Urinary bladder of rat: fine structure of normal and hypertrophic musculature

Summary The fine structure of the muscle of the urinary bladder in female rats is similar to that of other visceral muscles, although it is arranged in bundles of variable length, cross-section and orientation, forming a meshwork. When distended, the musculature is 100–120 m thick, with some variation and occasional discontinuity. Extended areas of cell-to-cell apposition with uniform intercellular space occur between muscle cells, whereas attachment plaques for mechanical coupling are less common than in other visceral muscles. There are no gap junctions between muscle cells. Many bundles of microfilaments and small elastic fibres run between the muscle cells. After chronic partial obstruction of the urethra, the bladder enlarges and is about 15 times heavier, but has the same shape as in controls; the growth is mainly accounted for by muscle hypertrophy. The outer surface of the hypertrophic bladder is increased 6-fold over the controls; the muscle is increased 3-fold in thickness, and is more compact. Mitoses are not found, but there is a massive increase in muscle cell size. There is a modest decrease in percentage volume of mitochondria, an increase in sarcoplasmic reticulum, and no appreciable change in the pattern of myofilaments. Gap junctions between hypertrophic muscle cells are virtually absent. Intramuscular nerve fibres and vesicle-containing varicosities appear as common in the hypertrophic muscle as in controls. There is no infiltration of the muscle by connective tissue and no significant occurrence of muscle cell death.     

A vascular network closely linked to the epithelium of the urinary bladder of the rat

Summary Scanning electron microscopy was used on the mucosa of the rat urinary bladder after digestion with strong alkali and microdissection. The underside of the epithelium (and the plane of the epithelium-tunica propria interface) is not smooth but is scored by grooves-10 m wide and 3–4 m deep—connected into a fine mesh. A net of blood capillaries located in the uppermost part of the tunica propria occupies these grooves. They measure 3–9 m in diameter, are separated from the epithelium by a gap of 0.3 m, often show fenestrations, and are accompanied by numerous and extensive pericytes and by some fibroblasts. We discuss these observations in the light of current knowledge of blood flow in the bladder, contraction and distension of the bladder wall and formation of mucosal folds, transport of solutes through the epithelium, and plasma extravasation from mucosal blood vessels in neurogenic inflammation.     

Phenotypic modulation of smooth muscle cells during the formation of neointimal thickenings in the rat carotid artery after balloon injury: an electron-microscopic and stereological study

The formation of neointimal thickenings in the rat carotid artery after balloon injury was studied by a combination of electron-microscopic and stereological methods. All smooth muscle cells in the normal media had a contractile phenotype, the cytoplasm being dominated by myofilaments. Seven days after endothelial denudation, the smooth muscle cells in the innermost part of the media had assumed a synthetic phenotype by loss of myofilaments and formation of a large endoplasmic reticulum and Golgi complex. These cells moved through fine openings in the internal elastic lamina and gave rise to a growing neointima by proliferation and secretion of extracellular matrix components. Fourteen days after the operation, the neointima had almost reached its final size, and mitoses were no longer noted. Nevertheless, the cells maintained a synthetic phenotype with prominent secretory organelles, although myofilaments had started to become more abundant again. They were surrounded by an extracellular matrix made up of collagen fibrils and coalescing patches of elastin. Thirty-five days after the operation, an endothelial cell layer had reformed and covered most of the luminal vessel surface. In parallel, the smooth muscle cells in the neointima had returned to a contractile phenotype with a cytoplasm dominated by myofilaments. These findings provide a morphological basis for further analysis of the cellular and molecular interactions involved in the formation of neointimal thickenings after endothelial injury, and for the search for agents interfering with this process.     

12-O-tetradecanoylphorbol-13-acetate induces selective desquamation of superficial cells in rat urinary bladder epithelium

Summary 12-O-tetradecanoylphorbol-13-acetate (TPA) is known to affect the proliferation and/or differentiation of several types of cells. We injected TPA directly into the lumen of rat bladder to determine, using scanning and transmission electron microscopy, its effects on the bladder epithelium in vivo. At 1 h after TPA injection (1g/ml), the superficial cells of the epithelium had changed their morphology, and large spherical vacuoles occupied their cytoplasm. In some areas, the underlying intermediate cells were exposed by the desquamation of the superficial cells. During the next few hours, TPA was excreted from the bladder lumen by voluntary micturition, but the desquamation of the superficial cells proceeded further. All the superficial cells were lost from the luminal surface by 24 h after TPA injection. The changes noted were specific for the superficial cells and were not observed in the intermediate or basal cells. After 24h, part of the epithelium had a three-layer structure, indicating that regeneration was taking place. These results demonstrate that TPA selectively affects and desquamates superficial cells in a short period of time. This experimental system may be useful for studying in vivo cell proliferation and/or differentiation.     

