Parasympathetic Neurotransmitter Research Articles

Highlights in basic autonomic neurosciences: Epileptic seizure and sudden death

People with epilepsy die unexpectedly for unknown pathological reasons at a rate 24 times greater than the general population. Such deaths are classified as sudden unexplained death in epilepsy (SUDEP). The etiology of the SUDEP is frequently unclear which prevents the development of a rational treatment of this problem. Mice lacking Kv1.1 Shaker-like potassium channels encoded by the Kcna1 gene exhibit severe seizures and die prematurely. In this article the authors postulated that Kv1.1-potassium deficiency may underlie primary neurogenic cardiac dysfunction and cause SUDEP. EEG–ECG recordings showed that Kcna1-null mice display atrioventricular (AV) conduction blocks, as well as bradycardia and premature ventricular contractions. During seizures the occurrence of AV conduction blocks increased, which might cause SUDEP. Then the authors used atropine and propranolol to block the autonomic nervous system and found that atropine ameliorated AV conduction block indicating excessive parasympathetic tone may cause a neurocardiac defect. There were no changes in Kv1.1-deficient cardiac structure, but extensive Kv1.1 expression in juxtaparanodes of the wild-type vagus nerve, the primary source of parasympathetic input to the heart. The authors concluded that Kv1.1 deficiency leads to impaired neural control of cardiac rhythmicity due in part to aberrant parasympathetic neurotransmission, making Kcna1 a strong candidate gene for causing human SUDEP.

Cholinergic Regulation of Airway Inflammation and Remodelling

Acetylcholine is the predominant parasympathetic neurotransmitter in the airways that regulates bronchoconstriction and mucus secretion. Recent findings suggest that acetylcholine regulates additional functions in the airways, including inflammation and remodelling during inflammatory airway diseases. Moreover, it has become apparent that acetylcholine is synthesized by nonneuronal cells and tissues, including inflammatory cells and structural cells. In this paper, we will discuss the regulatory role of acetylcholine in inflammation and remodelling in which we will focus on the role of the airway smooth muscle cell as a target cell for acetylcholine that modulates inflammation and remodelling during respiratory diseases such as asthma and COPD.

Neurotransmitter Influence on Human Meibomian Gland Epithelial Cells

A striking characteristic of the human meibomian gland is its rich sensory, sympathetic, and parasympathetic innervation, yet the functional relevance of these nerve fibers remains unknown. Acting on the hypothesis that neurotransmitters are released in the vicinity of the gland, act on glandular receptors, and influence the production, secretion, and/or delivery of meibomian gland secretions to the ocular surface, the goal in this study was to begin to determine whether neurotransmitters influence the meibomian gland. Immortalized human meibomian gland epithelial (SLHMG) cells were examined for the presence of vasoactive intestinal peptide (VIP) and muscarinic acetylcholine (mACh) receptor transcripts and proteins. Cells were also exposed to VIP, carbachol, forskolin, and/or 3-isobutyl-1-methylxanthine (IBMX) to determine whether these agents, alone or in combination, modulate the adenylyl cyclase pathway, the accumulation of intracellular free calcium ([Ca2+]i), or cell proliferation. Results demonstrate that SLHMG cells transcribe and translate VIP and mACh receptors; VIP, with either IBMX or forskolin, activates the adenylyl cyclase pathway, and the effect of VIP and forskolin together is synergistic; both VIP and carbachol increase intracellular [Ca2+] in SLHMG cells; and VIP with forskolin stimulates SLHMG cell proliferation. This study shows that parasympathetic neurotransmitters and their agonists influence the function of human meibomian gland epithelial cells. It remains to be determined whether this action alters the production, secretion, and/or delivery of meibum to the ocular surface.

