Postnatal development of excitatory innervations in longitudinal smooth muscle of the chicken anterior mesenteric artery

AimsThe anterior mesenteric artery of chickens contains a well-developed outer longitudinal smooth muscle layer in addition to an inner circular layer. Cholinergic and purinergic neurons play crucial roles in excitatory transmission at the longitudinal smooth muscle. The aim of this study was to clarify postnatal development of excitatory neurotransmission of the longitudinal smooth muscle.Main methodsMembrane potentials of smooth muscle were recorded with a microelectrode technique. Perivascular nerves were stimulated by applying electrical field stimulation (EFS).Key findingsHistological examination showed that longitudinal smooth muscles exist in the artery at birth. EFS failed to evoke membrane response in 1-day-old chickens, though it caused depolarization (excitatory junction potential; EJP) in 12-week-old chickens. However, exogenous application of acetylcholine (ACh) or ATP produced depolarization in longitudinal smooth muscle of 1-day-old chickens, suggesting that responsiveness of smooth muscle to excitatory neurotransmitters is already established at birth. In preparations isolated from 10-day-old chickens, EFS caused EJP, which was totally blocked by atropine but not by a non-specific purinoceptor antagonist, suramin. Several purinoceptor subtypes including P2Y1, which may be related to depolarizing response in smooth muscle of adult chickens, were expressed in the anterior mesenteric artery of 10-day-old chickens.SignificanceExcitatory innervation in longitudinal smooth muscle of the chicken anterior mesenteric artery is not established at birth but develops during the early postnatal period. Moreover, development of cholinergic excitatory innervation precedes that of purinergic excitatory innervation, although receptors that mediate purinergic control are already expressed in smooth muscle.     

Motor innervation by enteric nerve fibers containing both nitric oxide synthase and galanin immunoreactivities in the striated muscle of the rat esophagus

The relationship between nitric oxide synthase (NOS)- and galanin-immunoreactive nerve terminals and the origin of NOS-immunoreactive nerve terminals on the motor endplates in the striated muscles of the rat esophagus was investigated. Double immunohistochemical staining revealed a dual innervation of motor endplates by calcitonin gene-related peptide (CGRP)-immunoreactive axons and by axons that were immunoreactive for both NOS and galanin. On average, 91% of NOS terminals were galanin immunoreactive. NOS-immunoreactive fibers were revealed at 67% of endplates, identified by the presence of CGRP terminals. The left vagus and superior laryngeal nerve were cut and 15 days allowed for terminals to degenerate. This caused a significant loss of CGRP fibers, but did not affect the density of innervation of the striated muscle by NOS-immunoreactive fibers. Thus the NOS/galanin fibers are deduced to originate from ganglia in the esophageal wall. This is supported by our observation of numerous NOS-immunoreactive nerve cell bodies in the myenteric ganglia of the esophagus, 74% of which were galanin immunoreactive. There were no CGRP-immunoreactive nerve cell bodies in the wall of the esophagus.     

Signal transduction and protein phosphorylation in smooth muscle contraction

Smooth muscles are important constituents of vertebrate organisms that provide for contractile activity of internal organs and blood vessels. Basic molecular mechanism of both smooth and striated muscle contractility is the force-producing ATP-dependent interaction of the major contractile proteins, actin and myosin II molecular motor, activated upon elevation of the free intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, whereas striated muscles display a proportionality of generated force to the [Ca²⁺]_i level, smooth muscles feature molecular mechanisms that modulate sensitivity of contractile machinery to [Ca²⁺]_i. Phosphorylation of proteins that regulate functional activity of actomyosin plays an essential role in these modulatory mechanisms. This provides an ability for smooth muscle to contract and maintain tension within a broad range of [Ca²⁺]_i and with a low energy cost, unavailable to a striated muscle. Detailed exploration of these mechanisms is required to understand the molecular organization and functioning of vertebrate contractile systems and for development of novel advances for treating cardiovascular and many other disorders. This review summarizes the currently known and hypothetical mechanisms involved in regulation of smooth muscle Ca²⁺-sensitivity with a special reference to phosphorylation of regulatory proteins of the contractile machinery as a means to modulate their activity.     

