Increase In Acetylcholine Sensitivity Research Articles

Mild hyperbaric oxygen exposure protects heart during ischemia/reperfusion and affects vascular relaxation.

Mild hyperbaric oxygen therapy (mHBOT) is an adjuvant therapy used in conditions where tissue oxygenation is reduced and is implemented using pressures less than 1.5 ATA and 100% O2 (instead of the classical HBOT at 1.9-3 ATA) which results in cheaper, easier to implement, and equally effective. mHBOT is offered for wellness and beauty and as an anti-aging strategy, in spite of the absence of studies on the cardiovascular system. Consequently, we investigated the impact of mHBOT on the cardiovascular system. Mechanical and energetic parameters of isolated heart submitted to ischemia/reperfusion injury and arterial contractile response from mHBOT-exposed rats were evaluated. In the heart, mHBOT increased pre-ischemic velocity of contraction and ischemic end-diastolic pressure and developed pressure and contractile economy during reperfusion. mHBOT decreased infarct size and increased the plasma nitrite levels. In the artery, mHBOT increased acetylcholine sensitivity. mHBOT protects the heart during ischemia/reperfusion and affects vascular relaxation.

Deletion of protein tyrosine phosphatase 1B (PTP1B) prevents type 1 diabetes‐induced endothelial dysfunction.

PTP1B dephosphorylates receptors tyrosine kinase and acts as a brake on various signaling pathways. Deletion of PTP1B protects against metabolic dysfunction by increasing insulin sensitivity, but little is known about its role in the control of vascular function. We hypothesized that PTP1B deletion prevents vascular dysfunction in the absence of insulin. Insulin secretion was abolished in Balb/C and PTP1B‐deficient mice with streptozotocin (STZ) injection. 24 days after treatment, aortic reactivity was assessed via myography. STZ induced similar increases in glycemia in Balb/C and PTP1B KO mice (384±24 vs 444±21 mg/dL, Balb/C vs PTP1B). Phenylephrine (PE)‐induced constriction was increased by STZ treatment in Balb/C mice only (115±13 vs 136±6% of KCl, vehicle vs STZ). Endothelial function assessed via acetylcholine (ACh)‐mediated relaxation was not affected by PTP1B deficiency but was reduced by STZ in Balb/C mice (63±5 vs 43±5% of precontraction, vehicle vs STZ). In PTP1B KO mice, STZ treatment did not affect maximum relaxation (56±7 vs 58±7% of preconstriction, vehicle vs STZ) but increased ACh sensitivity (logEC50 7.4±0.3 vs 8.2±0.2, vehicle vs STZ). Western blot analysis revealed a trend towards increased baseline Akt activation without changes in eNOS expression or activation. These data indicate that PTP1B deletion protects against the harmful vascular effects of type I diabetes.

Nicotinic receptors in circuit excitability and epilepsy.

Neuronal nicotinic acetylcholine receptors belong to the family of excitatory ligand-gated channels and result from the assembly of five subunits. Functional heteromeric nictonic receptors are present in the hippocampus and neocortex, thalamus, mesolimbic dopamine system and brainstem motor nuclei, where they may play a role, respectively, in memory, sensory processing, addiction and motor control. Some forms of autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) have been found to be associated with mutations in the genes coding for the alpha 4 or beta2 subunits of the nicotinic receptor. Mutant receptors display an increased acetylcholine sensitivity with respect to normal receptors. Since the thalamus and the cortex are strongly innervated by cholinergic neurons projecting from the brainstem and basal forebrain, an unbalance between excitation and inhibition, brought about by the presence of mutant receptors, could generate seizures by facilitating and synchronizing spontaneous oscillations in thalamo-cortical circuits.

How mutations in the nAChRs can cause ADNFLE epilepsy.