Purinergic P2X 3 heteroreceptors enhance parasympathetic motor drive in isolated porcine detrusor, a reliable model for development of P2X selective blockers for detrusor hyperactivity

Various forms of low urinary tract symptoms (LUTS) seem dependant upon dysregulation of the purinergic pathway which produces sensory- or motor-activated incontinence. A body of evidence in human urinary bladders supports a link between up-regulation of purinergic activity and the pathogenesis of detrusor instability. This study investigated the potential role of adenosine 5′-triphosphate (ATP) in the control of detrusor motor drive in a model of porcine urinary bladder. The involvement of ATP on excitatory activity was assessed by measuring neurally-evoked [ 3H]-acetylcholine (ACh) release and smooth muscle contraction in detrusor strips. Epithelium-deprived preparations were used to minimize the influence of non-neural sources of ACh and ATP on parasympathetic neurotransmission. ACh release and smooth muscle contractility were not significantly affected by neural ATP in normal detrusor, but markedly enhanced when ATP hydrolysis was reduced by ectoATPase inhibitors, as well as by α,β-methylene-ATP (ABMA), agonist resistant to ecto-enzymes degradation. Prejunctional P2X receptors located on cholinergic nerves are involved in such potentiating effect. These purinergic heteroreceptors were characterized as P2X 3 subunits by means of the putative antagonists: NF449 (P2X 1,3 selective), NF023 (P2X 1,3 selective), PPNDS (P2X 1 selective) and A-317491 (P2X 3 selective). In porcine detrusor, P2X 3 receptors are functionally expressed at neural site facilitating neurogenic ACh release. When purine breakdown is experimentally down-regulated to mimicking the impaired purinergic pathway observed in pathological human bladders, endogenous ATP can markedly enhance detrusor contractility through activation of these receptors. Since P2X 3 blockade represents a potential therapeutic approach for diseases of the urinary tract, isolated porcine detrusor represents a reliable model for development of novel selective P2X 3 antagonists beneficial in the treatment of detrusor hyperactivity.

Cholinergic modulation of dendritic cell function

Dendritic cells (DCs) are highly specialized antigen-presenting cells with a unique ability to activate resting T lymphocytes. Acetylcholine (ACh) is the primary parasympathetic neurotransmitter and also a non-neural paracrine factor produced by different cells. Here, we analyzed the expression of the cholinergic system in DCs. We found that DCs express the muscarinic receptors M 3, M 4 and M 5, as well as the enzymes responsible for the synthesis and degradation of ACh, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), respectively. Differentiation of DCs in the presence of the cholinergic agonist carbachol, the synthetic analog of ACh, resulted in an increased expression of HLA-DR and CD86 and the stimulation of TNF-α and IL-8 production. All these effects were prevented by atropine, a muscarinic ACh receptor (mAChR) antagonist. Carbachol, was also able to modulate the function of DCs when added after the differentiation is accomplished; it increased the expression of HLA-DR, improved the T cell priming ability of DCs, and stimulated the production of TNF-α but not IL-12 or IL-10. By contrast, carbachol significantly inhibited the stimulation of HLA-DR expression and the enhancement in the T cell priming ability of DCs triggered by LPS. Interestingly, the TNF-α antagonist etanercept completely prevented the increased expression of HLA-DR induced by carbachol, suggesting that it promotes the phenotypic maturation of DCs by stimulating the production of TNF-α. ACh induced similar effects than carbachol; it stimulated the expression of HLA-DR and the production of TNF-α, while inhibiting the stimulation of HLA-DR expression and IL-12 production triggered by LPS. Similarly, neostigmine, an inhibitor of AChE, also stimulated the expression of HLA-DR and the production of TNF-α by DCs while inhibiting the production of TNF-α and IL-12 triggered by LPS. These results support the existence of an autocrine/paracrine loop through which ACh modulates the function of DCs.

