Innervation of the intestinal muscular coat

Summary Nerve bundles have an uneven distribution in the muscularis externa of the guinea-pig ileum. They are absent in the longitudinal muscle layer, and are mainly concentrated between the two muscle layers and between the bulk of the circular layer and its innermost portion. The latter is formed by muscle cells which are smaller and more electron dense than the ordinary smooth muscle cells. In cross sections of the circular layer there are 5425 intramuscular axons (of which 921 contain vesicles) per 10000 smooth muscle cells, and they are grouped in bundles, mainly formed by 10–40 axons. The gap between axons and smooth muscle cells is generally hundreds of nanometres; only a few junctions with a gap smaller than 20 nm occur. Interstitial cells have a cytoplasm occupied mainly by smooth reticulum and ribosomes, and processes containing filaments. These processes are closely associated with smooth muscle cells and nerve bundles. Interstitial cells have no basal lamina. Nexuses are found in the circular but not in the longitudinal muscle layer. Attachment plaques and invaginating processes are found in both muscle layers. More complex interdigitations appear between contacting muscle cells. There are 84 intramuscular blood vessels per 10 000 cross-sectioned smooth muscle cells (or 1700 per mm²).     

Light and electronmicroscopy of the bowel in an unusual form of Hirschsprung's disease

In a histological and histochemical study of multiple biopsies of unaffected segments of the bowel from 15 patients with Hirschsprung's Disease (H.D.), the AChE or non-specific esterase and the NADPH tetrazolium reductase enzyme reactions proved to be useful in identification of myenteric plexus islands; and acid phosphatase for the delineation of individual neurones. In the affected segment (usually aganglionic), this myenteric plexus tissue was not reactive for esterase, but individual nerve fibres among muscle fibres of the two muscle coats showed the enzyme product in a third of the cases. Fine structural study of biopsies from a typical case of H.D., showed normal looking axons and Schwann cytoplasm with terminals bearing both andrenergic and cholinergic vesicles in the unaffected colon, smooth muscle fibres with normal fine structure in all parts of the bowel, and loss of neurons with myenteric plexus replaced by nerve fibre groups in the affected rectosigmoid. One patient clinically presenting as a case of severe H.D., with histologically and histochemically normal myenteric and submucous ganglion cells, and not responding to resection of the bowel, showed degeneration of the unmyelinated axons with prominent Schwann cytoplasm, depleted cholinergic but persistent adrenergic vesicles, and markedly thinned and degenerating smooth muscle fibres and myofilaments, suggesting either a primary disorder of muscle tissue of the colon or, less likely, a denervation atrophy with secondary degeneration of the smooth muscle fibres.     

The fine structure of the interstitial tissue of the rat prostate

The structure of the interstitial tissue of the rat prostate has been studied using the light and electron microscopes in an attempt to determine the role of the fibromuscular stroma in the normal functioning of the gland. Smooth muscle cells and fibroblasts are the most numerous cell types. They are accompanied by macrophages, mast cells, and undifferentiated cells of low electron density. Smooth muscle cells have cytoplasmic protrusions that extend into corresponding depressions in adjacent muscle cells, and, at these points, the intercellular space is narrowed to 150–2000 Å. Smooth muscle cells and fibroblasts are arranged in parallel in septa between adjacent epithelial alveoli and form a sheath around each alveolus. Proceeding peripherally from the epithelium into the interstitial tissue, this sheath is composed of a layer of one or two highly flattened fibroblasts and a parallel layer of smooth muscle cells, followed subsequently by additional layers of fibroblasts and smooth muscle that merge into the remainder of the interstitial tissue. Most of the capillaries have an uninterrupted endothelium, but in some regions endothelial fenestrations are present. Unmyelinated axons contain aggregations of small granular and agranular vesicles. Vesicles are found in axons at distances up to several thousand angstroms from muscle cells and in axons that approach to within 150–200 Å of smooth muscle cells. In some cases an axon lies in a deep depression in the surface of a muscle cell. The type of innervation and variety of intercellular contact between muscle cells is discussed in relation to probable physiological characteristics of the tissue.     