The linkage between autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and neuronal nicotinic acetylcholine receptor has been strongly reinforced by the report of five distinct mutations in the two genes coding for the major brain alpha4beta2 nicotinic acetylcholine (ACh) receptors. As a first step toward understanding the basic mechanisms underlying this genetically transmissible neurologic disorder, we examined the similarities and differences of the functional properties displayed by naturally occurring mutant forms of this ligand-gated channel. Functional studies of neuronal nicotinic ACh receptors reconstituted in Xenopus oocytes were designed to analyze the common traits displayed by the different mutations associated with ADNFLE. Coexpression of the control and mutated alleles harboring the alpha4S248F mutation obtained from patient DNAs yielded ACh-evoked currents of amplitude comparable to the control responses but with a higher sensitivity and desensitization to the natural agonist. Alternatively, the other mutants (alpha4L776ins3, alpha4S252L, and beta2V287M) displayed an increased ACh sensitivity without pronounced desensitization. In addition, whereas a reduction of calcium permeability was observed for the mutants (alpha4S248F and alpha4L776ins3), no significant modification of ionic selectivity could be detected in the alpha4S252L mutation. Hence increase in ACh sensitivity is the only common characteristic so far observed between the four naturally occurring mutant receptors investigated. Analyses of functional properties of four nAChR mutants associated with ADNFLE indicate that a gain of function of these mutant receptors may be at the origin of the neuronal network dysfunction that causes the epileptic seizures. These data are discussed in the context of our latest knowledge of the pyramidal cell function.

Mutations of the neuronal nicotinic acetylcholine receptors and their association with ADNFLE

Elucidating the origin of epileptic seizures represents one of the many ways by which today’s scientists are approaching this devastating neurological disorder. Although epilepsies have several different origins ranging from head trauma to genetically transmissible affections, common neuronal network dysfunction can be recognised between these many forms of the disease. Thus, understanding the basic mechanisms underlying some genetically transmissible epilepsies should bring new and important knowledge that is readily applicable to other types of epilepsies. In this work we review our current knowledge of one genetically transmissible form of nocturnal epilepsy, the ADNFLE. In the light of the most recent findings obtained on five mutants of the neuronal nicotinic acetylcholine receptors associated with ADNFLE, we discuss the effects of these spontaneous genome alterations on the receptor function. The only common trait identified so far between these mutant receptors is an increase in acetylcholine sensitivity. Based on our understanding of the receptor distribution in the different brain areas, their development and the neuronal network circuitry, we hypothesise that increased acetylcholine sensitivity causes an unbalance in the fine tuning of the cortico-reticular thalamic and thalamo-cortical loops. In addition, we illustrate how spontaneous mutations in the gene coding for a receptor provoke a change in its pharmacological profile and thereby might account for the inter-individual therapeutic sensitivity.

CHRNB2 Is the Second Acetylcholine Receptor Subunit Associated with Autosomal Dominant Nocturnal Frontal Lobe Epilepsy

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is an uncommon, idiopathic partial epilepsy characterized by clusters of motor seizures occurring in sleep. We describe a mutation of the beta2 subunit of the nicotinic acetylcholine receptor, effecting a V287M substitution within the M2 domain. The mutation, in an evolutionary conserved region of CHRNB2, is associated with ADNFLE in a Scottish family. Functional receptors with the V287M mutation are highly expressed in Xenopus oocytes and characterized by an approximately 10-fold increase in acetylcholine sensitivity. CHRNB2 is a new gene for idiopathic epilepsy, the second acetylcholine receptor subunit implicated in ADNFLE.