Ultrastructure and differentiation of the larval esophageal muscle cells of the starfish Pisaster ochraceus

The muscle cells that cause constriction of the starfish larval esophagus (esophageal muscle cells) are one of the first cell types to express their differentiated morphological characteristics during development. Ultrastructurally these muscle cells resemble vertebrate and invertebrate smooth muscles. They contain a nucleus, a Golgi apparatus, contractile myofilaments, hemidesmosome-like structures, and what appears to be a simple sarcoplasmic reticulum. In asteroid embryos, this muscle layer originates during mouth formation when mesenchyme cells migrate from the tips of the coeloms to the esophagus. Once there, they elongate, forming processes. Over the next few days, the processes become filled with arrays of longitudinally arranged thick and thin myofilaments and thin sacs of smooth endoplasmic reticulum. The latter appear between the bundles of contractile filaments and the cell membranes. Contractile activity begins at approximately this time. The cisternae may represent a sarcoplasmic reticulum that is required for contraction. The majority of the esophageal muscle cell processes extend around the circumference of the developing esophagus, but occasional cells may be oriented in other directions. The latter cells are always farther away from the basal lamina and probably have little or no contact with it. Contact with basal lamina may serve to direct the migration of the cells and the orientation of the processes. J. Morphol. 237:1–18, 1998. © 1998 Wiley-Liss, Inc.     

甲壳动物横纹肌的收缩蛋白质与调节机制

甲壳动物横纹肌肌原纤维的肌丝陈列,收缩蛋白质和收缩的Ca²⁺依赖性调节机制与脊椎动物横纹肌有不少差异.脊椎动物横纹肌、甲壳动物快肌与慢肌的粗丝与细丝的数量比依次为1:2,1:3和1:6,肌丝阵列各异.甲壳动物粗肌丝由肌球蛋白和副肌球蛋白组成,其分子装配与脊椎动物不同.细肌丝含有肌动蛋白、原肌球蛋白和肌钙蛋白,肌钙蛋白-T分子量较高,肌钙蛋白-C仅1个Ca²⁺结合位点.甲壳动物横纹肌兼有细肌丝调节与粗肌丝调节.     

Neurocalcin-like immunoreactivity in the rat esophageal nervous system

Neurocalcin is a newly identified neuronal calcium-binding protein. We tried here to investigate the immunohistochemical distribution of neurocalcin in the rat esophagus. Nerve cell bodies having neurocalcin immunoreactivity were found throughout the myenteric plexus. In the myenteric ganglia, two types of nerve terminals showed neurocalcin immunoreactivity. One was varicose terminals containing numerous small clear vesicles and forming a synapse with nerve cells. The other terminals were characterized by laminar or pleomorphic structure and many mitochondria. These laminar terminals were supposed to be sensory receptors of the esophageal wall. In the motor endplates of the striated muscles, nerve terminals containing many small clear vesicles and mitochondria also had neurocalcin immunoreactivity. After left vagus nerve cutting under the nodose ganglia, the number of immunopositive thick nerve fibers, laminar endings and nerve terminals on the striated muscles decreased markedly. Retrograde tracing experiments using Fast Blue showed extrinsic innervation of esophagus from ambiguus nucleus, dorsal motor nucleus of vagus, superior cervical ganglia, celiac ganglia, nodose ganglia and dorsal root ganglia. In the celiac ganglia, nodose ganglia and dorsal root ganglia, retrogradely labeled nerve cells were neurocalcin-immunoreactive. Neurons in the celiac ganglia may project varicose terminals, while nodose and dorsal root neurons project laminar terminals. Although cell bodies of motoneurons in the ambiguus nucleus lacked neurocalcin immunoreactivity, these neurons may contain neurocalcin only in the nerve terminals in the motor endplates. Neurocalcin immunoreactivity is distributed in many extrinsic and intrinsic neurons in the esophagus and this protein may play important roles in regulating calcium signaling in the neurons.     

Structural proteins in the myofilaments and regulation of contraction in vertebrate smooth muscle.