Development and ageing of intestinal musculature and nerves: the guinea-pig taenia coli

The fine structure of taenia coli was studied by electron microscopy in guinea-pigs from birth to old age (over 2 years old). Smooth muscle cells are approximately 1,000 microm(3) in volume at birth, 2,200 microm(3) in young adults and 4,500 microm(3) in old age. Muscle growth and muscle cell enlargement continue throughout life, an increase in muscle volume of about 240 times. Differentiated muscle cells divide during development and in adults. Because mitoses are found in any part of the muscle, the tissue grows from within, rather than by addition at the ends or borders. There is progressive increase in nucleus volume, and decrease in surface-to-volume ratio and in nucleus-cell volume ratio in muscle cells. At all ages the taenia consists of a uniform population of muscle cells (apart from dividing cells); there are no undifferentiated cells, no precursor cells or myoblasts, and no degenerating cells. Interstitial cells and fibroblasts are observed at all ages with only small variations in relative number. The amount of intramuscular collagen increases in old age. There is roughly one capillary for every 170 muscle cell profiles at birth, and one for every 200 in adults and in old age. The innervation is dense and reaches all parts of the muscle. In adults there are approximately 1,300 axons per 10,000 microm(2) of sectional area, or between 8,000 and 38,000 axons in a full cross section of taenia; this amounts to approximately 2% of the muscle volume. An answer to the question of why there are so many nerves in the taenia was not found. Expanded axon profiles are part of typical varicose fibres. Varicosities are packed with small clear vesicles and lie at the surface of nerve bundles. Absence of strong, constant patterns indicating specialized contacts of the nerve terminals is a feature of these nerves at all ages. Some varicosities are closest to interstitial cells; more commonly they are close to muscle cells at sites that strongly suggest a neuro-muscular junction. The additional possibility that some varicosities are part of afferent fibres is discussed. The innervation is well developed at birth and the highest density of innervation is found around day 4 when 4% of the taenia consists of nervous tissue. The innervation of immature taenia is characterized by close juxtaposition of axons and muscle cells. Axon profiles packed with vesicles, varicosities and presumptive neuro-muscular junctions are present at birth. The extent of Schwann cells in intramuscular nerves is markedly less than in adults, and virtually all the axons have maximal membrane-to-membrane contact with other axons. In taenia of aged guinea-pigs, the density of innervation is reduced. There is no actual loss of nerve tissue; the total amount of nerve tissue is greater than in young adults, and the apparent reduction reflects a more intense growth of muscle cells. The Schwann cell component becomes more conspicuous than in young adults and there is a greater number of axons fully wrapped by a Schwann cell. Presumptive neuro-muscular junctions are common and probably commoner than in young adults. Growth of muscle cells, changes in their cytological features and in the stroma occur throughout life, including old age. Nerves too continue to grow and undergo structural changes in pattern of distribution, relation with Schwann cells and effector cells.     

An analysis of the transmission of excitation from autonomic nerves to smooth muscle

1. An analysis has been made of the transmission of excitation from the hypogastric nerve to the smooth muscle cells of the guinea-pig vas deferens.2. Depolarization of single muscle cells with current pulses from an intracellular electrode gave local depolarizations of the cell membrane which were not propagated. The total membrane resistance after 100 msec of depolarization was 15 MOmega for depolarizations between 10 and 40 mV.3. Depolarization of some cell membranes with a current pulse during the excitatory junction potential (E.J.P.) decreased the amplitude of the E.J.P. from about 10 mV at 20 mV depolarization, to nearly zero at 60 mV depolarization. In some cells the E.J.P. was unchanged during depolarizations of 50 mV.4. The action of transmitter on the smooth muscle cell membrane continued for the duration of the E.J.P. Action potentials which occurred at various times during the E.J.P. failed to remove the remaining phases of the E.J.P.5. It was shown that the slow time course of the E.J.P. could not be due to the instantaneous and simultaneous release of transmitter from a number of relatively distant sources.6. It was shown that each smooth muscle cell was innervated by several axons. The serial sections examined with the electron microscope showed that a smooth muscle had either a single axon terminating within 200 A of the muscle or no axons terminating on it at all. Therefore transmitter must be released along the length of the axons as well as at the terminations of the axons.     