Pituitary Adenylate Cyclase-Activating Polypeptide Activates a Phospholipase C-Dependent Signal Pathway in Chick Ciliary Ganglion Neurons that Selectively Inhibits α7-Containing Nicotinic Receptors

Neuropeptide receptors couple via G-proteins to two principal signaling pathways that elevate cAMP through adenylate cyclase (AC) or mobilize intracellular Ca(2+) through phospholipase C (PLC)-stimulated inositol phosphate (IP) turnover and production of inositol 1,4,5-trisphosphate (IP(3)). We showed previously that high-affinity receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) are present on chick ciliary ganglion neurons and that receptor occupation increases cAMP production, resulting in enhanced acetylcholine sensitivity. After we suppressed AC activity and cAMP production with 2'-5' dideoxyadenosine, however, PACAP no longer increased acetylcholine sensitivity but instead reduced it, suggesting that an AC-independent signal pathway activated by PACAP inhibits some nicotinic acetylcholine receptors (AChRs). We now use fast-perfusion, imaging, and biochemical methods to identify the AChRs modulated by PACAP and to characterize the signal pathway responsible for their inhibition. Without previous AC block, both the rapidly desensitizing, alpha-bungarotoxin (alphaBgt)-sensitive alpha7-AChRs and the slowly desensitizing, alphaBgt-insensitive alpha3*-AChRs on the neurons were potentiated by PACAP. After AC blockade, however, PACAP inhibited alpha7-AChRs but left alpha3*-AChRs unaffected. The selective inhibition of alpha7-AChRs appeared to use a PLC signaling pathway because it was not seen after lowering PLC activity or buffering intracellular Ca(2+) and was mimicked by dialyzing neurons with an IP(3) receptor agonist. PACAP also induced IP turnover and increased [Ca(2+)](i) assessed directly with Fluo-3AM imaging. Given our previous findings that PACAP receptors couple to AC, the present results demonstrate a remarkable ability of a single neuropeptide to activate two signaling pathways and in so doing selectively regulate two classes of downstream ion channel targets.

Developmental changes in the acetylcholine influence on heart muscle of Rana catesbeiana: in situ and in vitro effects.

The influence of acetylcholine (ACh) on cardiac performance of larval (Taylor Kollros [TK] stages II-XVIII) and postmetamorphic (3-609 g) Rana catesbeiana was analyzed in situ (circulatory system intact) and in vitro (isolated heart or ventricular strip preparations). Topical application of ACh to the heart in situ resulted in a dose-dependent decrease in heart rate and in a slight decrease in systolic ventricular pressure in all developmental stages. Injection of acetylcholine into the ventricle lumen in situ caused a dose-dependent transient decrease in systolic ventricular pressure, with little heart rate effect. Intraventricular ACh injection also changed the hemodynamic coupling between ventricle and conus arteriosus, generating a biphasic pressure profile in the conus due to sequential contractions of the ventricle and of the conus. In situ the sensitivity of the ventricle to ACh decreased during larval development, with the lowest sensitivity in small postmetamorphic adults. ACh applied in vitro to cardiac muscle strips or small hearts produced a negative inotropic effect. The ACh dose necessary to induce a 50% reduction in muscle strip contraction force in vitro decreased substantially during larval development, indicating an increase in ACh sensitivity with development. The effects of ACh both in vitro and in situ were diminished or eliminated by topical application or injection of atropine, suggesting the presence of muscarinic cholinergic receptors. After preincubation with the acetylcholinesterase blocker eserine, injection of ACh into the conus arteriosus decreased systolic ventricular pressure with a delay of 4-10 seconds, probably representing the minimum blood circulation time.(ABSTRACT TRUNCATED AT 250 WORDS)

Degradation of acetylcholine in human airways: role of butyrylcholinesterase

1. Neostigmine and BW284C51 induced concentration-dependent contractions in human isolated bronchial preparations whereas tetraisopropylpyrophosphoramide (iso-OMPA) was inactive on airway resting tone. 2. Neostigmine (0.1 microM) or iso-OMPA (100 microM) increased acetylcholine sensitivity in human isolated bronchial preparations but did not alter methacholine or carbachol concentration-effect curves. 3. In the presence of iso-OMPA (10 microM) the bronchial rings were more sensitive to neostigmine. The pD2 values were, control: 6.05 +/- 0.15 and treated: 6.91 +/- 0.14. 4. Neostigmine or iso-OMPA retarded the degradation of acetylcholine when this substrate was exogenously added to human isolated airways. A marked reduction of acetylcholine degradation was observed in the presence of both inhibitors. Exogenous butyrylcholine degradation was prevented by iso-OMPA (10 microM) but not by neostigmine (0.1 microM). 5. These results suggest the presence of butyrylcholinesterase activity in human bronchial muscle and this enzyme may co-regulate the degradation of acetylcholine in this tissue.

