto® and no changes with Erigo®. CONCLUSIONS: This preliminary prospective randomized study shows that the mobilization of patients with severe brain injuries by means of Erigo® does not increase the production of catecholamines. It means that Erigo® is a well-tolerated method of mobilization and can be considered a safe system of early mobilization of these patients. Further studies are required to validate our conclusions. TRIAL REGISTRATION: The study was registered in the ISRCTN registry with the trial registration number ISRCTN56402432 . Date of registration: 08.03.2016. Retrospectively registered.

Cardiovascular response, feeding behavior and locomotor activity in mice lacking the NPY Y1 receptor.

Neuropeptide Y (NPY) is a 36-amino-acid neurotransmitter which is widely distributed throughout the central and peripheral nervous system. NPY involvement has been suggested in various physiological responses including cardiovascular homeostasis and the hypothalamic control of food intake. At least six subtypes of NPY receptors have been described. Because of the lack of selective antagonists, the specific role of each receptor subtype has been difficult to establish. Here we describe mice deficient for the expression of the Y1 receptor subtype. Homozygous mutant mice demonstrate a complete absence of blood pressure response to NPY, whereas they retain normal response to other vasoconstrictors. Daily food intake, as well as NPY-stimulated feeding, are only slightly diminished, whereas fast-induced refeeding is markedly reduced. Adult mice lacking the NPY Y1 receptor are characterized by increased body fat with no change in protein content. The higher energetic efficiency of mutant mice might result, in part, from the lower metabolic rate measured during the active period, associated with reduced locomotor activity. These results demonstrate the importance of NPY Y1 receptors in NPY-mediated cardiovascular response and in the regulation of body weight through central control of energy expenditure. In addition, these data are also indicative of a role for the Y1 receptor in the control of food intake.

Angiotensin converting enzyme inhibitor induced angio-oedema: a review of the pathophysiology and risk factors.

Angio-oedema (AE) is a known adverse effect of angiotensin converting enzyme inhibitor (ACE-I) therapy. Over the past several decades, evidence of failure to diagnose this important and potentially fatal reaction is commonly found in the literature. Because this reaction is often seen first in the primary care setting, a review was undertaken to analyse and document the keys to both diagnostic criteria as well as to investigate potential risk factors for ACE-I AE occurrence. A general review of published literature was conducted through Medline, EMBASE, and the Cochrane Database, targeting ACE-I-related AE pathomechanism, diagnosis, epidemiology, risk factors, and clinical decision making and treatment. The incidence and severity of AE appears to be on the rise and there is evidence of considerable delay in diagnosis contributing to significant morbidity and mortality for patients. The mechanism of AE due to ACE-I drugs is not fully understood, but some genomic and metabolomic information has been correlated. Additional epidemiologic data and clinical treatment outcome predictors have been evaluated, creating a basis for future work on the development of clinical prediction tools to aid in risk identification and diagnostic differentiation. Accurate recognition of AE by the primary care provider is essential to limit the rising morbidity associated with ACE-I treatment-related AE. Research findings on the phenotypic indicators relevant to this group of patients as well as basic research into the pathomechanism of AE are available, and should be used in the construction of better risk analysis and clinical diagnostic tools for ACE-I AE.

Interaction between neuropeptide Y (NPY) and brain-derived neurotrophic factor in NPY-mediated neuroprotection against excitotoxicity: a role for microglia.