Alterations of Vasoactive Intestinal Polypeptide Receptors in Allergic Rhinitis

Vasoactive intestinal peptide (VIP) is one of the parasympathetic neurotransmitters involved in the regulation of airway mucus secretion. The biological functions of VIP are mediated through two receptors (vasoactive intestinal peptide receptor type 1 [VPAC1R] and type 2 [VPAC2R]). The purpose of this study was to determine the distribution of VIP receptors and to compare the level of VIP receptor expression in the nasal mucosa of patients with allergic rhinitis and normal controls. Inferior turbinate mucosal samples were obtained from 20 normal subjects and 20 patients with allergic rhinitis. VPAC1R and VPAC2R mRNA was extracted from the nasal mucosa, and then a reverse-transcription-polymerase chain reaction was performed. Sections were immunostained using specific antibodies for VIP receptors. Western blot analysis was used to analyze differences in the level of expression of VPAC1R and VPAC2R protein between patients with allergic rhinitis and normal controls. The level of expression of VIP receptor mRNA and protein in patients with allergic rhinitis was significantly increased compared with normal nasal mucosa. VIP receptor immunoreactivity was detected on the nasal epithelium and submucosal glands in nasal specimens from both normal controls and patients with allergic rhinitis. In the epithelium from patients with allergic rhinitis, VIP receptor immunoreactivity was strong, whereas in the nasal epithelium from normal subjects it was faint. These results suggest that an increased expression level of VIP receptors is one possible explanation for nasal hyperresponsiveness in patients with allergic rhinitis.

Kv1.1 Potassium Channel Deficiency Reveals Brain-Driven Cardiac Dysfunction as a Candidate Mechanism for Sudden Unexplained Death in Epilepsy

Mice lacking Kv1.1 Shaker-like potassium channels encoded by the Kcna1 gene exhibit severe seizures and die prematurely. The channel is widely expressed in brain but only minimally, if at all, in mouse myocardium. To test whether Kv1.1-potassium deficiency could underlie primary neurogenic cardiac dysfunction, we performed simultaneous video EEG-ECG recordings and found that Kcna1-null mice display potentially malignant interictal cardiac abnormalities, including a fivefold increase in atrioventricular (AV) conduction blocks, as well as bradycardia and premature ventricular contractions. During seizures the occurrence of AV conduction blocks increased, predisposing Kv1.1-deficient mice to sudden unexplained death in epilepsy (SUDEP), which we recorded fortuitously in one animal. To determine whether the interictal AV conduction blocks were of cardiac or neural origin, we examined their response to selective pharmacological blockade of the autonomic nervous system. Simultaneous administration of atropine and propranolol to block parasympathetic and sympathetic branches, respectively, eliminated conduction blocks. When administered separately, only atropine ameliorated AV conduction blocks, indicating that excessive parasympathetic tone contributes to the neurocardiac defect. We found no changes in Kv1.1-deficient cardiac structure, but extensive Kv1.1 expression in juxtaparanodes of the wild-type vagus nerve, the primary source of parasympathetic input to the heart, suggesting a novel site of action leading to Kv1.1-associated cardiac bradyarrhythmias. Together, our data suggest that Kv1.1 deficiency leads to impaired neural control of cardiac rhythmicity due in part to aberrant parasympathetic neurotransmission, making Kcna1 a strong candidate gene for human SUDEP.

Cholinergic autoantibodies from primary Sjögren’s syndrome modulate submandibular gland Na+/K+‐ATPase activity via prostaglandin E2 and cyclic AMP

We demonstrate that patients with primary Sjögren's syndrome (pSS) produce functional IgG autoantibodies that interact with the glandular M(3) muscarinic acetylcholine receptors (mAChRs). These autoantibodies act as a partial muscarinic agonist, increasing prostaglandin E(2) (PGE(2)) and cyclic AMP production through modifying Na(+)/K(+)-ATPase activity, but also interfere with the secretory effect of the parasympathetic neurotransmitter. The IgG from patients with pSS has two effects on the submandibular gland. On the one hand, it may act as an inducer of the proinflammatory molecule (PGE(2)) that, in turn, inhibits Na(+)/K(+)-ATPase activity. On the other hand, it plays a role in the pathogenesis of dry mouth, abolishing the Na(+)/K(+)-ATPase inhibition and the net K(+) efflux stimulation of the salivary gland in response to the authentic agonist pilocarpine, decreasing salivary fluid production.