Increased acetylcholine sensitivity in Duchenne muscular dystrophy myotubes

Monolayer cultures were established from explants of muscle obtained from 6 patients with Duchenne muscular dystrophy (DMD) and 9 controls. Electrophysiological studies were made after 3-4 weeks in vitro, when many myotubes had formed. An intracellular electrode was used to record cell membrane potential, and acetylcholine was applied by ionophoresis. The myotubes grown from Duchenne muscle showed greater acetylcholine sensitivity than controls.

Delayed response to denervation in muscles of C57BL/Ola mice

Muscle fibre areas and numbers, tetanic force, resident macrophage numbers and acetylcholine sensitivity were measured in normal and denervated soleus muscles of C57BL/Ola mice for comparison with data from outbred and other inbred strains. Serum creatine kinase levels were also measured. The muscles of normal C57BL/Ola mice had more fibres, generated more tension, had fewer resident macrophages and had a lower acetylcholine sensitivity than muscles of other strains. The normal level of serum creatine kinase was lower in C57BL/Ola mice than in Balb/c mice. Following denervation, the mean cross-sectional area of muscle fibres in Balb/c mice started to fall from day 1 but a fall was not seen in muscles from C57BL/Ola mice until day 5. The development of increased acetylcholine sensitivity was slower in the C57BL/Ola mice although the values in all mouse strains had converged by five days. The levels of serum creatine kinase also rose more slowly following denervation in C57BL/Ola mice. The differences between the data from C57BL/Ola mice and others are ascribed to the slow rate of Wallerian degeneration in that strain. The results support the view that the effects of denervation on muscle are due to both inactivity and an inflammatory effect contributed by nerve degeneration. The mutation that causes slow nerve degeneration may, however, also affect muscle directly, making it more resistant to catabolism.

Functional properties and developmental regulation of nicotinic acetylcholine receptors on embryonic chicken sympathetic neurons

Measurement of acetylcholine (ACh)-induced currents indicates that the sensitivity of embryonic sympathetic neurons increases following innervation in vivo and in vitro. We have used single-channel recording to assess the contribution of changes in ACh receptor properties to this increase. Early in development (before synaptogenesis), we detect three classes of ACh-activated channels that differ in their conductance and kinetics. Molecular studies indicating a variety of neuronal receptor subunit clones suggest a similar diversity. Later in development (after innervation), changes in functional properties include increases in conductance and apparent mean open time, the addition of a new conductance class, as well as apparent clustering and segregation of channel types. These changes in channel function are compatible with the developmental increase in ACh sensitivity.

Acetylcholine receptor α-, β-, γ-, and δ-subunit mRNA levels are regulated by muscle activity

Denervation of adult skeletal muscle results in increased sensitivity to acetylcholine in extrajunctional regions of the muscle fiber. This increase in acetylcholine sensitivity is accompanied by a large increase in the level of mRNAs coding for the α-, β-, γ-, and δ-subunits of the acetylcholine receptor. To determine whether muscle activity is sufficient to regulate expression of extrajunctional acetylcholine receptor mRNA levels, denervated muscles were stimulated with extracellular electrodes. Direct stimulation of denervated muscle suppresses both the increase in extrajunctional acetylcholine sensitivity and the expression of mRNA encoding the α-, β-, γ-, and δ-subunits of the acetylcholine receptor. These results show that muscle activity regulates the level of extrajunctional acetylcholine receptors by regulating the expression of their mRNAs.