The neuroprotective effect of neuropeptide Y (NPY) receptor activation was investigated in organotypic mouse hippocampal slice cultures exposed to the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Exposure of 2-week-old slice cultures, derived from 7-day-old C57BL/6 mice, to 8 microm AMPA, for 24 h, induced degeneration of CA1 and CA3 pyramidal cells, as measured by cellular uptake of propidium iodide (PI). A significant neuroprotection, with a reduction of PI uptake in CA1 and CA3 pyramidal cell layers, was observed after incubation with a Y(2) receptor agonist [NPY(13-36), 300 nm]. This effect was sensitive to the presence of the selective Y(2) receptor antagonist (BIIE0246, 1 microm), but was not affected by addition of TrkB-Fc or by a neutralizing antibody against brain-derived neurotrophic factor (BDNF). Moreover, addition of a Y(1) receptor antagonist (BIBP3226, 1 microm) or a NPY-neutralizing antibody helped to disclose a neuroprotective role of endogenous NPY in CA1 region. Cultures exposed to 8 microm AMPA for 24 h, displayed, as measured by an enzyme-linked immunosorbent assay, a significant increase in BDNF. In such cultures there was an up-regulation of neuronal TrkB immunoreactivity, as well as the presence of BDNF-immunoreactive microglial cells at sites of injury. Thus, an increase of AMPA-receptor mediated neurodegeneration, in the mouse hippocampus, was prevented by neuroprotective pathways activated by NPY receptors (Y(1) and Y(2)), which can be affected by BDNF released by microglia and neurons.

Implication of dipeptidylpeptidase IV activity in human bronchial inflammation and in bronchoconstriction evaluated in anesthetized rabbits.

BACKGROUND: Decreased dipeptidylpeptidase IV (DPPIV) activity within the human nasal mucosa has previously been shown to contribute to the severity of chronic inflammatory rhinosinusitis. OBJECTIVE: To investigate and correlate the role of DPPIV activity with regard to bronchial inflammation. METHODS: DPPIV/CD26 activity/concentration was investigated in the bronchial tissue of human subjects suffering from chronic bronchial inflammation. In addition, the effect of a recombinant Aspergillus fumigatus DPPIV (fuDPPIV) was investigated on histamine-induced bronchoconstriction in anesthetized rabbits. RESULTS AND CONCLUSIONS: DPPIV/CD26 was present in submucosal seromucous glands, in leukocytes and to a very low degree in endothelial cells of human bronchi. DPPIV activity was correlated with tissue CD26 content measured by immunoassay. As previously reported for the nasal mucosa, DPPIV/CD26 activity was inversely correlated with the degree of airway inflammation. Systemic pretreatment with recombinant fuDPPIV markedly reduced the increase in histamine-induced airway resistance in rabbits. In conclusion, DPPIV activity modulates lower airway tone by degrading unknown peptidic substrates released by histamine in response to an allergen. Contrasting with our observations in the nose, this modulation is apparently not mediated via a neurokinin (NK1) receptor.

BDNF and DPP-IV in polyps and middle turbinates epithelial cells.

HYPOTHESIS: Neuropeptides released from sensory nerves may contribute to airway inflammation, particularly if their metabolism is impaired through defective inactivation and/or increased production. In the airways, neuropeptides are subjected to degradation by enzymes such as dipeptidyl peptidase (DPP-IV), and are upregulated by neurotrophins such as brain derived neurotrophic factor (BDNF). We therefore assessed in primary human nasal epithelial cells the expression of DPP-IV and BDNF under basal and inflammatory conditions. METHODS: Human epithelial cells were isolated from nasal polyps and middle turbinates, and grown on collagen-coated polycarbonate filters with an air liquid-interface. After three weeks of culture, constitutive cellular expression of DPP-IV and BDNF was assessed by measuring its activity and by ELISA, respectively. To mimick in vivo inflammatory conditions, cells were exposed apically and basolaterally to 50 ng/ml of TNFalpha, IL-1beta, and IFN-gamma for two days. DPP-IV activity and BDNF protein expression were measured in cell lysates and in the apical and basolateral media. RESULTS: Constitutive DPP-IV activity was similar in polyps and turbinates cells. In contrast, polyps epithelial cells expressed higher amounts of BDNF compared to turbinates derived cells. On the other hand, TNFalpha, IL-1beta, and IFN-gamma did not affect DPP-IV activity but significantly increased the cellular expression and the basolateral secretion of BDNF. CONCLUSIONS: Our data show for the first time that primary human airway epithelial cells produced DPP-IV and BDNF under basal conditions. Furthermore, the production and secretion of BDNF were markedly increased in response to pro-inflammatory cytokines, confirming the involvement of BDNF in airway inflammation.

Interleukin-8 is produced in neoplastic and infectious diseases of the human central nervous system.