A study of excitatory neuromuscular transmission in the bovine trachea

1. The excitatory innervation of bovine tracheal smooth muscle has been studied with the sucrose-gap apparatus.2. Single 2 ms electrical stimuli applied to the whole tissue excited intrinsic nerves, and produced a small transient depolarization of the smooth muscle, the excitatory junction potential (e.j.p.). The e.j.p. caused a twitch-type contraction; twitches and e.j.p.s summated during repetitive stimulation but facilitation was not observed, and action potentials were never elicited.3. The effects of electrical stimulation could be abolished by atropine (5 x 10(-7) mol/l) and augmented by neostigmine (4 x 10(-6) mol/l), and were mimicked by exogenous acetylcholine (1.0 mug/ml).4. With the electron microscope, the density of innervation was found to be low (one axon per ninety smooth muscle cells). Axons were found in small groups in the clefts between bundles of cells, but no axons penetrated within the muscle bundles. Naked axon varicosities containing agranular vesicles were seen, but no axon approached within 200 nm of a smooth muscle cell.5. It is difficult to reconcile the sparsity of innervation with the dependence of the tissue on nerve excitation to initiate activity.     

T-2 toxin effect on rat aorta: cellular changes in vivo and growth of smooth muscle cells in vitro

Rats were injected intraperitoneally with T-2 toxin and their aortas were studied by light and electron microscopy. The growth of smooth muscle cell explants taken from the tunica media of aortas of similarly treated animals was observed. A single large dose (2 mg/kg) or four injections of 0.3 mg/kg T-2 toxin caused damage and occasional necrosis of endothelial cells, accumulation of basement membrane-like material in the intima, and swelling and activation of smooth muscle cells in the tunica media. Three or more weeks after the last injection of 0.3 mg/kg T-2 toxin the endothelial cells were normal but an excess of fragmented intimal basement membrane-like material persisted and smooth muscle cells were still activated. Outgrowths from explants of aortic tunica media taken within 1 week of the last dose of T-2 toxin showed marked inhibition of smooth muscle cell growth. Three or more weeks after the toxin, the explants showed significantly increased outgrowths. These findings suggest that T-2 toxin causes early endothelial and smooth muscle cell injury accompanied by inhibition of smooth muscle cell growth in culture. This is followed by stimulation of the proliferative capacity of smooth muscle cells in vitro. If a similar mechanism is operative in vivo, it could explain the chronic vascular changes observed after limited exposure to T-2 toxin.     

An ultrastructural analysis of the developing enteric nervous system of the guinea-pig small intestine

Summary The developing enteric nervous system of the guinea-pig has been analysed ultrastructurally. In addition, electron microscope autoradiography, following incubation with tritiated 5-hydroxytryptamine ([³H]5-HT) or tritiated norepinephrine ([³H]NE) was used to locate the developing axons of enteric serotoninergic and adrenergic neurons respectively. Observations have been correlated with previous studies of the development of the various types of enteric neuron and the onset of intestinal neuromuscular function. Prior to 25 days of gestation no neurons can be recognized morphologically. Neurons first appear at 25 days' gestation, together with a primitive neuropil in neural islands within the outer gut mesenchyme. Ganglion cell precursors are primitive at first and resemble the cells in the surrounding mesenchyme. Growth cones are abundant but there are no terminal varicosities or synapses. The circular muscle also begins to form at this time. At 32 days' gestation the longitudinal layer of smooth muscle can be discerned and, within the myenteric plexus, terminal axonal varicosities appear containing small (about 50 nm in diameter) electron-lucent synaptic vesicles. The submucosal plexus appears to be derived from neurons and neurites that reach it from the earlier-developing myenteric plexus. The submucosal plexus can be recognized at 38 days of gestation but is not well developed until day 42. Synapses on ganglion cell somata first appear in the myenteric plexus on gestational day 38 and are numerous on day 42 when the first axo-dendritic synapses can be seen. Between days 42 and 48 the developing neural tissue and growing smooth muscle cells interdigitate but after day 48, the plexus becomes ensheathed by supporting cells and connective tissue and this interdigitation is lost. Prior to day 48 most varicosities contain small electron-lucent synaptic vesicles; however, after this time a variety of terminals appears. Between days 48 and 53 of gestation evidence of degenerating neuronal processes is common, indicating that cell death may occur. Electron microscopic autoradiography with [³H]5-HT reveals labelling of axons in the neuropil of the myenteric plexus at day 32 of gestation. Some primitive appearing cell bodies, however, are also labelled and these cells seem to be entering the myenteric plexus from the surrounding mesenchyme. After 42 days of gestation [³H]5-HT labels only axons of both nerve plexuses. Often, labelled terminals are apposed to ganglion cells or dendrites. In contrast, significant labelling by [³H]NE is not found until gestational day 48. Axons are labelled by [³H]NE and these tend to be located at the interface between the myenteric plexus and the surrounding connective tissue.     