Neurotransmitters in Airway Parasympathetic Neurons Altered by Neurotrophin-3 and Repeated Allergen Challenge

Changes in airway nerves associated with chronic inflammation may underlie the pathogenesis and symptoms of lower airway diseases, such as asthma. The molecules most likely causing such alterations are neurotrophins (NTs) and/or related neurokines. In several species, including humans, lower airway parasympathetic postganglionic neurons that project axons to airway smooth muscle are either cholinergic or nonadrenergic noncholinergic (NANC), the latter synthesizing vasoactive intestinal peptide and nitric oxide, but not acetylcholine. In guinea pig trachealis smooth muscle, cholinergic nerve terminals arise from ganglionic neurons located near the tracheal smooth muscle, whereas the source of NANC nerve fibers is from neurons in ganglia located in the adjacent myenteric plexus of the esophagus, making this an ideal species to study regulation of parasympathetic neurotransmitter phenotypes. In the present study, we determined that, 48 hours after repeated allergen challenge, the NANC phenotype of airway parasympathetic ganglionic neurons changed to a cholinergic phenotype, and NT-3 mimicked this change. Nerve growth factor, brain-derived neurotrophic factor, leukemia inhibitory factor, or IL-1β had no effect on either phenotype, and they did not induce these neurons to synthesize substance P or tyrosine hydroxylase. These results indicate a role for inflammation and NT-3 in regulating biochemical and anatomical characteristics of principal neurons in adult airway parasympathetic ganglia.

Enhanced Cholinergic Response in Pancreata of Nonhuman Primates with Impaired Glucose Tolerance Shown on [18F]Fluorobenzyltrozamicol Positron Emission Tomography

Islet cell adaptation to insulin resistance in type 2 diabetes mellitus may be due in part to increased stimulation of beta cells by the autonomic nervous system. The parasympathetic neurotransmitter acetylcholine (ACh) mediates insulin release via M3 muscarinic receptors on islet beta cells. The vesicular ACh transporter (VAChT) receptor correlates with cholinergic activity in vivo. The positron emission tomography (PET) radiotracer (+)-4-[18F]fluorobenzyltrozamicol ([18F]FBT) binds to the VAChT receptor on presynaptic cholinergic neurons and can be quantified by PET. In this study, we utilize [18F]FBT PET to demonstrate pancreatic cholinergic activity before and after dextrose infusion in nonhuman primates with normal (NGT) and impaired (IGT) glucose tolerance. Seven adult female vervet (Chlorocebus aethiops) monkeys were maintained on an atherogenic Western diet. They were divided into two groups: four with NGT and three with IGT. Each subject underwent [18F]FBT PET twice: first, a baseline PET under fasting conditions; and second, PET under fasting conditions but after intravenous infusion of dextrose solution. Quantitative analysis of pancreatic uptake at 60 min post-injection was performed. There was no difference in pancreatic uptake of [18F]FBT on baseline scans between the two groups. Pancreatic uptake of [18F]FBT increased in every subject after dextrose infusion (P = 0.03). On post-dextrose PET scans, pancreatic uptake of [18F]FBT was significantly higher in IGT subjects compared with NGT subjects (P = 0.03). The post-dextrose to pre-dextrose uptake ratios were higher in IGT subjects (P = 0.08). Acute increases in pancreatic cholinergic activity in vivo were detected in the pancreata of nonhuman primates with NGT and IGT after intravenous dextrose infusion on [18F]FBT PET. In subjects with IGT, this activity was significantly higher, suggesting increased autonomic nervous system stimulation of the pancreatic islets in insulin-resistant subjects.