Selective elimination of motor nerve terminals in the rat soleus muscle during development.

1. The pattern of innervation and either frequency of miniature end-plate potentials (m.e.p.p.s) or acetylcholine (ACh) sensitivity was examined in individual fibres of the soleus muscle in new-born rats to elucidate the mechanism of elimination of motor nerve terminals. 2. the number of motor nerve terminals innervating a muscle fibre was judged by the difference in latencies of end-plate potentials evoked by stimulation of the ventral roots L4 and L5. 3. At early developmental stages, about 50% of soleus muscle fibres were usually singly innervated by the L5 ventral root and this nerve to fibre relation did not change throughout the developmental days. In contrast to the L5 root, the L4 root innervated all the muscle fibres polyneuronally at day 6. In day 16 or older animals, the remaining half of the fibres were singly innervated by the L4 ventral nerves. Therefore, it is concluded that the elimination of motor nerve terminals in the soleus muscle occurs selectively only for the L4 ventral nerves. 4. The number of polyneuronally innervated fibres was increased, to some extent, within 2 days after transection of the spinal cord at day 12. It is suggested that the increased polyneuronal innervation following the inactivity produced by cordotomy may reflect the recovery of terminals which had lost function only a short time previously during elimination. 5. Though frequency of m.e.p.p.s increased from day 6 to adult age as previously reported, there was no significant difference in the frequencies between singly and polyneuronally innervated fibres at any age. Cord transection at day 12 caused no change in the frequency. 6. ACh sensitivity observed in singly innervated fibres was similar to that in polyneuronally innervated fibres. Furthermore, cordotomy on day 12 did not induce an increase in ACh sensitivity around the end-plate. It is concluded that the elimination of terminals is not causally related to changes in ACh sensitivity. 7. The possible mechanisms of selective elimination of terminals and the process of elimination are discussed.

Fluctuation of Acetylcholine Sensitivity in Skeletal Muscles with Development, Denervation and Glycerol Treatment

The variation of acetylcholine (ACh) sensitivity induced by development, denervation and glycerol treatment was studied using skeletal muscles of frogs and rats. In the course of metamorphosis from tadpole to adult, sensitivities (affinity) of rectus abdominis muscles of frogs to ACh and n-butyltrimethylammonium (BTMA) increased, as did the maximum responses (intrinsic activity) to BTMA, whereas the sensitivities to ACh-competitive antagonist decreased. The increasing phenomena were similar to the supersensitivity seen with denervation. The slope of dose-inhibitory response curve for curare-like agents changed from 1.0 to 1.5 with development of muscles. These results suggest changes in ACh receptors. With 4 or 10% glycerol treatment of chronically denervated diaphragm muscles, the responses to ACh decreased, whereas those to ATP increased. The maximum responses and sensitivity of ACh dose-response curves on glycerinated muscles decreased dose dependently with glycerol. ACh log dose-response curves of muscles treated with 2 % glycerol shifted in parallel with d-tubocurarine application and pA2 values were observed to be similar to those of non-treated muscles. The responses of glycerinated muscles to ACh were potentiated by 1.5 µM neostigmine less than those seen in non-treated muscles. The increase in ACh sensitivity with development may be in close analogy to the supersensitivity seen with denervation, and the pattern of the increase in ACh sensitivity seen with development or denervation was the reverse of that seen with glycerol treatment. The sensitivity of ACh receptor is probably closely related with the glycerol treatment.