Two types of sympathetic axon innervating the juxtaglomerular arterioles of the rabbit and rat kidney differ structurally from those supplying other arteries

Summary Ultrastructural analyses of serial thin sections have revealed two structurally different types of sympathetic axon innervating the afferent and efferent juxtaglomerular arterioles and the intralobular arteries in the outer cortex of the rabbit kidney. Both types of axon have also been found in association with an afferent arteriole in rat kidney. One axon type consists of relatively large diameter unmyelinated axons bearing varicosities in the form of slight expansions. The varicosities have a distinct structural zonation: synaptic vesicles occupy the expansion which faces the smooth muscle cells, whereas the rest of the axon is filled with numerous microtubules. The other axon type has varicosities containing vesicles and mitochondria but few microtubules. The varicosities are generally small and the intervaricosities very thin. The relationship of both axon types with support cells and/or basal lamina is sometimes poorly defined. Both axon types are catecholaminergic as their vesicles take up 6-hydroxydopamine and both types form junctions with arteriolar smooth muscle cells. As well as differing from each other, both types of intrarenal axon differ in several respects from those which innervate other arterial vessels.     

The fine structure of vascular sympathetic neuromuscular contacts in the rat

The fine structure of the neuromuscular contacts of arterioles and small arteries from the jejunum and mesentery of the rat has been studied. There is a rich innervation of these vessels, consisting of bundles of axons in the adventitia around the vessels. In the typical sympathetic neuromuscular contact on a small blood vessel, the axon bundle is loose, the Schwann cell cover is deficient, the neuromuscular distance is less than 1,500 Å, the basement membranes of axon and smooth muscle cell are fused together, and the axon tends to follow the contour of the smooth muscle cell. However, it is not possible to be certain that these criteria are essential for a true functional neuromuscular contact. In particular, it appears from the structural findings that a neuromuscular distance of 2,000–3,000 Å or more could well be compatible with effective neuromuscular transmission. Smooth muscle cells in arterioles often have multiple innervation, and an axon probably innervates more than one smooth muscle cell. Occasional axons contain mainly or solely vesicles of the “large granular” type.     

Fine structure of nerve terminals in the human gut

An electron microscopical investigation was made of neuronal terminals in operatively removed human duodenum and jejunum. No intraepithelial neuronal elements were found. The lamina propria mucosae, submucosa and muscular layer possessed, in addition to large bundles of unmyelinated nerve fibers, small ones consisting of three or four axons wrapped by infoldings of one Schwann cell. Some of these axons were light; others contained mitochondria and clear vesicles measuring 200–500 Å across. Still other axons had dense-cored vesicles with a diameter ～ 700 Å. The plasma membranes of vesiculated axons were not thickened and only in part invested by the Schwann cell, but were covered by a basement membrane. Such profiles were interpreted as nerve terminals, and were separated from fibers of the muscularis mucosae, bases of epithelial cells and submucous capillaries by interspaces measuring 5,000–6,800 Å. The gap between neuronal terminals and smooth muscle in the muscular wall was smaller (900 Å, more often 1,500–3,000 Å). The myenteric plexus contained nerve bundles consisting of the usual axons and of preterminals; some of the latter contained small clear vesicles, others predominantly larger granulated vesicles, and still others a mixture of both types of vesicles. The similarities and differences of neuronal terminals occurring in animals and in man were pointed out. The significance of the findings of this study was discussed in the light of current concepts of the physiology of autonomic nerve transmission.     