Anandamide injected into the lateral ventricle of the brain inhibits submandibular salivary secretion by attenuating parasympathetic neurotransmission

Our objective was to determine the effect of arachidonylethanolamide (anandamide, AEA) injected intracerebroventricularly (icv) into the lateral ventricle of the rat brain on submandibular gland (SMG) salivary secretion. Parasympathetic decentralization (PSD) produced by cutting the chorda tympani nerve strongly inhibited methacholine (MC)-induced salivary secretion while sympathetic denervation (SD) produced by removing the superior cervical ganglia reduced it slightly. Also, AEA (50 ng/5 microL, icv) significantly decreased MC-induced salivary secretion in intact rats (MC 1 microg/kg: control (C), 5.3 +/- 0.6 vs AEA, 2.7 +/- 0.6 mg; MC 3 microg/kg: C, 17.6 +/- 1.0 vs AEA, 8.7 +/- 0.9 mg; MC 10 microg/kg: C, 37.4 +/- 1.2 vs AEA, 22.9 +/- 2.6 mg). However, AEA did not alter the significantly reduced salivary secretion in rats with PSD, but decreased the slightly reduced salivary secretion in rats with SD (MC 1 microg/kg: C, 3.8 +/- 0.8 vs AEA, 1.4 +/- 0.6 mg; MC 3 microg/kg: C, 14.7 +/- 2.4 vs AEA, 6.9 +/- 1.2 mg; P < 0.05; MC 10 microg/kg: C, 39.5 +/- 1.0 vs AEA, 22.3 +/- 0.5 mg; P < 0.001). We showed that the inhibitory effect of AEA is mediated by cannabinoid type 1 CB1 receptors and involves GABAergic neurotransmission, since it was blocked by previous injection of the CB1 receptor antagonist AM251 (500 ng/5 microL, icv) or of the GABA A receptor antagonist, bicuculline (25 ng/5 microL, icv). Our results suggest that parasympathetic neurotransmission from the central nervous system to the SMG can be inhibited by endocannabinoid and GABAergic systems.

Abstract: 67 CHARACTERISTICS OF CIRCULATING VLDL RESULTING FROM INSULIN RESISTANCE AND FATTY LIVER

Although fasting induces hepatic triglyceride (TG) accumulation in both rodents and humans, little is known about the underlying mechanism. Because parasympathetic nervous system activity tends to attenuate the secretion of very-low-density-lipoprotein-triglyceride (VLDL-TG) and increase TG stores in the liver, and serum cholinesterase activity is elevated in fatty liver disease, the inhibition of the parasympathetic neurotransmitter acetylcholinesterase (AChE) may have some influence on hepatic lipid metabolism. To assess the influence of AChE inhibition on lipid metabolism, the effect of physostigmine, an AChE inhibitor, on fasting-induced increase in liver TG was investigated in mice. In comparison with ad libitum-fed mice, 30 h fasting increased liver TG accumulation accompanied by a downregulation of sterol regulatory element-binding protein 1 (SREBP-1) and liver-fatty acid binding-protein (L-FABP). Physostigmine promoted the 30 h fasting-induced increase in liver TG levels in a dose-dependent manner, accompanied by a significant fall in plasma insulin levels, without a fall in plasma TG. Furthermore, physostigmine significantly attenuated the fasting-induced decrease of both mRNA and protein levels of SREBP-1 and L-FABP, and increased IRS-2 protein levels in the liver. The muscarinic receptor antagonist atropine blocked these effects of physostigmine on liver TG, serum insulin, and hepatic protein levels of SREBP-1 and L-FABP. These results demonstrate that AChE inhibition facilitated fasting-induced TG accumulation with up regulation of the hepatic L-FABP and SREBP-1 in mice, at least in part via the activation of muscarinic acetylcholine receptors. Our studies highlight the crucial role of parasympathetic regulation in fasting-induced TG accumulation, and may be an important source of information on the mechanism of hepatic disorders of lipid metabolism.

