Apamin and nonadrenergic inhibition of guinea pig trachealis

Apamin has been shown to antagonize the nonadrenergic, noncholinergic (NANC) inhibitory system in guinea pig taenia coli. We have examined the effects of apamin on the nonadrenergic noncholinergic inhibitory system and its putative transmitters in isolated guinea pig trachea. Electrical field stimulation (ES) of isolated trachea pretreated with atropine and propranolol evoked reproducible relaxations that were blocked by tetrodoxin, but were unaffected by apamin. Vasoactive intestinal peptide (VIP), adenosine (AD), and adenosine triphosphate (ATP) produced concentration-dependent inhibition of histamine (H)-induced contractions of isolated trachea but the inhibitory actions of these agents were not significantly affected by apamin. In contrast, apamin virtually abolished ES-evoked relaxations in guinea pig isolated taenia caeci, and reduced the inhibition of H-induced contraction by ATP from 40% to 1%. We conclude that neither the NANC inhibitory system in the guinea pig trachea nor its putative mediators VIP, AD, and ATP are antagonized by apamin, in contrast to taenia caeci.Supported by the Medical Research Council of Canada.Scholar of the Medical Research Council of Canada.     

Demonstration of a nonadrenergic inhibitory nervous system in the trachea of the guinea pig

A nonadrenergic inhibitory nervous system has been demonstrated in the guinea pig trachea. Electrical field stimulation of this system, in the presence of adrenergic and cholinergic blockade, resulted in relaxation of tracheal rings contracted by the mediators of immediate hypersensitivity or histamine. The relaxation was blocked by tetrodotoxin, which indicated that nerve stimulation was responsible for the relaxation. The gastrointestinal tract, which has a similar embryological origin to the respiratory tract, also has a nonadrenergic inhibitory system. In the gastrointestinal tract, this system is thought to be responsible for the relaxation phase of peristalsis, and absence of this system, in the colon and the rectum, is thought to be an explanation for the spastic bowel in Hirschsprung's disease. It is possible that an abnormality of the respiratory nonadrenergic inhibitory system may play a role in the pathogenesis of the hyperreactive airways in asthma. The airways, due to a lack of inhibition, may be either partially contracted or unable to relax, and thus appear hyperreactive to stimuli.     

Evidence for myosin-linked regulation in guinea pig taenia coli muscle

Summary The ATPase activity of actomyosin prepared from taenia coli muscle of guinea pig was found to increase upon adding rabbit skeletal heavy meromyosin (HMM) in the absence of Ca²⁺. SDS-gel electrophoresis of muscle homogenates did not reveal the presence of troponin. Ca²⁺-regulation in taenia coli muscle thus appears to be myosin-linked.The glycerinated muscles which did not develop any tension in the presence of EGTA contracted after irrigation with rabbit skeletal myosin.Skeletal HMM could also cause tension generation in strips of glycerinated taenia coli in the presence of EGTA. The tension developed by the muscles in the presence of Ca²⁺ was increased if HMM was added. The HMM-induced tension was associated with a marked increase in ATPase activity both in the presence and in the absence of Ca²⁺. No HMM-associated tension could be detected when inactivated HMM was employed or when MgATP was substituted with Mg-pyrophosphate or Mg-AMP-PNP.The mechanical effect of HMM probably results from a mechanochemical interaction between the added HMM and muscle actin.This work was supported by a Katzir-Katchalsky fellowship (to J. B.) and by a grant from the Muscular Dystrophy Association of America (to A. O.)     