Fast-conducting skeletofusimotor axons supplying intrafusal chain fibers in the cat peroneus tertius muscle.

1. In six experiments on cat peroneus tertius muscle, from 12 to 23 motor axons with conduction velocities above 85 m/s were repetitively stimulated so as to produce glycogen depletion in the muscle fibers they innervated. 2. The whole muscle was then quick-frozen, serially cut, stained to demonstrate glycogen, and examined for intrafusal glycogen depletion. 3. Zones of glycogen depletion were found in 27 of the 99 examined spindles: they were almost invariably located on chain fibers and specifically on the longest of the chain fibers in affected spindles. 4. Since it was shown that there are no purely fusimotor fast axons in the motor supply to peroneus tertius, it is concluded that skeletofusimotor axons are present among the fastest motor axons to this muscle.     

“Fast” and “slow” skeleto-fusimotor innervation in cat tenuissimus spindles; a study with the glycogen-depletion method

The glycogen-depletion method was used to investigate the motor supply to tenuissimus with respect to the presence of fast β axons and to assess the total proportion of both fast and slow β-innervated spindles in this muscle. In a first series of 5 expts., groups of motor axons with conduction velocities higher than 85 m/s were repetitively stimulated so as to produce glycogen depletion in the muscle fibres they innervated. The whole muscle was then quick-frozen, serially cut, stained to demonstrate glycogen and examined for intrafusal glycogen depletion. Zones of glycogen depletion were found in 16 of the 46 examined spindles; they were most frequently located in the longest of the chain intrafusal muscle fibres. Since it is known that there are no purely fusimotor axons to tenuissimus with conduction velocities above 50 m/s, it was concluded that β axons are present among the fastest axons to this muscle. In a second series of 5 expts. as many motor axons as possible with conduction velocities above 60 m/s were stimulated. Zones of glycogen depletion were found in 19 of the 47 examined spindles. They affected chain fibres in about half of the instances and bag₂ fibres in the others. As this latter location is characteristic of slow dynamic β axons, it was concluded that both slow and fast β axons occur regularly in the motor supply to tenuissimus. β-innervation is present in at least 40% of tenuissimus spindles with almost no convergence of fast and slow β axons onto the same spindle.     

Intrastriatal grafts of rat colonic smooth muscle lacking myenteric ganglia stimulate axonal sprouting and regeneration

Grafts of living or freeze-killed freshly dissected colonic smooth muscle from young inbred Fischer rats were implanted into the corpus striatum of adult Fischer rats. Sections of brain were examined electron microscopically 3 and 6 wk after implantation. At both times, living grafts were vascularised and contained healthy differentiated smooth muscle cells, fibroblasts, interstitial cells of Cajal and some macrophages. Large bundles of small nonmyelinated axons, identified as CNS axonal sprouts, could be observed in the brain at and near the interface between the living smooth muscle and the CNS tissue. Bundles of regenerating CNS axons, often associated with astrocyte processes, had grown into the grafts. Some axons within the grafts had matured, enlarged and become myelinated by oligodendrocyte processes or Schwann cells. In some cases, smooth muscle cells were observed in close and intricate association with axons. In contrast to the living grafts, grafts of freeze-killed smooth muscle, examined 3 and 6 wk after implantation, contained macrophages, fibroblasts, collagen and large amounts of cellular debris, but no living muscle cells, astrocytes or Schwann cells. The striatal neuropil around freeze-killed grafts did not contain large bundles of CNS axonal sprouts and bundles of axons were not observed within the freeze-killed graft. This study demonstrates that cells from the smooth muscle layers of the colon, in the absence of myenteric ganglia, can stimulate a vigorous regenerative response from CNS axons when implanted into the corpus striatum of adult rats.     