Muscarinic M3 receptor stimulation increases cigarette smoke-induced IL-8 secretion by human airway smooth muscle cells

Acetylcholine is the primary parasympathetic neurotransmitter in the airways and is known to cause bronchoconstriction and mucus secretion. Recent findings suggest that acetylcholine also regulates aspects of remodelling and inflammation through its action on muscarinic receptors. In the present study, we aimed to determine the effects of muscarinic receptor stimulation on cytokine production by human airway smooth muscle cells (primary and immortalised cell lines). The muscarinic receptor agonists carbachol and methacholine both induced modest effects on basal interleukin (IL)-8 and -6 secretion, whereas the secretion of RANTES, eotaxin, vascular endothelial growth factor-A and monocyte chemoattractant protein-1 was not affected. Secretion of IL-8 and -6 was only observed in immortalised airway smooth muscle cells that express muscarinic M3 receptors. In these cells, methacholine also significantly augmented IL-8 secretion in combination with cigarette smoke extract in a synergistic manner, whereas synergistic effects on IL-6 secretion were not significant. Muscarinic M3 receptors were the primary subtype involved in augmenting cigarette smoke extract-induced IL-8 secretion, as only tiotropium bromide and muscarinic M3 receptor subtype selective antagonists abrogated the effects of methacholine. Collectively, these results indicate that muscarinic M3 receptor stimulation augments cigarette smoke extract-induced cytokine production by airway smooth muscle. This interaction could be of importance in patients with chronic obstructive pulmonary disease.

Dual Radiotracer Analysis of Cholinergic Neuronal Changes in Prediabetic Mouse Pancreas

Pancreatic neuronal changes associated with beta cell loss in type 1 diabetes mellitus are complex, involving, in part, parasympathetic mechanisms to compensate for preclinical hyperglycemia. The parasympathetic neurotransmitter acetylcholine (ACh) mediates insulin release via M3 muscarinic receptors on islet beta cells. The vesicular ACh transporter (VAChT) receptor has been shown to be a useful marker of cholinergic activity in vivo. The positron emission tomography (PET) radiotracer (+)-4-[(18)F]fluorobenzyltrozamicol ([(18)F]FBT) binds to the VAChT receptor on presynaptic cholinergic neurons and can be quantified by PET. The compound 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), available in a tritiated form, binds to M3 muscarinic receptors on beta cells and is a potential target for assessing pancreatic beta cell mass. In this study, we investigate the feasibility of dual radiotracer analysis in identifying neurofunctional changes that may signify type 1 diabetes mellitus in its early preclinical state. Ex vivo determinations of pancreatic uptake were performed in prediabetic nonobese diabetic mice and controls after intravenous injection of [(18)F]FBT or 4-[(3)H]DAMP. Beta cell loss in prediabetic mice was confirmed using immunohistochemical methods. [(18)F]FBT uptake was significantly higher in prediabetic pancreata than controls: 3.22 +/- 0.81 and 2.51 +/- 1.04, respectively (P < 0.03). 4-[(3)H]DAMP uptake was significantly lower in prediabetic pancreata than controls: 0.612 +/- 0.161 and 0.968 +/- 0.364, respectively (P = 0.01). These data suggest that a combination of radiotracer imaging agents that bind to neuronal elements intimately involved in insulin production may be an effective method of evaluating changes associated with early beta cell loss using PET.

Neuronal nitric oxide synthase gene transfer decreases [Ca 2+] i in cardiac sympathetic neurons

