Development and ultrastructure of the neuromuscular junction in bronchial smooth muscle tissue

At various stages of pre- and postnatal ontogenesis ultrastructure of contacts of smooth myocytes and nervous terminals in the white mice bronchial wall has been investigated. Nervous fibers grow into the forming tissue of the lung beginning from the 11th day of embryogenesis. By the end of the prenatal development the nervous fibers fasciculi with varicosites and having vesicles are localized at the distance of 100-300 nm from the developing myocytes. Formation of dense neuromuscular junctions with the distance of 35-60 nm between the axonal membranes and the myocyte is observed on the 10-15 day after birth. In mature animals combination of various types of neuromuscular connections is revealed; they ensure local and distant neurotrophic regulation. In the bronchial smooth musculature afferent connections are revealed, as well as connections of myocytic processes with the effectors. Terminals of the cholinergic type predominate, adrenergic effectors occur very seldom. There are terminals, in which combination of vesicles having various structure and diameter are observed.     

Adrenergic and cholinergic components of the innervation of the arteries of the base of the bird brain

By means of Falck's and Koelle's methods adrenergic and cholinergic structures were studied in the arteries in the cerebral basis of blue rock pigeons and of hens, white leghorn stock. The number of nerve transmitters was estimated per 1 mm2 of the vessel surface. The arteries of the basis in pigeon brain are surrounded with developed adrenergic and cholinergic nerve plexuses, their density decreasing in the following order: nasal branch of the internal carotid artery, middle, nasal cerebral and basilar arteries. A little more cholinergic transmitters occur on the middle cerebral artery, while on the other vessels, concentration of cholinergic and adrenergic fibers is equal. In hens, the density in the arrangement of adrenergic nerve transmitters is higher in the nasal branch of the internal carotid and in the nasal cerebral arteries than in the basilar artery. At the same time, chromaffin cells forming numerous conglomerations in some places are found on the latter. In pigeons, the density of adrenergic fibers arrangement on the arteries of the cerebral basis is higher than in hens.     

Muscular force transmission: a unified, dual or multiple system? A review and some explorative experimental results

Structures contributing to force transmission in muscle are reviewed combining some historical and relatively recently published experimental data. Also, effects of aponeurotomy and tenotomy are reviewed shortly as well as some new experimental results regarding these interventions that reinforce the concept of myofascial force transmission. The review is also illustrated by some new images of single muscle fibres from Xenopus Laevis indicative of such transmission and some data about locations of insertion of human gluteus maximus muscle. From this review and the new material, emerges a line of thought indicating that mechanical connections between muscle fibres and intramuscular connective tissue play an important role in force transmission. New experimental observations are presented for non-spanning muscle (i.c., rat biceps femoris muscle), regarding the great variety of types of intramuscular connections that exist i n addition to myo-tendinous junctions at the perimuscular ends of muscle fibres. Such connections are classified as (1) tapered end connections, (2) Myo-myonal junctions, (3) myo-epimysial junctions and (3) Myo-endomysial junctions. This line of thought is followed up by consideration of a possible role of connections of intra- and extramuscular connective tissue in force transmission out of the muscle. Experimental results of an explorative nature, regarding the interactions of extensor digitorum longus (EDL), tibialis anterior (TA) and hallucis longus (HAL) muscles within a relatively intact dorsal flexor compartment of the rat hind leg, indicate that: (1) length force properties of EDL are influenced by TA activity in a length dependent fashion. Depending on TA length, force exerted by EDL, kept at constant origin insertion distance, is variable and the effect is influenced by EDL length itself as well; (2) Force is transmitted from muscle to extramuscular connective tissue and vice versa. As a consequence force exerted at proximal and distal tendons of a muscle are not always equal. The difference being transmitted by extramuscular connective tissue and may appear at the tendons of other muscles or may be transmitted via connective tissue directly to bone. It is concluded that the system of force transmission from skeletal muscle should be considered as a multiple system.     

