Bovine Bladder Research Articles

The presence of NHE1 and NHE3 Na+-H+ exchangers and an apical cAMP-independent Cl- channel indicate that both absorptive and secretory functions are present in calf gall bladder epithelium.

We investigated the transport systems that can sustain Na+ and Cl- movements across bovine gall bladder epithelium, focusing on the Na+-H+ exchanger (NHE) family and chloride conductive pathways. Experiments conducted using the fluorescent probe acridine orange (AO) with brush-border membrane vesicles (BBMV) or vesicles obtained from the total epithelium (EMV) demonstrated the presence of a Na+-H+ exchange in both preparations. The use of specific inhibitors indicated the presence of an apical NHE3 exchanger and a NHE1 isoform which should reside in the basolateral membrane. Using reverse transcriptase (RT) PCR, we identified cDNA fragments corresponding to the NHE1, NHE3, Cl--HCO3- (AE2a) transporters and to the CFTR channel. Using the patch-clamp technique, we investigated Cl- conductances on cultured epithelial cells. We found a 5 pS Cl- channel with a voltage-independent open probability, insensitive to stilbenes (SITS), Zn2+ and cAMP. The results suggest that absorption and secretion coexist in calf gall bladder epithelium. A Na+-H+-Cl--HCO3- double exchange may, at least partially, sustain the absorptive function, and a Cl- apical conductive pathway may be involved in secretion. The conductance we observed does not seem to be cAMP-regulated, unlike other mammalian gall bladders.

Terminal glycosylation of bovine uroplakin III, one of the major integral-membrane glycoproteins of mammalian bladder

Uroplakin III (UPIII) is one of the major transmembrane glycoproteins exposed at the luminal face of mammalian bladder. We investigated the terminal glycosylation of bovine UPIII in order to ascertain whether it contains the α2,3-sialylated sequence thus potentially serving as a receptor for uropathogenic Escherichia coli expressing type S adhesins. We report the occurrence of sialic acid in α2,3- and α2,6-linkage to galactose in bovine UPIII glycans as evidenced by the sensitivity of UPIII to both Vibrio cholera and Newcastle disease virus neuraminidase and by the colocalization of UPIII antigen and material detected by lectins of Sambucus nigra and Maackia amurensis on the luminal face of the bladder. We also present evidence that UPIII glycans are capped by Gal-α1,3-Gal epitope. Consistently, α2,3- and α2,6-sialyltransferase, as well as α1,3-galactosyltransferase were found to be present in the cells detached from the luminal side of bovine bladder, which are responsible for the UPIII biosynthesis. The putative role of UPIII sialylated glycans in enhancing the uropathogenicity of E. coli expressing type S adhesins is discussed.

Roles of the lamina propria and the detrusor in tension transfer during bladder filling.

In this study, structural changes within the lamina propria and detrusor layers were analysed during development as a function of bladder filling. Second-, third- and full-term foetal bovine bladders were filled to 0%, 25%, 50%, 75% and 100% of their total capacity and snap frozen. The bladders were analysed histochemically and the relative thicknesses of the lamina propria and detrusor were measured. In all gestational stages examined, the total thickness of the bladder wall decreased during bladder filling. The lamina propria of the full-term bladder thinned at a consistently faster rate than did the detrusor. The lamina propria of second and third trimester bladders followed the same thinning pattern, except when the bladders were filled from 25% to 50% of their capacities. At these gestational stages, the detrusor thinned at a faster rate than the lamina propria. Our results demonstrate that the detrusor layer carries tension only during a specific portion of the filling cycle and only during the second and third trimesters. We conclude that the lamina propria acts as the capacitance layer, while the detrusor functions as the "limiting" or "girding" layer to prevent over-distension of the bladder wall.

Paraneoplastic pemphigus in a dog.

This report presents a case of paraneoplastic pemphigus in a 7-year-old female Bouvier. The dog initially showed extensive oral ulcerations that exacerbated upon treatment with trimethoprim-sulfadiazine. Subsequently, the dog developed vesiculobullous and ulcerative lesions on the ear margins, the nose, periocular, and at the nail beds. Due to complete therapy resistance and a deteriorating general condition, the dog was euthanized. During post-mortem examination a thymic lymphoma was found. While an early biopsy of the oral cavity revealed features of erythema multiforme, skin lesions at necropsy were typical of pemphigus vulgaris. Indirect immunofluorescence of patient serum revealed an antikeratinocyte membrane pattern typical for pemphigus. The serum was also positive on bovine bladder epithelium. In a Western blot, autoantibodies to a 210 and a 190 kDa protein were detected.