The contractile systems of vertebrate smooth and striated muscles are compared. Smooth muscles contain relatively large amounts of actin and tropomyosin organized into thin filaments, and smaller amounts of myosin in the form of thick filaments. The protein contents are consistent with observed thin:thick filament ratios of about 15-18:1 in smooth compared to 2:1 in striated muscle. The basic characteristics of both types of contractile proteins are similar; but there are a variety of quantitative differences in protein structures, enzymatic activities and filament stabilities. Biochemical and X-ray diffraction data generally support recent ultrastructural evidence concerning the organization of the myofilaments in smooth muscle, although a basic contractile unit comparable to the sarcomere in striated muscle has not been discerned. Myofilament interactions and contraction in smooth muscle are controlled by changes in the Ca2+ concentration. Recent evidence suggests the Ca2+-binding regulatory site is associated with the myosin in vertebrate smooth muscle (as in a variety of invertebrate muscles), rather than with troponin which is the regulatory protein associated with the thin filament in vertebrate striated muscle.     

Rescue of skeletal muscle α-actin–null mice by cardiac (fetal) α-actin

Skeletal muscle α-actin (ACTA1) is the major actin in postnatal skeletal muscle. Mutations of ACTA1 cause mostly fatal congenital myopathies. Cardiac α-actin (ACTC) is the major striated actin in adult heart and fetal skeletal muscle. It is unknown why ACTC and ACTA1 expression switch during development. We investigated whether ACTC can replace ACTA1 in postnatal skeletal muscle. Two ACTC transgenic mouse lines were crossed with Acta1 knockout mice (which all die by 9 d after birth). Offspring resulting from the cross with the high expressing line survive to old age, and their skeletal muscles show no gross pathological features. The mice are not impaired on grip strength, rotarod, or locomotor activity. These findings indicate that ACTC is sufficiently similar to ACTA1 to produce adequate function in postnatal skeletal muscle. This raises the prospect that ACTC reactivation might provide a therapy for ACTA1 diseases. In addition, the mouse model will allow analysis of the precise functional differences between ACTA1 and ACTC.     

Morphofunctional and histochemical characteristics of the hindlimb muscles of the Rana temporaria in ontogeny

Mixed muscles of adult frogs respond to the increase in external potassium and to Ach by polyphasic contracture which is due to asynchronous activity of various groups of muscle fibers (fast phasic, intermediate and tonic ones). In the developing in vivo hindlimb muscles, the predominance of phasic contractile response and relatively weak tonic one were noted. In contrast to definitive muscles, in which maximum potassium and acetylcholine contractures are identical, growing muscles produce weak contractile reaction to Ach. Ach sensitivity of the developing muscles (as revealed by the contracture) is lower than in the definitive ones. Histochemical (studies on the lipid content and the activity of succinate dehydrogenase) and morphometric (the ratio of muscle fibers of different types at different stages of development, comparison of their diameters, relative size of tonic bundle, etc.) studies indicate that the development of morphological substrate for tonic contractions (tonic and intermediate muscle fibers) takes place at a lower rate as compared to the development of the substrate for phasic contractions. However, histochemically tonic fibers may be revealed already at the stage of myotubes.     

Cholinergic stimulation induces asynchrony between the circular and longitudinal muscle contraction during esophageal peristalsis