Alpha-1, alpha-2, and beta adrenergic signal transduction in cultured uterine myocytes

Summary The following studies were undertaken to develop a cultured uterine myocyte model which would allow further clarification of the adrenergic signal transduction mechanisms utilized by these myocytes. After mechanical removal of the endometrium, rabbit uterine myoctes were isolated by an overnight enzymatic disaggregation using collagenase and DNase I. The isolated myocytes were maintained in culture in 75-cm² flasks containing Waymouth's MB 751/1 medium-10% fetal bovine serum along with 10⁻⁸ M estradiol, penicillin, streptomycin, and Fungizone. The phase contrast and electron micrographic appearance of these cells was consistent with that previously reported for smooth muscle myocytes in culture. Immunocytochemical studies utilizing monoclonal anti-alpha-smooth muscle actin antibodies confirmed the presence of smooth muscle actin in these cultured myocytes. Western blot studies similarly confirmed the presence of alpha-smooth muscle actin in rabbit myometrial tissue and the cultured myocytes, both the primary and F1 generation. After prelabeling the myocytes with [³H]inositol, adrenergic stimulation experiments demonstrated alpha-1 receptor mediated stimulation of inositol phosphates. Beta receptor stimulation experiments confirmed cAMP production in these cultured myocytes, and the ability of clonidine, an alpha-2 agonist, to inhibit forskolin stimulated cAMP production confirmed the presence of functional alpha-2 adrenergic receptors in these myocytes. In conclusion, these cultured rabbit uterine myocytes have provided an in vitro model which can be utilized to further clarify the adrenergic receptor signal transduction mechanisms in genital tract smooth muscle. This research was supported by grant HD-22063 from the National Institutes of Health, Bethesda, MD.     

Determination in the system of the neuromuscular relations of the coronary arteries at stages in human postnatal development

By means of micromorphometric method in transversal sections of electively stained right and left coronary arteries the data, characterizing the area of the middle and external sheaths, amount of myelin fibers in nerves, accompanying the artery have been obtained in men of 8 age groups (from birth up to old age), who died from causes not connected with the cardiovascular pathology. The summary amount of all classes of myelin fibers increases synchronously with an increase of the muscle sheath area from newborn up to 35-45 years of age. After this age the amount of the myelin fibers decreases together with decreasing area of the middle sheath. Minimal values of these indices are observed in the old age (85-90 years). The external sheath area increases permanently from birth up to the old age. The correlative analysis reveals a positive and significant connection of various degree between the myelin fibers number and value of the middle arterial sheath in persons of juvenile, mature and elderly age: amount of the muscular layer is adequate to the myelin fibers amount. In other age groups the correlative connections are either small, or not significant. Alternative, corresponding to each age quantitative gradations of the nervous connections are determined by means of the peripheral substrate. Its role in the vessels investigated is performed, to a definite degree, by the muscle sheath.     

Effect of combined exposure to hypokinesia and gravitational stress on the structure of the wall of the renal vein

Renal veins from 25 rabbits were investigated by means of imbedding in paraffin with subsequent hematoxylin--eosin staining, after Van Gieson and with fuchselin after Hart. Prolonged hypokinesia (over 3 weeks) with subsequent single application of gravitational overloading was stated to produce some structural changes in the wall of the renal vein. They are manifested as loosening of the middle vascular sheath with vacuolization of myocytes and outgrowth of its elastic elements. Hypokinesia of longer duration (8 weeks) with subsequent gravitational overloading produces atrophy of smooth muscle tissue in the middle sheath of the venous wall that brings about decreasing in tonus of the wall of the renal vein.     

Interstitial Cajal-like cells (ICLC) in atrial myocardium: ultrastructural and immunohistochemical characterization

We have previously reported (Hinescu & Popescu, 2005) the existence of interstitial Cajal-like cells (ICLC), by transmission electron microscopy, in human atrial myocardium. In the present study, ICLC were identified with non-conventional light microscopy (NCLM) on semi-thin sections stained with toluidine blue and immunohistochemistry (IHC) for CD117/c-kit, CD34, vimentin and other additional antigens for differential diagnosis. Quantitatively, on semi-thin sections, ICLC represent about 1-1.5% of the atrial myocardial volume (vs. approximately 45% working myocytes, approximately 2% endothelial cells, 3-4% for other interstitial cells, and the remaining percentage: extracellular matrix). Roughly, there is one ICLC for 8-10 working atrial myocytes in the intercellular space, beneath the epicardium, with a characteristic (pyriform, spindle or triangular) shape. These ICLC usually have 2-3 definitory processes, emerging from cell body, which usually embrace atrial myocytes (260 nm average distance plasmalemma/sarcolemma) or establish close contact with nerve fibers or capillaries (approximately 420 nm average distance to endothelial cells). Cell prolongations are characteristic: very thin (mean thickness = 0.15+/-0.1 microm), very long for a non-nervous cell (several tens of microm) and moniliform (uneven caliber). Stromal synapses between ICLC and other interstitial cells (macrophages) were found (e.g. in a multicontact type synapse, the average synaptic cleft was approximately 65 nm). Naturally, the usual cell organelles (mitochondria, smooth and rough endoplasmic reticulum, intermediate filaments) are relatively well developed. Caveolae were also visible on cell prolongations. No thick filaments were detected. IHC showed that ICLC were slightly and inconsistently positive for CD117/c-kit, variously co-expressed CD34 and EGF receptor, but appeared strongly positive for vimentin, along their prolongations. Some ICLC seemed positive for a-smooth muscle actin and tau protein, but were negative for nestin, desmin, CD13 and S-100. In conclusion, we provide further evidence of the existence of ICLC in human atrial myocardium, supporting the possible ICLC role in pacemaking, secretion (juxta- and/or paracrine), intercellular signaling (neurons and myocytes). For pathology, ICLC might as well be 'players' in arrhythmogenesis and atrial remodeling.     