The presence of interleukin-8 (IL-8), a leukocyte chemotactic factor, was examined in primary and metastatic central nervous system tumors and in nonneoplastic acute meningoencephalitides. In vitro: (a) 11 of 12 glioblastoma cell lines constitutively expressed IL-8 mRNA; (b) 5 of 6 of these cell lines secreted IL-8 protein as detected by enzyme-linked immunosorbent assay and a glucosaminidase release bioassay; and (c) IL-1 beta or tumor necrosis factor was able to augment both IL-8 mRNA steady state levels and protein secretion of all cell lines tested except IN-319. IL-8 was also found in vivo. (a) IL-8 poly A+ mRNA was detected in 2 of 2 low grade astrocytomas, 1 of 2 anaplastic astrocytomas, and 6 of 6 glioblastomas. (b) IL-8 protein was present in the cyst fluid of 1 of 4 low grade astrocytomas, 1 anaplastic astrocytoma, 2 of 2 glioblastomas, 1 oligodendroglioma grade III, and one central nervous system cervical carcinoma metastasis. (c) The cerebrospinal fluid of 3 of 4 metastatic lymphomas, 2 of 16 glioblastomas, 1 of 2 low grade astrocytomas, but none of 3 anaplastic astrocytomas and none of 9 meningiomas contained IL-8. The presence of IL-8 was not restricted to central nervous system tumors as 2 of 2 bacterial meningitis and 5 of 5 acute viral meningitis patients contained considerable IL-8 levels in the cerebrospinal fluid. (d) Immunohistochemical analysis showed IL-8 immunoreactivity in perivascular tumor cells in 11 of 15 glioblastoma sections. These data suggest that IL-8 secretion could be a key factor involved in the determination of the lymphoid infiltrates observed in brain tumors and the development of cerebrospinal fluid pleocytosis in meningoencephalitides.

The role of neuropeptide Y in cardiovascular regulation.

Neuropeptide Y (NPY) is a 36 amino acid peptide present in the brain, the adrenal medulla and peripheral sympathetic nerves. The localization and mode of release of NPY led to the proposal that this peptide plays an important role in modulating the contribution of the sympathetic nervous system to blood pressure control. In this paper Bernard Waeber and colleagues review the current knowledge about the mechanisms involved in NPY signal transduction and the different mechanisms whereby NPY, released by the peripheral nervous system, may influence vascular tone and cardiac function.

Interplay between galanin and leptin in the hypothalamic control of feeding via corticotropin-releasing hormone and neuropeptide Y.

Over long periods, feeding and metabolism are tightly regulated at the central level. The total amount of nutrients ingested is thought to result from a delicate balance between orexigenic and anorexigenic factors expressed and secreted by specialized hypothalamic neuronal populations. We have developed a system of perifused hypothalamic neurons to characterize the relationships existing between the orexigenic peptide galanin and two other physiological modulators of feeding: neuropeptide Y (NPY) and corticotropin-releasing hormone (CRH). We demonstrated that galanin stimulates CRH and NPY secretion from hypothalamic neurons in a dose-dependent manner. Exposure to leptin for 24 h before galanin stimulation decreased NPY secretion by 30%, leaving the responsiveness of CRH neurons intact. These results suggest that CRH and NPY neurons participate to the intrahypothalamic signaling pathway of galanin, an observation that can explain the lower potency of galanin to stimulate food intake in vivo compared with NPY. The differential effects exerted by leptin on CRH and NPY suggest that there exists a subset of NPY neurons that are exquisitely sensitive to marked variations in leptin levels, and that the CRH neurons are less responsive to increases in leptin concentrations.

Urinary and plasma catecholamines and metanephrines in dogs with pheochromocytoma, hypercortisolism, nonadrenal disease and in healthy dogs.