Evidence for inhibitory amino acid receptors on guinea pig medial vestibular nucleus neurons in vitro

There is little evidence to indicate the identity of the inhibitory receptors which mediate inhibitory interaction between the two medial vestibular nuclei ('brainstem commissural inhibition'). In the present study we tested the hypothesis that medial vestibular nucleus (MVN) neurons have gamma-aminobutyric acid (GABA) or glycine receptors by recording from single MVN neurons in isolated guinea pig MVN slices maintained in vitro while superfusing with GABA (10(-8) M) and the non-competitive GABAA antagonist picrotoxin (10(-6) M or 2 x 10(-6) M), or glycine (10(-6) M) and the competitive glycine antagonist strychnine (10(-6) M). Forty-four % (16/36) of the neurons tested with GABA showed a decrease in firing; in 7 out of 8 cases in which a decrease in firing occurred, the addition of the antagonist picrotoxin completely blocked the effect of the GABA alone. Fifty % (7/14) of the neurons tested with glycine showed a decrease in firing; in 4 out of 6 cases where a decrease occurred, the addition of the antagonist strychnine completely blocked the effect of the glycine alone. In one case only did a cell respond both to GABA and glycine (8 neurons tested with both). These results are consistent with the hypothesis that some MVN neurons have GABA or glycine receptors (but in most cases not both), which may mediate brainstem commissural inhibition.     

Evidence for galanin as an inhibitory neuropeptide on myenteric cholinergic neurons in the guinea pig small intestine

The effect of galanin on the [3H]ACh release from myenteric plexus-longitudinal muscle strips of the guinea pig small intestine was studied. While galanin did not alter the basal spontaneous efflux of ACh, it significantly depressed the ACh release evoked by electrical stimulation or caused by VIP and substance P. These results suggest an important neuromodulatory role for galanin in the enteric nervous system.     

Properties of inhibitory junctional transmission in smooth muscle of the guinea pig lower esophageal sphincter

Inhibitory neurotransmission in guinea pig lower esophageal sphincter (LES) muscles was investigated by using electrophysiological methods. Transmural nerve stimulation (TNS) initiated an inhibitory junction potential (i.j.p.); the amplitude increased 35% by atropine (10(-6) M) and converted to a muscarinic excitatory junction potential (e.j.p.) by apamin (10(-7) M) plus Nomega-nitro-L-arginine (L-NNA, 10(-5) M). In atropinized tissue, the i.j.p. amplitude was reduced 58% by guanethidine (5 x 10(-6) M), 41% by L-NNA (10(-5) M), 57% by suramin (10(-4) M), and it was abolished by apamin (10(-7) M), suggesting that this potential was produced by ATP and nitric oxide (NO) released from adrenergic and nitrergic nerves, respectively, through the activation of Ca2+-sensitive K+ channels.Hyperpolarizations produced by ATP and NO were inhibited by apamin. The i.j.p. amplitude was reduced after desensitizing the membrane with ATP. In atropinized tissue, TNS produced a relaxation that was reduced 15% by guanethidine (5 x 10(-6) M), 50% by L-NNA (10(-5) M), and 30% by apamin (10(-7) M). Thus the LES receives cholinergic excitatory and adrenergic and nitrergic inhibitory innervations; the latter two components contribute evenly to the i.j.p. generation. The relaxation is mainly produced by NO in a membrane potential-independent way.     

Neuropeptide Y-immunoreactive nerves in the uvea of guinea pig and rat

Neuropeptide Y (NPY)-immunoreactivity has been localized to a rich network of fibres in the uvea of guinea pig and rat. In the iris, NPY-immunoreactive nerves were present in iridial smooth muscle and around blood vessels; similarly throughout the choroid a dense network of NPY-immunoreactive fibres was found surrounding the choroidal vascularization. Following sympathectomy a marked decrease of NPY-immunoreactivity, in particular in the iris and less marked in the choroid, was noted, indicating that these fibres may originate at least in part from neuronal cell bodies in the superior cervical ganglion. The close relationship of the NPY-immunoreactive fibres to blood vessels in both anterior and posterior uvea is consistent with the established vasoconstrictor action of this peptide.     

Vagal origin of substance P-containing nerves in the guinea pig lung

This study reports on the possible origin and putative sensory nature of substance P-containing fibres in the lung. Following vagal ligation experiments, carried out on adult guinea pigs, the vagi, nodose ganglia and lungs were investigated by immunocytochemistry and radioimmunoassay. After ligation the lung showed a marked depletion of substance P-immunoreactivity; concomitantly there was an accumulation of immunoreactive material proximal to the ligature, indicating a peripheral direction of the peptide axonal flow. Several substance P-immunoreactive neuronal cell bodies were observed in the nodose ganglion. These results suggest that substance P-immunoreactive fibres in the lung might originate from primary sensory neurones in the nodose ganglion, which is compatible with other evidence that substance P has a sensory role.     