Ketamine blocks Ca2+-activated K+ channels in rabbit cerebral arterial smooth muscle cells

Although ketamine and Ca2+-activated K+ (KCa) channels have been implicated in the contractile activity regulation of cerebral arteries, no studies have addressed the specific interactions between ketamine and the KCa channels in cerebral arteries. The purpose of this study was to examine the direct effects of ketamine on KCa channel activities using the patch-clamp technique in single-cell preparations of rabbit middle cerebral arterial smooth muscle. We tested the hypothesis that ketamine modulates the KCa channel activity of the cerebral arterial smooth muscle cells of the rabbit. Vascular myocytes were isolated from rabbit middle cerebral arteries using enzymatic dissociation. Single KCa channel activities of smooth muscle cells from rabbit cerebral arteries were recorded using the patch-clamp technique. In the inside-out patches, ketamine in the micromolar range inhibited channel activity with a half-maximal inhibition of the ketamine concentration value of 83.8 +/- 12.9 microM. The Hill coefficient was 1.2 +/- 0.3. The slope conductance of the current-voltage relationship was 320.1 +/- 2.0 pS between 0 and +60 mV in the presence of ketamine and symmetrical 145 mM K+. Ketamine had little effect on either the voltage-dependency or open- and closed-time histograms of KCa channel. The present study clearly demonstrates that ketamine inhibits KCa channel activities in rabbit middle cerebral arterial smooth muscle cells. This inhibition of KCa channels may represent a mechanism for ketamine-induced cerebral vasoconstriction.     

Ultrastructural features of the interrelationship between nerve fibers and the surrounding tissue in the major arteries of rabbits

The fine structure of nerve plexus in the rabbit abdominal aorta, ear and coronary arteries has been studied. Four types of the nerve fibre organization corresponding to different levels of sectioning their preterminal and terminal zones have been determined. Axons form large bundles which lose Schwann sheathes and divide into smaller and even single axons as they approach the distal end. Single axons make up contacts with effector cells without forming special synaptic structures. The smalles distance found between axon membranes and smooth muscle cells is 20 nm and 50 nm for the ear and coronary arteries, respectively. In the abdominal aorta, axons lie at a distance of several microns from the muscle sheath. Such arrangement supports the hypothesis of a "distant" nervous influence on the smooth muscle and indicates that the space of nervous influence comprises the tissue surrounding the blood vessel     

Glutamatergic innervation of rat skeletal muscle by supraspinal neurons: a new paradigm in spinal cord injury repair

Acetylcholine is the specific chemical code of spinal nerve terminal transmission at the mammalian neuromuscular junction (NMJ), whereas nicotinic acetylcholine receptors inserted into the membrane of muscle fibres mediate signalling for the muscle response. Glutamate has a primary role in neuromuscular transmission of organisms that are phylogenetically distant from mammals, the invertebrates, including insect and molluscs. Recent research has shown that diverting descending glutamatergic fibres in the spinal cord to rat skeletal muscle by means of a peripheral nerve graft causes the cholinergic synapse to switch to the glutamatergic type. These data demonstrate that under appropriate surgical manipulation supraspinal neurons can directly target muscle fibres and specify the postsynaptic receptors to achieve a functional glutamatergic NMJ.     