In healthy subjects, a close temporal correlation exists between contractions of the circular muscle (CM) and longitudinal muscle (LM) layers of the esophagus. Patients with nutcracker esophagus show disassociation between the peak of contractions of the CM and LM layers and the peak of contraction 1-3 s apart (Jung HY, Puckett JL, Bhalla V, Rojas-Feria M, Bhargava V, Liu J, Mittal RK. Gastroenterology 128: 1179-1186, 2005). The purpose of the present study was to evaluate the effect of acetylcholinesterase inhibitor (edrophonium) and acetylcholine receptor antagonist (atropine) on human esophageal peristalsis in normal subjects. High-frequency intraluminal ultrasound imaging and manometry were performed simultaneously during swallow-induced peristalsis in ten normal subjects. Standardized 5-ml water swallows were recorded 2 cm above the lower esophageal sphincter under three study conditions: control, edrophonium (80 microg/kg iv), and atropine (10 microg/kg iv). A close temporal correlation exists between the peak pressure and peak wall thickness during the control period. The mean time lag between the peak LM and peak CM contraction was 0.03 s. After edrophonium administration, the mean contraction amplitude increased from 101 +/- 9 mmHg to 150 +/- 20 mmHg (P < 0.05) and mean peak muscle thickness increased from 3.0 +/- 0.2 mm to 3.6 +/- 0.3 mm (P < 0.01), and duration of both CM and LM contractions were also increased. Furthermore, the mean time difference between the peak LM and CM was increased to 1.1 s, (ranging 0.2 to 3.4 s) (P < 0.0001). We conclude that cholinomimetic agent induces discoordination between the two muscle layers of the esophagus.     

Further pharmacological study on the cholinergic and glutaminergic properties of the radula muscles of a mollusc,Rapana thomasiana

1. In the radula protractor of the prosobranch mollusc Rapana thomasiana, both twitch contractions and acetylcholine contractions were markedly depressed or blocked by propantheline (10⁻⁵ M) and strychnine (10⁻⁵ M), but in the radula retractor, only acetylcholine contraction was markedly affected by the antagonists,2. Glutamate contractions of both of the muscles were little or slightly affected by the drugs.3. Twitch contraction of the protractor was slowly depressed when the muscle was immersed in concanavalin A (0.3 mg/ml), while that of the retractor was first potentiated and then slowly depressed in it.4. In both of the muscles, glutamate contractions were markedly enhanced by the lectin, but acetylcholine contractions were not affected.5. These results support the notion that the principal excitatory neurotransmitter in the protractor is acetylcholine, whereas that in the retractor is glutamate.     

Effect of strontium on the contractile properties of postnatally developing rat heart ventricles

The effects of substitution of calcium (Ca) by an equimolar concentration of strontium (Sr) on isometric contractions of isolated ventricular muscle from postnatally developing rat heart were studied. The duration of contraction and the time-to-peak tension were increased in all age groups although much less in the adult rats than in the neonates. The contractile force was increased in the muscles of rats between 1 and 14 days of age but was depressed in the older animals. The prominent rest-twitch potentiation of neonatal rat heart in Ca-Tyrode was totally eliminated by Sr, whereas a clear rest-twitch potentiation was induced by this cation in the adult rat heart, in which tissue the potentiation is normally absent in Ca-Tyrode. The maximal twitch potentiation by rest in Ca-Tyrode and the positive inotropic effect of Sr substitution grew from birth up to day 9 and from then gradually declined towards the level of adult rat heart by the end of the 3rd postnatal week. The phase of increasing rest-twitch potentiation coincides fairly well with the known development of sarcoplasmic reticulum and the phase of decline with the appearance of the T system of the sarcolemma. It is suggested that the qualitative changes in the contractile properties of developing rat heart during the 3rd postnatal week are due to the more efficient utilization of intracellular calcium stores, owing to the development of the T system.     

Validity of Estimation of Pelvic Floor Muscle Activity from Transperineal Ultrasound Imaging in Men

Purpose

To investigate the relationship between displacement of pelvic floor landmarks observed with transperineal ultrasound imaging and electromyography of the muscles hypothesised to cause the displacements.

Materials and Methods

Three healthy men participated in this study, which included ultrasound imaging of the mid-urethra, urethra-vesical junction, ano-rectal junction and bulb of the penis. Fine-wire electromyography electrodes were inserted into the puborectalis and bulbocavernosus muscles and a transurethral catheter electrode recorded striated urethral sphincter electromyography. A nasogastric sensor recorded intra-abdominal pressure. Tasks included submaximal and maximal voluntary contractions, and Valsalva. The relationship between each of the parameters measured from ultrasound images and electromyography or intra-abdominal pressure amplitudes was described with nonlinear regression.