Slow muscle fibers in the tensor tympani muscle of the guinea pig

The tensor tympani muscle of the adult guinea pig was studied by light and electron microscopy. Extrafusal muscle fibers with centrally located nuclei were found. On the basis of morphological criteria, they were considered to be slow muscle fibers and others, with typical peripheral nuclei, twitch muscle fibers.     

豚鼠前列腺Cajal间质细胞与交感神经及平滑肌的形态与功能学联系

目的:探索前列腺Cajal间质细胞(interstitial cells of Cajal,ICCs)与交感神经及平滑肌细胞之间的形态及功能学联系。方法:(1)制作豚鼠前列腺组织冰冻切片并进行双重免疫荧光染色。使用酪氨酸激酶受体(c-Kit)作为ICCs的标记物,酪氨酸羟化酶(tyrosine hydroxylase,TH)和多巴胺β羟化酶(dopamineβ-hydroxylase,DβH)作为交感神经纤维的标记物,α肌动蛋白(α-actin)作为平滑肌细胞的标记物。另外,分别对α1-肾上腺素受体与c-Kit以及缝隙连接蛋白Connexin43(Cx43)与c-Kit进行免疫荧光双重标记。(2)将前列腺肌条固定于灌流槽中,记录去甲肾上腺素(norepinephrine,NE)诱发的肌条收缩幅度和频率,以及加入Glivec后肌条收缩幅度和频率的变化。结果:(1)免疫荧光组织化学标记结果显示:ICCs与交感神经纤维、平滑肌细胞平行分布,ICCs边缘与交感神经、平滑肌细胞边缘密切接触,c-Kit标记的ICCs上存在α1-肾上腺素受体和缝隙连接蛋白Cx43的共表达。(2)在去甲肾上腺素的刺激下前列腺平滑肌肌条收缩幅度为(0.98±0.16)g,收缩频率为(2.54±0.28)次/min。使用Glivec后肌条收缩幅度显著下降,50μmol/L组为(0.56±0.07)g(P0.01),200μmol/L组为(0.25±0.05)g(P0.01),收缩频率未见明显改变,50μmol/L组为(2.67±0.32)次/min(P0.05),200μmol/L组为(2.75±0.31)次/min(P0.05)。结论:豚鼠前列腺ICCs具有介导前列腺交感神经信号,支配平滑肌活动的形态和功能学基础。     

The effects of calcium and magnesium on inhibitory junctional transmission in smooth muscle of guinea pig small intestine

Summary The membrane potential of smooth muscle cells in the circular layer of the guinea pig ileum was recorded using intracellular electrodes. Transmural stimulation, in the presence of atropine, caused a transient hyperpolarization, an inhibitory junction potential (IJP). IJP's are thought to result from the action of transmitter released from intramural inhibitory nerves. It has been reported that, in the guinea pig jejunum, the amplitude of the IJP resulting from field stimulation is not altered by changes in the calcium and magnesium ion concentration in the bathing solution. Experiments reported here have shown that the IJP amplitude decreased markedly on reducing the calcium ion concentration and or increasing the magnesium ion concentration. Indirect evidence is presented suggesting that the decrease in amplitude of the IJP is due to a decrease in the amount of transmitter released.     