BACKGROUND: Diagnosis of pheochromocytoma (PC) is based on a combination of clinical suspicion, finding an adrenal mass, increased plasma, and urine concentrations of catecholamine metabolites and is finally confirmed with histopathology. In human medicine, it is controversial whether biochemically testing plasma is superior to testing urine. OBJECTIVES: To measure urinary and plasma catecholamines and metanephrines in healthy dogs, dogs with PC, hypercortisolism (HC), and nonadrenal diseases (NAD) and to determine the test with the best diagnostic performance for dogs with PC. ANIMALS: Seven PC dogs, 10 dogs with HC, 14 dogs with NAD, 10 healthy dogs. METHODS: Prospective diagnostic clinical study. Urine and heparin plasma samples were collected and stored at -80°C before analysis using high-pressure liquid chromatography (HPLC) coupled to electrochemical detection or tandem mass spectrometry were performed. Urinary variables were expressed as ratios to urinary creatinine concentration. RESULTS: Dogs with PC had significantly higher urinary normetanephrine and metanephrine:creatinine ratios and significantly higher plasma-total and free normetanephrine and plasma-free metanephrine concentrations compared to the 3 other groups. There were no overlapping results of urinary normetanephrine concentrations between PC and all other groups, and only one PC dog with a plasma normetanephrine concentration in the range of the dogs with HC and NAD disease. Performances of total and free plasma variables were similar. Overlap of epinephrine and norepinephrine results between the groups was large with both urine and plasma. CONCLUSION AND CLINICAL IMPORTANCE: Measurement of normetanephrine is the preferred biochemical test for PC and urine was superior to plasma.

Neuropeptide Y expression and regulation in a differentiated rat insulin-secreting cell line.

Neuropeptide-Y (NPY) is a 36-amino acid peptide known to inhibit glucose-stimulated insulin secretion in various animal models in vitro and in vivo. NPY is thought to be one of the mediators of sympathetic action in the pancreas through nerve endings surrounding the islets, and it has recently been shown to be synthesized within the islets of Langerhans. To elucidate the potential role of NPY in the endocrine pancreas, we studied the expression and regulation of NPY secretion in a rat insulinoma cell line (INS-1). NPY mRNA and peptide are highly expressed and secreted by INS-1 cells. NPY levels were determined by a sensitive and specific two-site amplified enzyme-linked immunosorbent assay. Incubation of INS-1 cells with various glucose concentrations did not modify NPY secretion; however, stimulation of adenylate cyclase by forskolin induced a dose- and time-dependent increase in NPY release in the medium. The glucagon-like peptide-I-(7-36) amide (GLP-1), a known gluco-incretin in humans, induced at low concentration (10(-9) M) a similar expression of NPY mRNA and peptide secretion in INS-1 cells. On the other hand, the inhibition of cAMP accumulation by the alpha 2-adrenergic agonist clonidine decreased NPY secretion. In conclusion, 1) high levels of gene expression and secretion of NPY are found in a rat insulinoma cell line (INS-1). 2) Accumulation of cAMP induced by forskolin or a gluco-incretin (GLP-1) induces a further increase in NPY gene expression and release. 3) NPY secretion is not modulated by low or high glucose concentrations in the medium. 4) Induction of NPY, a known inhibitor of insulin secretion, may represent a novel counterregulatory mechanism of insulin secretion, limiting the stimulatory effect of GLP-1 on insulin secretion.

Plasma neuropeptide Y concentrations in patients with neuroendocrine tumors.

In order to develop an immunoradiometric assay for human neuropeptide (hNPY), a recently discovered and potent vasoconstrictor 36-amino-acid peptide, we used hNPY and some of its subpeptides to prepare monoclonal anti-NPY antibodies. Two monoclonal antibodies with high affinity for hNPY, that is affinity constants in the range of 10(10) mol/L-1, which respectively, reacted with the 9-18 portion and the 32-36 portion of hNPY were used in the immunoradiometric system. The assay was highly specific, NPY-related peptides such as pancreatic polypeptide and peptide YY not being detected. The lower limit of sensitivity was 0.5 pmol/L. In 303 normal subjects, plasma NPY concentrations were less than 0.5 pmol/L in 67%, 0.5 to 5.0 pmol/L in 25% and 5.1 to 30 pmol/L in the remaining 8%. A value of 7.5 pmol/L (95th percentile value in the normal group) was considered as the upper limit of normal. Among 111 patients with various neuroendocrine tumors, elevated plasma NPY concentrations were found in patients with pheochromocytomas and neuroblastomas, the highest plasma levels being found in patients with malignant pheochromocytomas. We conclude that patients with neuroendocrine tumors, especially secreting and or malignant tumors of the sympathochromaffin system, often have elevated plasma NPY concentrations.