Temporal Expression of Elastic Fiber Components in Bladder Development

Fetal and postnatal bovine bladders were examined for expression of elastic fiber components by immunohistochemistry as well as by measurement of steady state mRNA levels. Expression of fibrillin-1, microfibril-associated glycoprotein (MAGP) and elastin during the fetal period were compared with that of postnatal two year old animals (heifers) and adults. Each bladder was separated into two distinct tissue samples: 1) the outer smooth muscle layer (detrusor) and 2) the inner epithelium (urothelium) lined lamina propria (urotherial-lamina propria). Each of these samples was analyzed separately. Distribution of the elastic fiber components, determined by immunohistochemistry with matrix-specific antibodies, was different depending upon the region of the bladder wall examined and its developmental stage. In particular, MAGP and fibrillin-1 were conspicuously present in the urothelium during the later fetal stages. RNA products of elastic fiber genes were detectable both in the detrusor smooth muscle and urothelial-lamina propria fractions. The highest level of expression occurred in the urothelial-lamina propria fraction during the late second—early third trimester. Elastin expression was different from that of MAGP and fibrillin-1. The highest levels of steady-state elastin mRNA occurred at the earliest developmental stages examined and then progressively decreased through term. A high level of elastin expression occurred within the inner or lamina propria layer of the bladder. Since this layer is the functional capacitance layer within the bladder, its flexibility is likely related to the structural integration of elastin and associated microfibrillar components.

Developmental Expression of Interstitial Collagen Genes In Fetal Bladders

Bovine bladders at 3 stages during fetal development were examined for expression of collagens by immunohistochemistry as well as by measurement of steady state mRNA levels. Expression of type I and type III collagens during the fetal period was compared with that of adult cows as well as young animal (heifer). Each bladder was separated into a detrusor and a urothelial-lamina propria sample which were then analyzed separately. Distribution and fiber arrangement of types I and III collagens were different depending upon the region of the bladder wall examined. Type III collagen, in particular, has a “coiled” appearance which is especially prominent within the lamina propria. Collagen gene expression showed a distinctive pattern which was different for both type I and type III collagen. While type I collagen gene expression peaked during the late second to early third trimester, type III collagen expression progressively decreased throughout the fetal period. In addition, expression of both collagens was greater in the urothelial-lamina propria fractions. These data demonstrate that the pattern of collagen gene expression in the developing bladder is developmentally regulated and is unique to each of the two major structural layers.

Active and passive compliance of the fetal bovine bladder.

Others have shown that the fetal bovine bladder is relatively noncompliant. Previous studies on compliance of fetal bovine bladders have demonstrated that the youngest fetal bladders had lowest and the oldest fetal bladders (near full-term) had greatest compliance. Our study was designed to determine the level of participation of active tension in the compliance of fetal bladders during gestation. Fetal bovine bladders were obtained immediately after maternal harvest and crown-to-rump length was measured to determine gestational age. The fetus was inspected for genitourinary anomalies and the bladder was immediately placed in chilled M199 media. Strips (1 x 0.5 cm.) were excised from the anterior sagittal plane of the bladder and subjected to length-tension analysis in oxygenated Tyrode's buffer at 37C. Tension was measured using a force transducer and length was increased using a micropositioner. Compliance refers to the length-tension studies performed in normal Tyrode's solution and consists of a combination of active (smooth muscle tone) and passive properties. Passive compliance refers to length-tension studies performed after inactivation of bladder smooth muscle tone. Compliance with muscle tone intact was determined by incrementally stretching the strips to twice resting length in physiological buffer and then permitting them to return to resting length. Passive compliance with muscle tone ablated was determined in the same fashion after overnight incubation in calcium-free Tyrode's buffer in the presence of 5 mM. egtazic acid and 10 mM. sodium azide. An exponential function was fit to the normalized length-tension curves, where the exponential coefficient (EC) is numerically inversely proportional to compliance. Passive compliance was greatest in the youngest bladders (EC = 0.5 in the first trimester) and gradually decreased with increasing fetal age (EC = 1.2 in the third trimester). Active compliance demonstrated the opposite pattern, since the younger bladders were more stiff (EC = 2.1 in the first and 1.6 in the third trimesters). These studies demonstrate that passive compliance is greatest in the youngest bladders and progressively decreases with gestation. However, active smooth muscle tone is greatest in the youngest bladders and decreases with gestation. Thus, high active smooth muscle tone in the youngest fetal bladders results in relatively poor compliance of the early stage fetal bladder.