Effects of (−)baclofen on inhibitory neurons in the guinea pig hippocampal slice

Intracellular recordings were made from electrophysiologically identified inhibitory neurons in the dentate hilus. (–)Baclofen (0.1–0.5 mol/l), applied by the bath, strongly hyperpolarized inhibitory neurons, reduced their input resistance and induced outward currents under voltage clamp at holding potential of –60 mV in cells recorded with KCl-filled electrodes. Increasing the (–)baclofen concentration (up to 1 mol/l) did not increase the amplitude of the outward current, but increased its duration. (–)Baclofen depressed Cl-dependent IPSPs evoked by perforant path stimulation in inhibitory neurones, granule cells and CA3 neurons. In the case of inhibitory neurons and CA3 neurons, depression of IPSPs, membrane hyperpolarization and increase in membrane conductance concurred. All effects were blocked by BaCl₂ (1 mmol/l) in the superfusate. In the case of granule cells, depression of IPSPs by (–)balcofen out-lasted an only small membrane hyperpolarization, conductance increase or outward current. High concentrations (up to 10 mol/l) of (–)baclofen depressed evoked IPSPs of granule cells for an extended period of time, but the other effects remained small and transient. IPSPs elicited in granule cells by microdrop application of glutamate to the dentate hilus were also blocked by (–)baclofen, but spontaneous IPSPs were only reduced in amplitude. We suggest that the blockade of GABA_A receptor-mediated IPSPs of hippocampal neurons by the GABA_B receptor agonist (–)baclofen can be explained by a K-dependent hyperpolarization of inhibitory neurons.     

Evidence for two distinct Fc receptors on guinea pig peritoneal macrophages

Fcreceptors of guinea pig peritoneal macrophages for homologous IgG1 and IgG2 antibodies were found to be solubilized by treatment of the cells with Nonidet P-40, since the soluble fraction obtained inhibited the binding reactions of complexes of IgG1 and IgG2 antibodies with ovalbumin to the intact cells. The solubilized Fc receptors for IgG1 and IgG2 antibodies were indistinguishable from one another by means of gel filtration on a column of Sepharose 6B in the presence or absence of the detergent. Both of the Fc receptors were almost completely removed by affinity chromatography on a column of IgG2-bound Sepharose 6B. Affinity chromatography on a column of IgG1-bound Sepharose 6B, however, showed that the Fc receptor for IgG1 antibody was also almost completely adsorbed, though about 50% of the Fc receptor for IgG2 antibody remained unbound. These results demonstrate the existence of two distinct Fc receptors on guinea pig macrophages; one binds to IgG2 antibody alone and the other bind IgG2 as well as IgG1 antibodies.     

Neck muscle activity after unilateral labyrinthectomy in the alert guinea pig

 In the guinea pig, lateral deviation of the head is a cardinal symptom of the vestibular syndrome caused by unilateral labyrinthectomy. In the course of recovery from this syndrome (vestibular compensation), lateral deviation of the head disappears completely in 2–3 days. Because this symptom is known to be due to the lesion of the horizontal semicircular canal system, and since obliquus capitis inferior (OCI) muscle is activated predominantly by yaw rotation (horizontal vestibulocollic reflex), we hypothesized that changes in the activity of this muscle could be at least in part responsible for the lateral head deviation caused by unilateral labyrinthectomy. In order to test this hypothesis, electromyographic (EMG) activities of the right and left OCI muscles, as well as eye movements, were recorded in 12 head-fixed alert guinea pigs at various times after left surgical labyrinthectomy (performed with the animals under halothane anesthesia). After the operation, a decrease in tonic EMG activity was observed in the right (contralateral to the lesion) OCI muscle while an increase in tonic EMG activity was detected in the left (ipsilateral) OCI muscle. In addition, phasic changes in EMG activity associated with ocular nystagmic beats occurred in the OCI muscles. These phasic changes were in the opposite direction to those of the tonic changes. There were bursts of activity in the right OCI and pauses in the left OCI. From measurements of rectified averaged EMG activities which took into account both parts (tonic and phasic) of the phenomenon, it was concluded that the labyrinthectomy-induced asymmetry between the activities of the left and right OCI muscles was high enough and lasted long enough to be an important mechanism in the lateral deviation of the head caused by unilateral labyrinthectomy.     