Neurotrophic regulation of resting membrane potential and acetylcholine sensitivity in rat extensor digitorum longus muscle

In this study the effect of brief exposure of rat sciatic nerve to various concentrations of colchicine was examined. Low concentrations (4 to 15 m m) did not affect the resting membrane potential of extensor digitorum longus muscles but increased their acetylcholine (ACh) sensitivity. Intermediate concentrations (30 to 60 m m) reduced the resting membrane potential and increased ACh sensitivity. High concentrations (120 to 240 m m) impaired neuromuscular transmission in addition to reducing the resting membrane potential and increasing ACh sensitivity. The changes caused by 30 to 60 m m colchicine were not correlated with any detectable change in muscle activity and were not due to a direct effect of colchicine on muscle because muscles from the treated and contralateral sides invariably contained the same concentration of colchicine whereas changes in the resting membrane potential and ACh sensitivity occurred only on the treated side. These changes were evident in 3 days, progressed to a maximum in 5 to 7 days, and returned almost to normal 16 days after 60 m m colchicine treatment. The block of axoplasmic transport caused by 60 m m colchicine was examined and behaved in a manner consistent with the concept that it is responsible for the decrease in resting membrane potential and the increase in ACh sensitivity. The block was most severe in 1 day, had recovered considerably in 7 days, and was essentially gone 17 to 21 days after colchicine application.

Nerve growth factor-induced increase in electrical excitability and acetylcholine sensitivity of a rat pheochromocytoma cell line

A cloned line of rat pheochromocytoma cells (PC12) which responds to nerve growth factor (NGF) by cessation of division and extension of neurone-like processes, is shown to develop electrical excitability and greatly increased acetylcholine sensitivity in the presence of NGF. This implies that NGF influences the physiological differentiation of certain neural crest derivatives.

Reappearance of extrajunctional acetylcholine sensitivity in denervated rat muscle after blockage with α-bungarotoxin

IN most vertebrate skeletal muscles only the endplate region of muscle membrane is sensitive to acetylcholine (ACh), but after denervation the entire extrajunctional membrane becomes sensitive1,2. In rat diaphragm fibres the degree of extrajunctional ACh sensitivity increases steadily during the 7–9 d following denervation and then remains relatively constant at a high level for at least several weeks. It has been shown that this increase in ACh sensitivity results from the appearance of new ACh receptors in the extrasynaptic membrane3,4, and an intriguing question is whether in already fully supersensitive fibres new ACh receptors continue to appear in the surface membrane. α-Bungarotoxin (α-BuTX) blocks the depolarising action of ACh on supersensitive muscle membrane4, and is thought to bind specifically and almost irreversibly to ACh receptors. Thus one can block all or most receptors of super-sensitive muscle fibres at a certain time. I report here experiments in which the reappearance of extrajunctional ACh sensitivity was followed after blocking sensitivity with α-BuTX, using denervated rat diaphragm muscle, maintained in organ culture5.

Role of Muscle Activity in Nerve-Muscle Interaction in vitro

CLONAL nerve and muscle cells interact in vitro to produce a nerve-muscle contact which is associated with an area of increased acetylcholine sensitivity on the muscle1,2. This association is reminiscent of the distribution of acetylcholine sensitivity on innervated skeletal muscle in vivo3,4. It is possible to use cultures of nerve and muscle cells to examine the requirements for localization in vitro. A previous report showed that cholinergic transmission is not required2. Here I discuss the requirement for muscle electrical and contractile activity in localisation of acetylcholine sensitivity.

Nerve-muscle interaction in vitro. Role of acetylcholine.

Nerve and muscle cells from clonal lines interact in vitro, resulting in the association on the muscle surface of an area of increased acetylcholine sensitivity with a site of nerve-muscle contact. This localization of acetylcholine sensitivity on the muscle cell to a site of contact between nerve and muscle was found to occur when acetylcholine receptors on the muscle had been blocked with alpha-neurotoxin. Localization was also found to occur when the nerve cell had been prevented from releasing acetylcholine. It is concluded that neither the presence of active acetylcholine receptors on the muscle, nor the release of acetylcholine from the nerve, was required for the events leading to the localization of acetylcholine sensitivity in vitro.