Angiotensin-II mediates norepinephrine and neuropeptide-Y secretion in a human pheochromocytoma.

The potential role of angiotensin-II in mediating catecholamine and neuropeptide-Y release in a human pheochromocytoma has been investigated. Angiotensin-II type I receptors are transcribed and translated into functional proteins in a surgically removed pheochromocytoma. Primary cell culture of the tumor has been studied in a perfused system. Angiotensin-II increased the release of norepinephrine and neuropeptide-Y by the pheochromocytes. Activation of the angiotensin-II type I receptors by angiotensin-II was associated with a rise in cytosolic free calcium. The renin-angiotensin system may, therefore, contribute to the secretion of catecholamines and NPY occurring in patients with pheochromocytoma and when stimulated trigger hypertensive crisis.

NPY regulates catecholamine secretion from human adrenal chromaffin cells.

The aim of the present work was to find out whether NPY synthesized in human adrenal chromaffin cells controls in an autocrine/paracrine fashion the release of catecholamines by these cells. Accordingly, the constitutive and regulated release of both NPY and catecholamines was measured simultaneously in cultured human chromaffin cells. In addition, by using both RT-PCR and a combination of specific agonists and antagonists, we characterized the expression of NPY receptors on these cells as well as their pharmacology. Our results were as follows. 1) Human chromaffin cells constitutively secrete NPY. 2) Nicotine elicits a rapid increase in the release of both catecholamines and NPY; this release of NPY is more sustained than that of catecholamines. 3) RT-PCR shows expression of Y1, Y2, Y4, and Y5 receptor mRNA by chromaffin cells; these receptors are functional, as various receptor specific agonists elicit an increase in intracellular calcium. 4) Peptide YY, in contrast to NPY, is not able to stimulate the release of catecholamines. This finding was corroborated by the observation that no receptor-specific antagonists were able to reduce constitutive catecholamine release, whereas an NPY-immunoneutralizing antibody markedly attenuated the secretion. Taken together, these data suggest that NPY originating from the adrenal medulla locally enhances the secretion of catecholamines, presumably by acting via the putative y3 receptor.

The immunological impairment of arcuate neuropeptide Y neurons by ricin A chain produces persistent decrease of food intake and body weight.

Neuropeptide Y is demonstrated as a potent orexigenic peptide when injected into the rat hypothalamic paraventricular nuclei. The neuropeptide Y innervation of paraventricular nuclei originates from both hypothalamic arcuate nuclei and brainstem neurons, whose specific role in the control of food intake is still under discussion. To assess the role of the arcuate neuropeptide Y in the regulation of food intake, we propose a new method for immunologically impairing the neuronal secretion of neuropeptide Y from a unique brain site. The monoclonal antibody to the neuropeptide Y precursor epitope, the C-flanking peptide, was microinjected with two cellular toxins (the ricin A chain and the monensin) into the hypothalamic arcuate nuclei or paraventricular nuclei. One microinjection into the arcuate nuclei reduced the food intake and body weight gain for 10 days. It prevented the food intake stimulation usually induced by a 12 h food deprivation. This decrease of food intake was not due to the aversive properties of monoclonal antibody or cellular toxins, or the immunoneutralization of the biologically active neuropeptide Y, because (i) the acute effect of the microinjection into the arcuate nuclei promoted a transient increase of the food intake likely induced by a strong release of neuropeptide Y from the arcuate neurons which were immunologically damaged, and (ii) the C-flanking peptide monoclonal antibody binds neither neuropeptide Y nor its receptors. The microinjection was inefficient when C-flanking peptide monoclonal antibody was replaced by non-specific rat immunoglobulins or when the C-flanking peptide monoclonal antibody/toxins mixture was injected into the paraventricular nuclei. The data bring further arguments in two domains.(ABSTRACT TRUNCATED AT 250 WORDS)

The subtype 2 of angiotensin II receptors and pressure-natriuresis in adult rat kidneys.