The effect of lanthanum on nerve terminals in goldfish muscle after paralysis with tetanus toxin

Lanthanum (1.9 mM) has previously been shown to produce a massive increase in the frequency of spontaneous miniature junction potentials at the neuromuscular junctions of goldfish fin muscles. In fins where transmission has been blocked by previous injection of tetanus toxin and where there are few (if any) spontaneous miniature potentials, lanthanum treatment is able to restore a modest frequency. The results of parallel experiments in which the ultrastructure of the nerve endings has been investigated by electron microscopy are reported. In normal goldfish muscles, the lanthanum-induced increase in frequency is accompanied by depletion of synaptic vesicles. In contrast, there is no depletion in tetanus toxin-paralysed nerve endings subjected to lanthanum treatment, which parallels the relative insensitivity of the endings to activation by lanthanum. Of particular interest is the finding that the lanthanum treatment of the toxin muscles apparently causes accumulation of vesicles in a row just inside the terminal membrane, both at synaptic and non-synaptic positions. The results are discussed with respect to the mechanisms of transmitter release and to the actions of tetanus toxin and lanthanum.     

Wound healing in the media of the normolipemic rabbit carotid artery injured by air drying or by balloon catheter de-endothelialization.

The response to air-dry injury to the carotid artery of the normolipemic rabbit was compared with the response to de-endothelialization with a balloon catheter. Air drying induced an inflammatory response that resembled arteritis rather than atherosclerosis. There was medial damage, neutrophil but not macrophage infiltration, and fibrin formation, limited smooth muscle proliferation, which regressed after 3 months, and no lipid deposition. Within 1 week the smooth muscle cells were mainly of the secretory phenotype, and a neointima had formed. At 4 weeks the neointimal proliferation continued, but most cells showed a contractile phenotype. By 3 months, the lesion consisted of fibromuscular thickening with few small smooth muscle cells. Balloon injury induced minimal medial damage and continuing intimal proliferation with no evidence of regression by 3 months. It is concluded that air drying the carotid artery induces smooth muscle damage as well as endothelial cell loss, and this stimulates a wound-healing mechanism that is different from the response to selective intimal injury.     

What is the hair cell transduction channel?

Rhythmic electrical activity is a feature of most smooth muscles but the mechanical consequences can vary from regular rapid phasic contractions to sustained contracture. For many years it was thought that spontaneous electrical activity originated in smooth muscle cells but recently it has become apparent that there are specialized pacemaker cells in many organs that are morphologically and functionally distinct from smooth muscle and that the former cells are the source of spontaneous electrical activity. Such a pacemaker function is well documented for the ICC of the gastrointestinal tract but evidence is accumulating that ICC-like cells play a similar role in other types of smooth muscle. We have recently shown that there are specialized pacemaking cells in the rabbit urethra which are spontaneously active when freshly isolated, readily distinguishable from smooth muscle cells under bright field illumination and relatively easy to study using patch-clamp and confocal imaging techniques. Recent results suggest that calcium oscillations in isolated rabbit urethral interstitial cells are initiated by calcium release from ryanodine sensitive intracellular stores, that oscillation frequency is very sensitive to the external calcium concentration and that conversion of the primary oscillation to a propagated calcium wave depends upon IP₃-induced calcium release.     

Proteomic profiling of extracellular vesicles released from vascular smooth muscle cells during initiation of phosphate-induced mineralization

ABSTRACT

Purpose/Aim: Elevated serum phosphate is one of the major factors contributing to vascular calcification. Studies suggested that extracellular vesicles released from vascular smooth muscle cells significantly contribute to the initiation and progression of this pathology. Recently, we have demonstrated that elevated phosphate stimulates release of extracellular vesicles from osteogenic cells at the initiation of the mineralization process. Here, we used MOVAS cell line as an in vitro model of vascular calcification to examine whether vascular smooth muscle cells respond to high phosphate levels in a similar way and increase formation of extracellular vesicles. Materials and Methods: Vesicles residing in extracellular matrix as well as vesicles released to culture medium were evaluated by nanoparticle tracking analyses. In addition, using mass spectrometry and protein profiling, protein composition of extracellular vesicles released by MOVAS cells under standard growth conditions and upon exposure to high phosphate was compared. Results: Significant increase of the number of extracellular vesicles was detected after 72 h of exposure of cells to high phosphate. Elevated phosphate levels also affected protein composition of extracellular vesicles released from MOVAS cells. Finally, the comparative analyses of proteins in extracellular vesicles isolated from extracellular matrix and from conditioned medium identified significant differences in protein composition in these two groups of extracellular vesicles. Conclusions: Results of this study demonstrate that exposure of MOVAS cells to high phosphate levels stimulates the release of extracellular vesicles and changes their protein composition.     