Comparative study of the cholinergic and adrenergic innervation of the cerebral vessels in humans and various laboratory animals

Choline- and adrenergic innervation of intracerebral branches of the middle and posterior cerebral arteries has been investigated histochemically and electron microscopically in slices, or after their preparation after W. Penfield. The vessels have been studied in the area of the fields 41, 17 of the cerebrum, trunk and spinal cord of the human being, cat and dog. When studying innervation of the intracerebral arteries (ICA), the preparation method has some advantages in comparison with investigation of these vessels in slices of the brain. Around most of the ICA from 200 up to 30 mcm in the diameter choline- and adrenergic nervous conductors are revealed. Using the method for calculating varicosities in nervous plexuses, it is demonstrated that degree of the ICA innervation is two times less than that in the arteries of similar caliber in the cerebral pia mater.     

Ultrastructure of intercellular contacts of smooth myocytes of the respiratory tract

By means of electron microscopy at various stages of pre- and postnatal ontogenesis in rats and mice intercellular connections of smooth myocytes, included into the bronchial wall composition, have been investigated. Interaction of the smooth myocytes is ensured with numerous and various types of specialized connections. Certain modifications of the nexus structure are described, they, perhaps, depend on the level of conformational changes of connexons--protein molecules, included into its composition and responsible for intensity of transfer of ions and metabolites. Between the smooth myocytes a contact, that was not previously described, is revealed. It is presented with a complex, formed by vesicular structures, concentrated in the area of oriented in parallel electron opaque membrane area of the interacting cells. Owing to the presence of punctate contacts, not only at early stages of development, but also in the definitive tissue, it is possible to consider them as an independent type of specialized connections, and not as immature forms of gap junctions. There are not any essential differences in the structure and distribution of the contacts during the time of the investigation.     

Cholinergic, adrenergic, and purinergic neuromuscular transmission.

A general model of the autonomic neuromuscular junction is proposed which emphasizes muscle effector bundles with gap junctions (or 'nexuses') forming the low resistance pathways allowing electrotonic coupling between neighboring cells, and extensive terminal varicose nerve fibers with 'en passage' release of transmitter. Some variations in autonomic neuromuscular geometry are discussed. Junctional clefts vary from 15nm in densely-innervated tissues such as vas deferens and iris to 2,000 nm in some large elastic arteries. Postjunctional specializations take the form of subsynaptic cysternae (in vas deferens and iris) and aggregations of plasmalemmal vesicles (in circular intestinal muscle). Current views of the synthesis, storage, release, and inactivation of transmitter during cholinergic, adrenergic, and purinergic transmission are summarized.     

Neural connections of the heart and superior vena cava in the rabbit and man

Innervational connections of the heart and the superior vena cava wall have been studied in the rabbit and the man. Besides, series of their embryos, impregnated with silver salts after Cajal-Favorsky have been investigated. Methods of Bielschowsky-Gros, Gomori and Karnovsky-Roots have also been applied. Adrenergic nervous elements have been revealed by means of incubation the slices in 2% solution of glyoxylic acid. Abundant cholinergic and adrenergic nervous plexuses are revealed on the wall of the superior vena cava, they are tightly connected with corresponding plexuses of the heart. Developmental of these nervous connections is followed, when embryogenesis of the cardiac nervous plexuses and large major vessels is studied in serial sections of embryos and fetuses of the rabbit and the man.     

Chronic hypoxia alters prejunctional alpha(2)-receptor function in vascular adrenergic nerves of adult and fetal sheep

The impact of development and chronic high-altitude hypoxia on the function of prejunctional alpha(2)-adrenoceptors was studied by measuring norepinephrine release in vitro from fetal and adult sheep middle cerebral and facial arteries. Blockade of prejunctional alpha(2)-adrenoceptors with idazoxan significantly increased stimulation-evoked norepinephrine release in normoxic arteries. This effect was eliminated after chronic hypoxia in cerebral arteries, with a tendency to decline in fetal facial arteries. After chronic hypoxia, the capacity to release norepinephrine declined in fetal middle cerebral arteries with a similar trend in facial arteries. Norepinephrine release was maintained in adult arteries. During development, stimulation-evoked norepinephrine release from middle cerebral and facial arteries was higher compared with adult arteries. In fetal arteries, adrenergic nerve function declined after chronic hypoxia. However, in adult arteries, adrenergic nerves adapted to chronic hypoxia by maintaining overall function. This differential adaptation of adrenergic nerves in fetal arteries may reflect differences in fetal distribution of blood flow in response to chronic hypoxic stress.     