The present work examined the effects of the subtype 2 of angiotensin II (AT2) receptors on the pressure-natriuresis using a new peptide agonist, and the possible involvement of cyclic guanosine 3', 5' monophosphate (cyclic GMP) in these effects. In adult anaesthetized rats (Inactin, 100 mg kg(-1), i.p.) deprived of endogenous angiotensin II by angiotensin converting enzyme inhibition (quinapril, 10 mg kg(-1), i.v.), T2-(Ang II 4-8)2 (TA), a highly specific AT2 receptor agonist (5, 10 and 30 microg kg(-1) min(-1), i.v.) or its solvent was infused in four groups. Renal functions were studied at renal perfusion pressures (RPP) of 90, 110 and 130 mmHg and urinary cyclic GMP excretion when RPP was at 130 mmHg. The effects of TA (10 microg kg(-1) min(-1)) were reassessed in animals pretreated with PD 123319 (PD, 50 microg kg(-1) min(-1), i.v.), an AT2 receptor antagonist and the action of the same dose of PD alone was also determined. Increases in RPP from 90 to 130 mmHg did not change renal blood flow (RBF) but induced 8 and 15 fold increases in urinary flow and sodium excretion respectively. The 5 microg kg(-1) min(-1) dose of TA was devoid of action. The 10 and 30 microg kg(-1) min(-1) doses did not alter total RBF and glomerular filtration rate, but blunted pressure-diuresis and natriuresis relationships. These effects were abolished by PD. TA decreased urinary cyclic GMP excretion. After pretreatment with PD, this decrease was reversed to an increase which was also observed in animals receiving PD alone. In conclusion, renal AT2 receptors oppose the sodium and water excretion induced by acute increases in blood pressure and this action cannot be directly explained by changes in cyclic GMP.

Intranasal administration of neuropeptide Y in man: systemic absorption and functional effects.

1. Exogenous neuropeptide Y (NPY, 10 nmol, 50 nmol and 100 nmol) and its vehicle (NaCl 0.9%) were administered in a double blind, randomized and controlled manner by intranasal spray in 7 healthy volunteers. Variations of plasma NPY concentration over time were measured during 120 min. Forty min after the administration of 50 nmol and 100 nmol of exogenous NPY, plasma NPY increased from 5.5 +/- 1.1 pM to 9.8 +/- 2.3 pM (P < 0.05) and from 9.06 +/- 5.1 pM to 20.8 +/- 6.16 pM (P < 0.001), respectively. There was no significant modification of the mean arterial blood pressure and no subjective discomfort was reported. 2. Nasal airway resistance (NAR) was measured by anterior rhinomanometry and was reduced by 25 +/- 3% and 32 +/- 5% after the spray of 50 nmol and 100 nmol, respectively, for about 90 min. 3. Double-blind, randomized, placebo-controlled and 3-way crossover design experiments were performed in 8 healthy volunteers to evaluate the influence of intranasal pretreatment with NPY (20 nmol) and the mixed alpha 1/alpha 2-adrenoceptor agonist oxymetazoline (20 nmol) on the functional effects of subsequent local irritation evoked by capsaicin (3.3 x 10(-4) mol). Subjective evaluation of NAR and local intensity of discomfort were evaluated by means of a visual analogue scale. Nasal secretions were collected and objective NAR was recorded by rhinomanometry. 4. Subjective NAR, nasal secretions and rhinomanometry recordings were not modified by intranasal application of saline, NPY or oxymetazoline. Subjective nasal obstruction, local discomfort, nasal secretions and NAR increase evoked by capsaicin were markedly reduced by NPY pretreatment (P < 0.05) when compared to saline or oxymetazoline. 5. It is concluded that intranasal application of exogenous NPY has very low systemic absorption but induced long lasting nasal vasoconstriction without cardiovascular effects. Pretreatment of the nasal mucosa with exogenous NPY reduces both secretagogue and vasodilator responses to subsequent application of capsaicin.

Influence of TASP-V, a novel neuropeptide Y (NPY) Y2 agonist, on nasal and bronchial responses evoked by histamine in anaesthetized pigs and in humans.