Gene transfer of neuronal nitric oxide synthase (nNOS) can decrease cardiac sympathetic outflow and facilitate parasympathetic neurotransmission. The precise pathway responsible for nitric oxide (NO) mediated inhibition of sympathetic neurotransmission is not known, but may be related to NO–cGMP activation of cGMP-stimulated phosphodiesterase (PDE2) that enhances the breakdown of cAMP to deactivate protein kinase A (PKA), resulting in a decrease in Ca 2+ influx mediated exocytosis of the neurotransmitter. We investigated depolarization evoked Ca 2+ influx in nNOS gene transduced sympathetic neurons from stellate ganglia with a noradrenergic cell specific vector (Ad.PRS-nNOS or empty vector), and examined how nNOS gene transfer affected cAMP and cGMP levels in these neurons. We found that targeting nNOS into these sympathetic neurons reduced amplitudes of voltage activated Ca 2+ transients by 44%. nNOS specific inhibition by N-[(4 S)-4-Amino-5-[(2-aminoetyl](amino] pentyl]- N′-nitroguanidine (AAAN) reversed this response. nNOS gene transfer also increased intracellular cGMP (47%) and decreased cAMP (29%). A PDE2 specific inhibitor Bay60-7557 reversed the reduction in cAMP caused by Ad.PRS-nNOS. These results suggest that neuronal NO modulates cGMP and PDE2 to regulate voltage gated intracellular Ca 2+ transients in sympathetic neurons. Therefore, we propose this as a possible key step involved in NO decreasing cardiac sympathetic neurotransmission.

Angiotensin II attenuates myocardial interstitial acetylcholine release in response to vagal stimulation

Although ANG II exerts a variety of effects on the cardiovascular system, its effects on the peripheral parasympathetic neurotransmission have only been evaluated by changes in heart rate (an effect on the sinus node). To elucidate the effect of ANG II on the parasympathetic neurotransmission in the left ventricle, we measured myocardial interstitial ACh release in response to vagal stimulation (1 ms, 10 V, 20 Hz) using cardiac microdialysis in anesthetized cats. In a control group (n = 6), vagal stimulation increased the ACh level from 0.85 +/- 0.03 to 10.7 +/- 1.0 (SE) nM. Intravenous administration of ANG II at 10 microg x kg(-1) x h(-1) suppressed the stimulation-induced ACh release to 7.5 +/- 0.6 nM (P < 0.01). In a group with pretreatment of intravenous ANG II receptor subtype 1 (AT(1) receptor) blocker losartan (10 mg/kg, n = 6), ANG II was unable to inhibit the stimulation-induced ACh release (8.6 +/- 1.5 vs. 8.4 +/- 1.7 nM). In contrast, in a group with local administration of losartan (10 mM, n = 6) through the dialysis probe, ANG II inhibited the stimulation-induced ACh release (8.0 +/- 0.8 vs. 5.8 +/- 1.0 nM, P < 0.05). In conclusion, intravenous ANG II significantly inhibited the parasympathetic neurotransmission through AT(1) receptors. The failure of local losartan administration to nullify the inhibitory effect of ANG II on the stimulation-induced ACh release indicates that the site of this inhibitory action is likely at parasympathetic ganglia rather than at postganglionic vagal nerve terminals.

Reply to Kreier and Buijs: no sympathy for the claim of parasympathetic innervation of white adipose tissue

to the editor: Kreier and Buijs ([16][1]) have questioned our work ([14][2]) demonstrating the lack of parasympathetic nervous system (PSNS) innervation of white adipose tissue (WAT) in laboratory rats and mice, as well as in Siberian hamsters. We appreciate the opportunity to clarify and extend our

Human hepatic progenitor cells express vasoactive intestinal peptide receptor type 2 and receive nerve endings

We recently showed that human hepatic progenitor cells (HPCs) express muscarinic acetylcholine (Ach) receptor subtype 3 and that--following liver transplantation--HPC numbers are significantly reduced. To further elaborate on this, we examined whether HPC also express receptors for vasoactive intestinal peptide (VIP), which, besides Ach, also is an important parasympathetic neurotransmitter. VIP expressing nerves are known to be present in the liver. We performed immunohistochemistry for VIP receptor subtypes 1 and 2 (VIPR1 and 2), on sections of normal and diseased human liver (n=17), and double staining for VIPR2 and known HPC markers. We performed RT-PCR for VIPR1 and 2 on total RNA from purified rat HPC. To document the probability of direct interaction, we also performed double immunostaining for nerve markers and HPC markers on human liver sections. VIPR2 immunostaining was clearly positive in HPC and reactive bile ductules on paraffin-embedded and frozen tissue sections. We could not demonstrate VIPR1 protein expression in the liver, with either of two VIPR1 antibodies tested. The presence of VIPR2 mRNA in HPC was confirmed by RT-PCR. Nerve endings were shown to abut on reactive bile ductules. We show here for the first time that HPC express VIPR2 and receive nerve endings. These features, and the fact that HPC numbers are influenced by the presence or absence of the autonomic innervation of the liver, suggest a direct interaction.