The origin of VIP-containing nerves in the urinary bladder of rat

The innervation of the urinary bladder is known to include a considerable number of nerves containing vasoactive intestinal polypeptide (VIP). The origin of such nerves in the bladder of rat was investigated in this study using the methods of immunocytochemistry and radioimmunoassay combined with surgical sectioning of the hypogastric and/or pelvic nerves to the bladder. Eight days after pelvic nerve sectioning proximal to the main pelvic ganglion, VIP-immunoreactive nerves and VIP content were markedly increased from the level in the sham-operated rat bladder. Sectioning of hypogastric or both nerve pathways led to a less significant increase. It was therefore postulated that the majority of VIP-immunoreactive nerves originate from ganglia located either close to the bladder or within the bladder wall. It is interesting that in these experiments the VIP content of the bladder nerves is inversely related to the changes in motility that would be expected to result from the nerve sections.     

Functional muscarinic M2 and M3 receptors and beta-adrenoceptor in cultured rat bladder smooth muscle

A highly purified rat urinary bladder smooth muscle cell culture was obtained by a modified enzymic isolation method, and the presence of functional muscarinic as well as beta-adrenergic receptors were subsequently determined. At 7-10 days of culture, cells became elongated and spindle-shaped showing a typical "hills and valleys" form. They were stained with anti-alpha-actin and anti-myosin antibodies. Radiolabeled ligand binding using [3H]N-methylscopolamine and [3H]CGP12177 showed that these cells expressed muscarinic and beta-adrenergic receptors. Stimulation of cultured cells with carbachol inhibited the forskolin-stimulated cyclic AMP formation, caused an elevation of intracellular Ca2+ concentration measured by fura-2 fluorometry. The latter response was almost completely blocked by 4-DAMP, a selective muscarinic M3 antagonist. On the other hand, stimulation of cultured cells with isoproterenol enhanced the basal cyclic AMP formation, which was reversed by carbachol. Therefore, the presence of functional muscarinic (both M2 and M3) as well as beta-adrenergic receptors was confirmed in pure culture of the rat bladder smooth muscle cells obtained by using an enzymic isolation method.     

Further observations on upper urinary tract smooth muscle

Summary Light and electron microscopic techniques have been employed to study the arrangement and distribution of two types of muscle in the upper urinary tract of the rat. An outer layer of cells has been identified in the wall of the renal calix and pelvis. These cells are separated by connective tissue but possess numerous processes which make close contacts with adjacent cells. A layer of similar cells has not been observed in the wall of the upper ureter. The inner layer of muscle in the calix and pelvis is composed of larger cells similar to and apparently continuous with ureteric muscle. These cells are closely related to one another without intervening connective tissue and possess numerous bundles of myofilaments which extend along the length of the cell. The two types of muscle are closely related and, in the junctional region, cells of the outer layer are arranged along the length and make close contacts with one or more of the inner smooth muscle cells. A quantitative estimation has been made of nerve bundles associated with smooth muscle forming the outer layer of the calix and pelvis and with the muscle of the ureter. The results have shown a five fold increase in nerves associated with the caliceal muscle when compared with the ureter. The results are discussed in relation to the concept of a ureteric pacemaker.The authors wish to thank Professor G. A. G. Mitchell for his useful advice and encouragement.     

Morphology of smooth muscle cells in the rat thoracic duct

Summary The three-dimensional cytoarchitecture and ultrastructure of the smooth muscle cells in the wall of the rat thoracic duct were investigated by scanning and transmission electron microscopy. The muscle layer basically consists of a single layer of circularly arranged cells. The smooth muscle cell is fusiform or ribbon-like in shape, as in veins or venules with a similar or smaller diameter. Connections by spinous processes are observed between adjacent muscle cells along their length. Spot-like membrane contacts frequently occur in areas where facing membranes are closely apposed. These are thought to be gap junctions and may be responsible for electrical coupling and mechanical attachment. Large invaginations arranged regularly in rows on the surface of the smooth muscle cells can be observed. These invaginations are closely associated with a flattened sarcoplasmic reticulum, and caveolae tend to open into the invaginations.     