Determination of extrinsic pathways of adrenergic nerves to the guinea-pig trachealis muscle using surgical denervation and organ-bath pharmacology

Intraluminal pressure changes were recorded in an isolated tracheal tube during electrical stimulation of the recurrent laryngeal nerve or transmural stimulation of the extra-thoracic trachealis muscle. The recurrent laryngeal nerve contained adrenergic nerve fibres running both anteriorly and posteriorly along this nerve and they caused an adrenergic inhibitory response of the trachealis muscle. The superior cervical ganglion was the source of the majority of the adrenergic fibres running both anteriorly and posteriorly within the recurrent laryngeal nerve. The antero-posterior adrenergic fibres reached the recurrent laryngeal nerve via an anastomosis with the superior cervical ganglion. The postero-anterior adrenergic fibres reached the recurrent laryngeal nerve via the vagus nerve, as ipsilateral vagotomy markedly reduced the inhibitory response of the trachealis muscle which had been due to the postero-anterior adrenergic fibres. Superior cervical ganglionectomy caused a significant reduction in the adrenergic response as a percentage of the total inhibitory response of the extra-thoracic trachealis muscle following transmural stimulation. The remaining response was due to stimulation of non-adrenergic non-cholinergic fibres and adrenergic fibres from another source.     

Spontaneous inhibitory postsynaptic potentials in guinea pig neocortex and olfactory cortex neurones

The membrane potential of olfactory cortex and neocortex neurones in vitro was recorded using conventional microelectrode techniques. During recordings with KCl- or CsCl-filled microelectrodes, spontaneous, subthreshold, transient membrane depolarizations were observed. These were abolished by the GABAA-receptor antagonist, bicuculline methiodide, and were prolonged by the barbiturate pentobarbitone. In most cells they were abolished by tetrodotoxin. It is concluded that these spontaneous depolarizations are inhibitory postsynaptic potentials arising from spontaneous activity in inhibitory interneurones.     

Capsaicin-sensitive stretch responses in ferret trachealis muscle.

1. Stretch-induced electrical and mechanical responses in segments of ferret trachealis muscle were studied. Stretches and post-stretch length changes were quantified by measuring distances between two marker spheres placed on the muscle surface. Electrical responses were determined by measuring membrane potential in the muscle cell syncytium. 2. Smooth muscle mechanical and electrical responses to the stretch manoeuvre were characterized by an initial shortening and depolarization phase and a reversal-repolarization phase. Both phases were resistant to atropine and tetrodotoxin. During the initial phase, the membrane depolarized to potentials as low as -20 mV. For stretches to 1.0 Lmax, from a holding length of 0.75 Lmax, 50% repolarization occurred at 6.8 +/- 0.4 min post-stretch; 50% reversal of shortening of the stretched segment occurred at 6.9 +/- 0.8 min post-stretch. 3. Depolarizing currents generated within muscle cells in the stretched segment spread into cells in non-stretched muscle. Space constants in the transverse and longitudinal directions averaged 480 +/- 46 and 146 +/- 50 microns, respectively. 4. During infusion of capsaicin (10 microM), muscle cells depolarized by 5.5 +/- 2.3 mV. Maximal depolarization was achieved after 15-20 min. After inhibition of neutral enkephalinase, capsaicin-evoked depolarization occurred more rapidly. Muscles depolarized by 11.2 +/- 2.1 mV after about 10 min of capsaicin and then slowly repolarized during continued treatment. When muscle segments were stretched during administration of capsaicin, the initial phase was similar to that observed before capsaicin, but the reversal-repolarization phase was prolonged. Following wash exposure to capsaicin, maximal stretch-induced depolarization was unchanged, but the time for 50% repolarization (t50-repolarization) decreased from the pre-capsaicin value of 8.4 +/- 1.3 to 4.1 +/- 0.5 min. The t50-reversal of stretch-evoked muscle shortening decreased to 54% of control values. 5. Short exposures (< 2 min) to substance P (SP, 1-7.5 microM) depolarized smooth muscle cells. Maximal depolarization was delayed, and occurred after [SP] had decreased to < 10 nM. Repolarization was delayed as long as 6 min following wash-out of SP. Stretches performed when SP-induced depolarization had nearly reversed showed no changes in the initial mechanical or electrical responses, but t50-repolarization increased to 162% of control values. 6. Immunochemical studies showed networks of neurones which react with SP antibodies. 7. These findings suggest that stretch induces SP release from capsaicin-sensitive C fibres, and that released SP affects smooth muscle ionic mechanisms which control and delay the reversal of stretch-induced membrane depolarization and shortening.