Results

Strong, non-linear relationships were calculated for each predicted landmark/muscle pair for submaximal contractions (R²–0.87–0.95). The relationships between mid-urethral displacement and striated urethral sphincter electromyography, and bulb of the penis displacement and bulbocavernosus electromyography were strong during maximal contractions (R²–0.74–0.88). Increased intra-abdominal pressure prevented shortening of puborectalis, which resulted in weak relationships between electromyography and anorectal and urethravesical junction displacement during all tasks.

Conclusions

Displacement of landmarks in transperineal ultrasound imaging provides meaningful measures of activation of individual pelvic floor muscles in men during voluntary contractions. This method may aid assessment of muscle function or feedback for training.     

Spatial and temporal organization of TrkB expression in the developing musculature of the mouse esophagus

TrkB expression was investigated immunocytochemically in the developing musculature of mouse esophagus using conventional and confocal laser scanning microscopy. To demonstrate spatial relationships of TrkB immunoreactive cells to striated and smooth muscle fibers we combined TrkB immunocytochemistry with fluorochrome-tagged alpha-bungarotoxin for labeling of nicotinic acetylcholine receptors, and alpha-smooth muscle actin for labeling of smooth muscle cells. At developmental stages E15 to P7, TrkB immunoreactive cells transiently occurred in a transformation zone where striated intermingled with smooth muscle fibers. This transformation zone started in the rostral esophagus at E15, moved caudally, and disappeared between P7 and P10 in the caudal esophagus. The first TrkB-immunoreactive cells appeared in the outer muscle layer at E15. No TrkB-positive cells exhibited acetylcholine receptor clusters or were positive for alpha-smooth muscle actin. A few showed slight alpha-bungarotoxin staining over their entire surface. Taken together, the appearance of TrkB-expressing cells in the transformation zone suggest a role in muscle transdifferentiation. Alternatively, these results, together with recent in vitro data, suggest that TrkB is expressed in a subpopulation of myoblasts in which acetylcholine receptor clustering may be inhibited through a TrkB-mediated pathway.     

Cholinergic innervation to the upper esophageal sphincter muscle in the eel,with special reference to drinking behavior

To elucidate innervation in the upper esophageal sphincter (UES) muscle of the eel, a key muscle in swallowing, repetitive electrical field stimulation (EFS; 30 mA, 40 V, 300 micros, 10 Hz, 10 trains) was employed. Anatomically, the eel UES muscle consists of striated fibers. The EFS-induced contraction of the UES was completely blocked by tetrodotoxin and curare, and abolished in Ca2+ -free Ringer solution. These results suggest that the EFS stimulates nerve fibers specifically and releases acetylcholine as a neurotransmitter. In fact, acetylcholine and carbachol constricted the UES in a concentration-dependent manner. Even after blocking neuronal firing with tetrodotoxin, acetylcholine constricted the UES muscle, suggesting the existence of acetylcholine receptors on the UES muscle cells. Both EFS- and carbachol-evoked contractions of the UES were blocked by curare at a lower concentration than by atropine or hexamethonium, suggesting that the acetylcholine receptor is nicotinic. Even in Ca2+ -free Ringer solution, a direct current stimulus (2 s duration) constricted the UES muscle to an extent similar to that in the presence of Ca2+, indicating that the muscle contraction itself does not need extracellular Ca2+, i.e., the muscle can be constricted by a release of Ca2+ from the sarcoplasmic reticulum.     

Variability in the muscle composition of rat esophagus and neural pathway of lower esophageal sphincter relaxation