1. In nine anaesthetized pigs we have studied the influence of intranasal or intrabronchial pretreatment with TASP-V, a neuropeptide Y (NPY) Y2 agonist formed by the attachment of NPY 21-36 to a template-assembled synthetic peptide (TASP), on the functional responses to subsequent intranasal or intrabronchial histamine challenge. 2. In a parallel study, subjective and objective nasal airway resistance (NAR) increase following intranasal histamine challenge was evaluated in 11 healthy volunteers after TASP-V or placebo pretreatment. 3. In pigs, increase in sphenopalatine blood flow induced by histamine dihydrochloride nasal spray (0.25 mg kg(-1) in 3 ml of saline) was significantly reduced by 65% (P<0.05) following intranasal pretreatment with 10 microg kg(-1) of TASP-V. Bronchoconstriction induced by histamine dihydrochloride nebulization (0.5 mg kg(-1) in 3 ml of saline) was significantly attenuated by 25 and 55% following aerosolized pretreatment with TASP-V analogue at 10 and 20 microg kg(-1), respectively. 4. In healthy volunteers, objective increase in NAR and reduction in nasal minimal cross section area (MCSA) induced by intranasal spray of histamine dihydrochloride (15 microg kg(-1) in 200 microl of saline) were significantly attenuated by 50% following local pretreatment with 1.275 microg kg(-1) of TASP-V when compared with saline. 5. It is concluded that intranasal or intrabronchial pretreatment with TASP-V reduced nasal obstruction and bronchoconstriction evoked by histamine challenge in the pig. In healthy human volunteers, this agent attenuated NAR increase and MCSA reduction induced by intranasal application of histamine.

High level expression of human neuropeptide Y receptors in mammalian cells infected with a recombinant vaccinia virus.

Neuropeptide Y (NPY) is a 36 amino acid peptide present in the central and peripheral nervous system. Numerous studies point to a role of NPY in cardiovascular regulation. NPY effects are mediated through stimulation of specific cell surface G protein-coupled receptors. To allow biochemical studies of the receptor and of its interaction with the ligand, we have developed a potent expression system for NPY receptors using a recombinant vaccinia virus. A human NPY receptor cDNA was fused to a strong vaccinia virus promoter and inserted into the viral genome by homologous recombination. Recombinant viruses were isolated and tested for their ability to induce NPY binding site expression following infection of mammalian cell lines. Using saturation and competition binding experiments we measured a Bmax of 5-10 x 10(6) NPY binding sites per cell. The Kd for the binding of NPY is about 20 nM. Labelling of infected cells with a fluorochrome-labelled NPY indicated that the recombinant protein integrates into the cell membrane.

Differential neuropeptide Y gene expression in post-mitotic versus dividing neuroblastoma cells driven by an adeno-associated virus vector.

The ability to express exogenous mammalian genes stably in post-mitotic cells such as neurons remains an important goal for those attempting to modulate neurotransmission through gene delivery. We therefore investigated how differentiation to a post-mitotic state affected the expression of an exogenous gene encoding for neuropeptide Y (NPY) following transfection with an adeno-associated virus (AAV) derived vector. This vector (pJDT95npy) was constructed with rat NPY cDNA (551 bp) inserted downstream from the indigenous AAV p5, p19 and p40 promoters to characterize their relative abilities to drive NPY mRNA expression. Transfection of dividing neuroblastoma CHP126 cells with pJDT95npy resulted in the differential expression of chimeric NPY mRNAs derived from each promoter. P40-driven species became dominant after 1 month post-transfection. Vector integration into chromosomal DNA was demonstrated by Southern blot analyses, indicating at least some region-selective integration. In dividing cell extracts, only a low level of pro-NPY immunoreactivity and no mature NPY immunoreactivity was recovered. However, after differentiation of the pJDT95npy-transfected CHP 126 cells to a post-mitotic state, significant levels of pro-NPY and mature NPY were recovered in the cells and media. Differentiation also had a time-dependent effect on mRNA expression: a spike of p5 driven expression on day 3 was followed predominantly by p40-driven expression on day 5. This study indicates that AAV-derived vectors using the p40 promoter may be used to express genes in post-mitotic cells such as neurons.