Ancient fossil specimens of extinct species are genetically more distant to an outgroup than extant sister species are

There exists a remarkable correlation between genetic distance as measured by protein or DNA dissimilarity and time of species divergence as inferred from fossil records. This observation has provoked the molecular clock hypothesis. However, data inconsistent with the hypothesis have steadily accumulated in recent years from studies of extant organisms. Here the published DNA and protein sequences from ancient fossil specimens were examined to see if they would support the molecular clock hypothesis. The hypothesis predicts that ancient specimens cannot be genetically more distant to an outgroup than extant sister species are. Also, two distinct ancient specimens cannot be genetically more distant than their extant sister species are. The findings here do not conform to these predictions. Neanderthals are more distant to chimpanzees and gorillas than modern humans are. Dinosaurs are more distant to frogs than extant birds are. Mastodons are more distant to opossums than other placental mammals are. The genetic distance between dinosaurs and mastodons is greater than that between extant birds and mammals. Therefore, while the molecular clock hypothesis is consistent with some data from extant organisms, it has yet to find support from ancient fossils. Far more damaging to the hypothesis than data from extant organisms, which merely question the constancy of mutation rate, the study of ancient fossil organisms here challenges for the first time the fundamental premise of modern evolution theory that genetic distances had always increased with time in the past history of life on Earth.     

Innervation of the ureterovesical junction musculature of man

The ultrastructure of the innervation of the human ureterovesical junction was studied. Three different nerve terminals were distinguished among the smooth muscle cells. 1. Nerve processes containing predominantly small granular vesicles (40--60 nm in diameter). 2. Other nerve fibres contained predominantly small round agranular vesicles (30--50 nm in diameter). 3. Processes with large granulated vesicles (80--120 nm in diameter). The first type may be adrenergic, the second cholinergic and the third may originate from the local nerve cells. The gap between the nerve fibres and muscle cells was 300 to 500 nm wide and no synaptic thickenings were observed. This suggests that the transmitter may influence several muscle cells, and the different nerve fibres may directly innervate the smooth muscle cells.     

Adrenergic stimulation of inositol-phosphate production in a genital tract smooth muscle cell line

Catecholamines are important in the modulation of smooth muscle contractile activity; this study was undertaken to evaluate adrenoceptor stimulation of intracellular inositol-phosphate production in a genital tract smooth muscle myocyte. DDT1 MF-2 smooth muscle myocytes, derived from a hamster ductus deferens leiomyosarcoma, were loaded with 3H-inositol, incubated in 10 mM LiCl, then stimulated with adrenergic agonists with and without antagonists. Subsequently, the inositol phosphates were isolated by anion-exchange chromatography. In the presence of norepinephrine (NE), inositol trisphosphate (IP3) was produced by 30 s and peaked at 2 min; inositol 1-phosphate was also apparent by 30 s, and continued to increase over 15 min. Clonidine (an alpha-2 agonist), isoproterenol, and NE in the presence of phentolamine or prazosin (an alpha-1 antagonist) failed to increase IP3. In contrast, NE in the presence of yohimbine (an alpha-2 antagonist) or propranolol stimulated IP3 production to levels comparable to that stimulated by NE alone. These studies provide evidence that inositol phosphate production is involved in alpha-1 adrenergic signal transduction in DDT1 MF-2 myocyte.     

The fine structure of nerve endings on rat thyroid follicular cells

Summary Axon profiles in thyroid glands obtained from adult male Wistar rats were studied electron-microscopically, using common and serial thin sections.Bouton profiles of nerve fibers, resembling the terminal or en passant type, often appeared closely associated with vascular smooth muscle cells via basement membranes. These structures are probably adrenergic, since they contained mainly small-core vesicles (mean diameter: 41.2 nm), in addition to a few large-core (mean diameter: 88.4 nm) and flattened vesicles.Nerve fibers containing microtubules and sometimes mitochondria and vesicles were seen lying between basement membranes and follicular cells. The incidence of nerve fiber contacts on profiles of follicular cells was 0.0177±0.0092 (S.D.). Using serial sections, follicles were seen to have up to two nerve endings, separated from the plasma membranes of the follicular cells by a gap of 22 nm. They contained mainly flattened vesicles and several large-core vesicles (mean diameter: 95.1 nm). Small-core vesicles were rarely seen in these nerve endings. Furthermore, subsurface cistern-like rough endoplasmic reticulum was found immediately under the plasma membranes of follicular cells facing membranes of nerve endings. These results suggest that the nerve fibers in contact with follicular cells are different from the adrenergic type.