Several studies from our laboratory show that axial stretch of the lower esophageal sphincter (LES) in an oral direction causes neurally mediated LES relaxation. Under physiological conditions, axial stretch of the LES is caused by longitudinal muscle contraction (LMC) of the esophagus. Because longitudinal muscle is composed of skeletal muscle in mice, vagal-induced LMC and LES relaxation are both blocked by pancuronium. We conducted studies in rats (thought to have skeletal muscle esophagus) to determine if vagus nerve-mediated LES relaxation is also blocked by pancuronium. LMC-mediated axial stretch on the LES was monitored using piezoelectric crystals. LES and esophageal pressures were monitored with a 2.5-Fr solid-state pressure transducer catheter. Following bilateral cervical vagotomy, the vagus nerve was stimulated electrically. LES, along with the esophagus, was harvested after in vivo experiments and immunostained for smooth muscle (smooth muscle α-actin) and skeletal muscle (fast myosin heavy chain). Vagus nerve-stimulated LES relaxation and esophageal LMC were reduced in a dose-dependent fashion and completely abolished by pancuronium (96 μg/kg) in six rats (group 1). On the other hand, in seven rats, LES relaxation and LMC were only blocked completely by a combination of pancuronium (group 2) and hexamethonium. Immunostaining revealed that the longitudinal muscle layer was composed of predominantly skeletal muscle in the group 1 rats. On the other hand, the longitudinal muscle layer of group 2 rats contained a significant amount of smooth muscle (P < 0.05). Our study shows tight coupling between axial stretch on the LES and relaxation of the LES, which suggests a cause and effect relationship between the two. We propose that the vagus nerve fibers that cause LMC induce LES relaxation through the stretch-sensitive activation of inhibitory motor neurons.     

The mechanical advantage of local longitudinal shortening on peristaltic transport

Whereas bolus transport along the esophagus results from peristaltic contractions of the circular muscle layer, it has been suggested that local shortening of the longitudinal muscle layer concentrates circular muscle fibers in the region where the highest contractile pressures are required. Here we analyze the mechanical consequences of local longitudinal shortening (LLS) through a mathematical model based on lubrication theory. We find that local pressure and shear stress in the contraction zone are greatly reduced by the existence of LLS. In consequence, peak contractile pressure is reduced by nearly 2/3 at physiological LLS, and this reduction is greatest when peak in LLS is well aligned with peak contractile pressure. We conclude that a peristaltic wave of local longitudinal muscle contraction coordinated with the circular muscle contraction wave has both a great physiological advantage (concentrating circular muscle fibers), and a great mechanical advantage (reducing the level of contractile force required to transport the bolus), which combine to greatly reduce circular muscle tone during esophageal peristalsis.     

OBLIQUELY STRIATED MUSCLE : III. Contraction Mechanism of Ascaris Body Muscle

Segments of the obliquely striated body muscle of Ascaris were fixed at minimum body length after treatment with acetylcholine and at maximum body length after treatment with piperazine citrate and then studied by light and electron microscopy. Evidence was found for two mechanisms of length change: sliding of thin filaments with respect to thick filaments such as occurs in cross-striated muscle, and shearing of thick filaments with respect to each other such that the degree of their stagger increases with extension and decreases with shortening. The shearing mechanism could account for great extensibility in this muscle and in nonstriated muscles in general and could underlie other manifestations of "plasticity" as well. In addition, it is suggested that the contractile apparatus is attached to the endomysium in such a way that the sarcomeres can act either in series, as in cross-striated muscle, or individually. Since the sarcomeres are virtually longitudinal in orientation and are almost coextensive with the muscle fiber, it would, therefore, be possible for a single sarcomere contracting independently to develop tension effectively between widely separated points on the fiber surface, thus permitting very efficient maintenance of isometric tension.     

A centrosomal inclusion (striped nebulous body) in pinealocytes of the golden hamster

Summary The postnatal maturation of regions of the epididymis and intragonadal segment of the deferens duct was studied in the rat by light-and transmission electron microscopy. Maturation of the genital duct starts in the distal cauda epididymidis and ductus deferens after one week of life, and one week later, in the more cranial segments of the epididymis. Epithelial principal cells and peritubular contractile cells are structurally mature 35 days after birth. The synchronous changes of these cells indicate that the same factors control their postnatal maturation. The epithelial principal cells obtain an endocytotic apparatus and long stereocilia, whereas peritubular cells acquire contractile features. These changes are associated with a progressive increase in the immunoreaction for smooth muscle actin in both cell types. Smooth muscle myosin is detected in the apical region of the epithelial cells and the peritubular cell cytoplasm by day one of postnatal development. The differentiation of contractile cells in the wall is accompanied by progressive organization of the pericellular matrix into a continuous basement membrane. Although fibronectin is visible at birth, it is gradually removed from the tubule wall.