Ontogeny of the ryanodine receptor in rabbit urinary bladder smooth muscle

Bladder smooth muscle contraction is mediated by both direct calcium entry through the cell membrane, and by calcium induced calcium release (CICR) from the sarcoplasmic reticulum (SR) storage sites. Ryanodine is a neutral plant alkaloid which binds to an ion channel located on the SR membrane. Its effects in cardiac skeletal muscle are well characterized where it inibits the efflux of intracellular calcium stores, and thus it serves as a negative inotrope. It has also been shown that in the develpping rabbit myocardium, there is a gradual increase in the expression of this ion channel. Little has been written about the expression and function of the ryanodine sensitive ion channel in smooth muscle. Recently we have shown that neonatal rabbit bladder smooth muscle is not very sensitive to ryanodine, while that from mature rabbits is extremely sensitive. This leads us to quantify the expression of the ryanodine sensitive ion channel. In this paper we demonstrate that the Kd values do not change to any significant degree with normal rabbit bladder development. However the Bmax values for 3 day, 2, 4, 6, and 8 week rabbit bladder smooth muscle are 7, 10, 15, 29, and 44 fmol specifically bound ryanodine/mg protein. The differences between the neonatal groups and the mature groups are significant (P<0.5). This increase in ryanodine sensitive ion channel expression with normal growth would suggest that with normal maturation, the bladder smooth muscle cell acquires an increased pool of sequestrered intracellular calcium. This would follow a similar pattern of development that has already been described in rabbit myocardium.     

Hypertrophic smooth muscle

Summary The ultrastructure of filaments is studied in the hypertrophic musculature of the small intestine of the guinea pig oral to an experimental stenosis. No structural change is observed in the thin and the thick myofilaments. However, there is a remarkable and consistent increase in the number of intermediate (10 nm) filaments; they are the predominant type of filament in many hypertrophic muscle cells. Experiments, in which the force developed in vitro by strips of control and hypertrophic musculature upon stimulation with carbachol, indicate that the force per unit sectional area is slightly less in the hypertrophic muscle than in the control tissue.The author thanks Miss Eva Franke for excellent technical assistance. This work was supported by grants from the Medical Research Council and the Central Research Funds of the University of London     

Effect of outlet obstruction on pyruvate metabolism of the rabbit urinary bladder

Bladder function is dependent upon cellular metabolism of substrates and the adequate generation of high-energy phosphate compounds. Partial outlet obstruction induces a marked decrease in bladder function which is associated with a significant decrease in the oxidative metabolism of glucose.The current investigation was designed to determine whether the time course of the decrease in mitochondrial oxidation in the hypertrophied urinary bladder is similar to the time course of the contractile dysfunction observed. In these studies we determined: 1) the rate of ¹⁴C-pyruvate metabolism to ¹⁴CO₂ in control and obstructed tissue (1, 3, 5 and 7 days), and 2) the mitochondrial enzymatic activities of malate dehydrogenase and citrate synthase.The results can be summarized as follows: 1) The rate of pyruvate metabolism decreases by over 50% within one day following partial outlet obstruction, and remains at this level for the seven day period of study. 2) Kinetic analysis demonstrates that the change in enzymatic activity is related to a decrease in Vmax; the K_d for pyruvate is similar for control and after all time periods of obstruction. 3) The enzymatic activity of malate dehydrogenase and citrate synthase is reduced by over 50% within one day following partial obstruction, and remains at this level throughout the 7 day study period. These metabolic results correlate in time and duration with the decreased ability of the bladder to empty following partial outlet obstruction.     

Distribution of neurons in the major pelvic ganglion of the rat which supply the bladder,colon or penis

Summary In male rats a large number of the postganglionic neurons which innervate the pelvic organs are located in the major pelvic ganglion. In the present study we have identified the location within this ganglion of neurons which project to either of three pelvic organs, the penis, colon or urinary bladder. Two fluorescent retrogradely-transported dyes, Fast Blue and Fluoro-Gold, were used. For most animals one dye was injected into the cavernous space of the penis, the wall of the distal colon or the wall of the urinary bladder. In a small number of animals two organs were injected, each with a different dye. One to six weeks after injection the major pelvic ganglia were fixed in buffered formaldehyde. The distribution of fluorescent dye-labelled cells was observed in whole mounts of complete ganglia and, in most cases, also in small accessory ganglia located between the ureter and the prostate. The studies showed a unique pattern of distribution for each organ-specific group of neurons. Most of the colon neurons are located in the major pelvic ganglion near the entrance of the pelvic nerve, whereas almost all of the penis neurons are near or within the penile nerve. Bladder neurons are relatively evenly distributed throughout the ganglion. These results demonstrate a distinct topographical organization of organ-specific neurons of the major pelvic ganglion of the male rat, a phenomenon which has also been observed in other peripheral ganglia.     