Electrophysiological events during neuroeffector transmission in the spleen of guinea-pigs and rats.

Intracellular recordings were made from smooth muscle cells of arterioles and the capsule of the spleen of guinea-pig and rat, and the responses to periarterial or subcapsular nerve stimulation were recorded. The innervation of the spleen was studied using fluorescence and immunohistochemical techniques. Catecholamine-containing axons were associated with smooth muscle of the splenic capsule, trabeculae, arterioles and amongst cells of the periarteriolar lymphoid sheath. Axons immunoreactive for neuropeptide Y (NPY) and tyrosine hydroxylase were distributed in an identical manner to catecholamine-containing axons, whereas axons immunoreactive for substance P or calcitonin gene-related peptide were present at a very low density in spleens from both species. In segments of arterioles, single transmural stimuli evoked excitatory junction potentials (EJPs) of 1-10 mV amplitude. EJPs facilitated during short trains of stimuli (1-10 Hz) and summated at 10 Hz, often initiating a muscle action potential. EJPs persisted in the presence of prazosin (1 microM) and idazoxan (1 microM), but were abolished by the P2x-purinoceptor antagonist suramin (1 mM). Spontaneous depolarizations were observed in smooth muscle cells of arterioles and capsule. Some events in arterioles were observed in the presence of suramin and so may originate postjunctionally independently of transmitter release. As single transmural stimuli failed to evoke a depolarization in capsular smooth muscle, spontaneous depolarizations in this tissue probably also arise postjunctionally. Short trains of high frequency stimuli (10-35 Hz) evoked biphasic depolarizations of capsular smooth muscle cells. The initial component peaked 2.5 s following the onset of stimulation; the second component peaked 15 s following the onset and decayed exponentially with a time constant of 15 s. By fitting a product of exponentials to the second component, it was possible to define the initial component, which decayed with a time constant of around 1.5 s. Neurally evoked depolarizations of capsular smooth muscle were abolished by 1 microM TTX. Blockade of alpha 1-adrenoceptors with prazosin reduced the initial component of the depolarization, whereas alpha 2-adrenoceptor blockade with idazoxan virtually abolished the second component. In some cells a small, faster depolarization persisted after alpha-adrenoceptor blockade. The slow alpha 2-adrenoceptor-mediated depolarization was identical to that recorded in the rat tail artery and in the guinea-pig mesenteric vein. The data indicate that sympathetic neuroeffector transmission from noradrenergic axons containing NPY to splenic arterial and capsular smooth muscle occur by different mechanisms.     

Expression of Vesicular Nucleotide Transporter in Rat Odontoblasts

Several theories have been proposed regarding pain transmission mechanisms in tooth. However, the exact signaling mechanism from odontoblasts to pulp nerves remains to be clarified. Recently, ATP-associated pain transmission has been reported, but it is unclear whether ATP is involved in tooth pain transmission. In the present study, we focused on the vesicular nucleotide transporter (VNUT), a transporter of ATP into vesicles, and examined whether VNUT was involved in ATP release from odontoblasts. We examined the expression of VNUT in rat pulp by RT-PCR and immunostaining. ATP release from cultured odontoblast-like cells with heat stimulation was evaluated using ATP luciferase methods. VNUT was expressed in pulp tissue, and the distribution of VNUT-immunopositive vesicles was confirmed in odontoblasts. In odontoblasts, some VNUT-immunopositive vesicles were colocalized with membrane fusion proteins. Additionally P2X₃, an ATP receptor, immunopositive axons were distributed between odontoblasts. The ATP release by thermal stimulation from odontoblast-like cells was inhibited by the addition of siRNA for VNUT. These findings suggest that cytosolic ATP is transported by VNUT and that the ATP in the vesicles is then released from odontoblasts to ATP receptors on axons. ATP vesicle transport in odontoblasts seems to be a key mechanism for signal transduction from odontoblasts to axons in the pulp.