Vasoactive Intestinal Polypeptide Evokes Only a Minimal Headache in Healthy Volunteers

The role of the parasympathetic nervous system in the pathogenesis of migraine is disputed. The headache-eliciting effect of the parasympathetic neurotransmitter, vasoactive intestinal polypeptide (VIP), and its effect on cerebral arteries and brain haemodynamics has not been systematically studied in man. We hypothesized that infusion of VIP might induce headache in healthy subjects and cause changes in cerebral haemodynamics. VIP (8 pmol/kg per min) or placebo (0.9% saline) was infused for 25 min into 12 healthy young volunteers in a crossover, double-blind design. Headache was scored on a verbal rating scale from 0 to 10, regional cerebral blood flow (rCBF) was measured with single-photon emission computed tomography and (133)Xe inhalation and mean flow velocity in the middle cerebral artery (V(meanMCA)) was measured with transcranial Doppler ultrasonography. The headache was very mild with a maximum score of 2 and described as a pressing or throbbing sensation. Five participants developed headache during VIP and one during placebo. During the infusion, a significant drop in V(meanMCA) was seen for VIP compared with placebo (P < 0.001), but the effect quickly waned and no difference was found when comparing the time between 30 and 120 min. In addition, no significant difference in the diameter of the MCA could be found during the infusion. No significant differences in rCBF (P = 0.10) were found between VIP and placebo. A marked dilation of the superficial temporal artery was seen (P = 0.04) after VIP in the first 30 min but no difference was found when comparing the time between 30 and 120 min. We found no difference in mean arterial blood pressure between VIP and placebo days but the heart rate increased significantly on a VIP day compared with a placebo day (AUC(0-30 min), P < 0.001). Plasma VIP was significantly higher on a VIP day compared with placebo (AUC(0-80 min), P < 0.001). These results show that VIP causes a decrease in V(meanMCA) without affecting rCBF. In spite of a marked vasodilator effect in the extracranial vessels and increased plasma VIP, healthy subjects developed only a very mild headache.

Altered neurogenic and mechanical responses to acetylcholine, ATP and substance P in detrusor from rat with outlet obstruction

The well-known side effects of anticholinergic compounds used to treat urinary incontinence caused by detrusor overactivity have addressed the interest on other pharmacological intervention. The purpose of the present work was to investigate the possible changes in purinergic and cholinergic components of parasympathetic neurotransmission in obstructed rat bladders with detrusor overactivity, and to examine the effect of the association of suramin, atropine and indomethacin on nerve-mediated responses to electrical field stimulation (EFS). Mechanical responses to exogenous acetylcholine, ATP and substance P were also evaluated. Altered sensitivities to acetylcholine and to the sensory neurotransmitter substance P, but unchanged sensitivity to the stable ATP analogue α,β-methyleneATP were observed in bladders from obstructed rats. Suramin and atropine inhibited purinergic and cholinergic components of the neurogenic responses evoked by EFS in detrusor strips from control and obstructed rats. Interestingly, suramin enhanced the antagonistic effect of atropine on neurogenic responses of detrusor strips at all frequencies of stimulation tested. Our results suggest that the association between an antimuscarinic drug and an antagonist of P2X purinoceptors such as suramin might be helpful to reduce the therapeutic dosage of the antimuscarinic drug, along with its side effects. This approach may be of interest in the therapy of patients with bladder incontinence caused by detrusor overactivity, which do not even respond to a maximal dosage of antimuscarinic drug.