Effect of ATP inhibitors on the translocation of luminal membrane between cytoplasm and cell surface of transitional epithelial cells during the expansion-contraction cycle of the rat urinary bladder

Summary Movement of asymmetric membrane plaques between the cytoplasm and surface of luminal urothelial cells was investigated during artificially induced contraction and expansion of untreated and ATP-depleted urinary bladders of the rat. Estimations of surface area, volume, and number of discoidal vesicles per unit volume of cytoplasm were determined by morphometric examination of electron micrographs. These values were compared in luminal cells from bladders incubated in control media or in media containing 0.15 mM 2,4-dinitrophenol and 0.02 mM sodium arsenate. The ATP inhibitors had no apparent effect upon the contraction of apical cells that had been incubated in an expanded state. In contrast, after distension of poisoned, contracted bladders, the orientation of intermediate filaments and the densities of discoidal vesicles were similar to the condition characterized by contracted cells. The results indicated that the normal reorientation of filaments, coincident with cell distension, had been suppressed by ATP inhibitors. This, in effect, impeded the filament-mediated translocation of membrane plaques to the surface. The reduction of surface area along the luminal border forced many cells to compensate by separating at their lateral margins.This work was supported by NIH Grant AM 32937     

Intracellular distribution of fumarase in rat skeletal muscle

The distribution of fumarase activity between the mitochondrial and cytoplasmic compartments of rat skeletal muscle was studied using the method of Fatania and Dalziel (Biochim. Biophys. Acta 631 (1980) 11–19), fractional extraction technique and a method based on the calculation of mitochondrial protein content in the tissue and on the determination of fumarase activity both in the tissue homogenate and in the isolated mitochondria. We found 10%, 5% and 0% of the total fumarase activity in the cytoplasm using these methods, respectively. The results suggest that no more than 10% of the total fumarase activity is present in the cytosolic fraction of rat skeletal muscle. The metabolic consequences of such distribution of fumarase in skeletal muscle are discussed.     

Origin and distribution of neuropeptide Y-, vasoactive intestinal polypeptide- and substance P-containing nerve fibers in the urinary bladder of the rat

Summary The origin and distribution in the urinary bladder of nerve fibers containing neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and substance P (SP) were investigated in rats. Experimental procedures comprised preganglionic decentralization or postganglionic denervation of the bladder and also chemical sympathectomy as well as capsaicin treatment of newborn rats.Nerve fibers containing NPY were richly distributed in the detrusor muscle and also in the pelvic ganglia. Numerous NPY-containing nerve cell bodies were found in pelvic ganglia. A rich occurrence of VIP fibers and a more sparse distribution of SP-containing fibers were also found in the bladder as well as a relatively rich representation of VIP- containing nerve cell bodies in the pelvic ganglia. After decentralization the intensity of VIP and NPY immunofluorescence increased in nerve cell bodies of the pelvic ganglia and in nerve fibers in the wall of the bladder. Postganglionic denervation, on the other hand, eliminated all peptides examined in the bladder wall. After postganglionic denervation the situation in the ganglia was approximately the same as after decentralization. Chemical sympathectomy (6-OHDA) did not seem to change significantly the frequency and distribution of VIP-, SP- and NPY-fibers in the muscle layer of the bladder or in the pelvic ganglia, while the NPY-containing nerve fibers in the submucosal layer and around blood vessels of the bladder disappeared. Adrenergic nerve fibers in the wall of the bladder (visualized by histofluorescence) were markedly reduced in number after administration of 6-OHDA. Capsaicin-treatment of newborn rats caused a loss of SP-fibers in the wall of the bladder, supporting the view that these fibers are sensory in nature in the urinary bladder. Although it cannot be entirely excluded that NPY-containing fibers in the wall of the bladder are adrenergic, the present results suggest that the NPY-fibers as well as the VIP-fibers of the bladder wall originate mainly in non-adrenergic cell bodies of the pelvic ganglia. However, perivascular NPY-containing nerve fibers are adrenergic in nature.     

Chloride-bicarbonate exchange in the urinary bladder of the turtle. Independence from sodium ion

The rates of Cl^? absorption and HCO^?₃ secretion were not different in turtle urinary bladders bathed in Na⁺-containing and solutions.These results in turtle bladder are inconsistent with Na⁺-anion cotransport but can be accounted for by a Cl^?/HCO^?₃ exchange system.     

Two mathematical models explain the variation in cystometrograms of obstructed urinary bladders

Overdistension of the urinary bladder, secondary to outlet obstruction, causes cellular changes in the bladder wall, including hypertrophy of the smooth muscle cells, which increase bladder mass. To investigate the effects of increased mass on the cystometrogram (CMG), we have developed two mathematical models. In the first model, we assume that mass is added such that the largest bladder volume at zero transmural pressure, the zero pressure volume (ZPV), is constant. It predicts increased pressures and decreased compliance in the CMG. In the second model, we assume that both mass and ZPV increase proportionally. It predicts unchanged pressures, increased compliance, and increased capacity in the CMG. These results allow us to divide animal experiments in the literature into two groups. Cystometrograms performed on animals that have had outlet obstruction induced by a cuff method, inducing a small increase in mass, belong to the first group: hypertrophy with no change in ZPV. Cystometrograms performed on animals that have had outlet obstruction induced by a ligature method, inducing a large increase in mass, belong to the second group: hypertrophy with increased ZPV. We conclude that increased ZPV results from a more severe obstruction which is indicated by the increased capacity and compliance.