Developmental expression of interstitial collagen genes in fetal bladders.

Bovine bladders at 3 stages during fetal development were examined for expression of collagens by immunohistochemistry as well as by measurement of steady state mRNA levels. Expression of type I and type III collagens during the fetal period was compared with that of adult cows as well as a young animal (heifer). Each bladder was separated into a detrusor and a urothelial-lamina propria sample which were then analyzed separately. Distribution and fiber arrangement of types I and III collagens were different depending upon the region of the bladder wall examined. Type III collagen, in particular, has a "coiled" appearance which is especially prominent within the lamina propria. Collagen gene expression showed a distinctive pattern which was different for both type I and type III collagen. While type I collagen gene expression peaked during the late second to early third trimester, type III collagen expression progressively decreased throughout the fetal period. In addition, expression of both collagens was greater in the urothelial-lamina propria fractions. These data demonstrate that the pattern of collagen gene expression in the developing bladder is developmentally regulated and is unique to each of the two major structural layers.

Active and Passive Compliance of the Fetal Bovine Bladder

Purpose Others have shown that the fetal bovine bladder is relatively noncompliant. Previous studies on compliance of fetal bovine bladders have demonstrated that the youngest fetal bladders had lowest and the oldest fetal bladders (near full-term) had greatest compliance. Our study was designed to determine the level of participation of active tension in the compliance of fetal bladders during gestation. Materials and Methods Fetal bovine bladders were obtained immediately after maternal harvest and crown-to-rump length was measured to determine gestational age. The fetus was inspected for genitourinary anomalies and the bladder was immediately placed in chilled M199 media. Strips (1 × 0.5 cm.) were excised from the anterior sagittal plane of the bladder and subjected to length-tension analysis in oxygenated Tyrode's buffer at 37C. Tension was measured using a force transducer and length was increased using a micropositioner. Compliance refers to the length-tension studies performed in normal Tyrode's solution and consists of a combination of active (smooth muscle tone) and passive properties. Passive compliance refers to length-tension studies performed after inactivation of bladder smooth muscle tone. Compliance with muscle tone intact was determined by incrementally stretching the strips to twice resting length in physiological buffer and then permitting them to return to resting length. Passive compliance with muscle tone ablated was determined in the same fashion after overnight incubation in calcium-free Tyrode's buffer in the presence of 5 mM. egtazic acid and 10 mM. sodium azide. An exponential function was fit to the normalized length-tension curves, where the exponential coefficient (EC) is numerically inversely proportional to compliance. Results Passive compliance was greatest in the youngest bladders (EC = 0.5 in the first trimester) and gradually decreased with increasing fetal age (EC = 1.2 in the third trimester). Active compliance demonstrated the opposite pattern, since the younger bladders were more stiff (EC = 2.1 in the first and 1.6 in the third trimesters). Conclusions These studies demonstrate that passive compliance is greatest in the youngest bladders and progressively decreases with gestation. However, active smooth muscle tone is greatest in the youngest bladders and decreases with gestation. Thus, high active smooth muscle tone in the youngest fetal bladders results in relatively poor compliance of the early stage fetal bladder.

Expression of microfibrillar proteins by bovine bladder urothelium

Objectives To determine the occurrence and potential function of proteins composing elastic microfibrils in the developing bovine bladder. Methods Monospecific antibodies, generated against two well-characterized microfibrillar proteins, microfibril-associated glycoprotein (MAGP) and fibrillin-1 (FBN1), were used in immunohistochemical analysis of full-thickness frozen sections of fetal bovine bladder. The localization of these two antibodies was compared with that of anti-type IV collagen antibody. Adjacent serial sections were stained for routine light microscopy. Cultured urothelial cells were fixed in 3.7% formaldehyde and permeabilized with 0.5% Triton X-100 before immunoanalysis. Control reactions used either preimmune serum or a monoclonal antibody to a nonmatrix protein. Poly(A+) ribonucleic acid was isolated from cultured urothelial cells and subjected to Northern analysis using specific complementary deoxyribonucleic acid probes for MAGP and FBN1. Results Both MAGP and FBN1 are expressed by the urothelium and are found in association with the underlying basement membrane, as visualized by their co-localization with type IV collagen. Furthermore, urothelial cells in culture continue to express both microfibrillar proteins. Conclusions The developing bovine urothelium expresses major microfibrillar protein components. The role of these microfibrils in the urothelium remains to be determined, but they may have an important anchoring function.

Time-Dependent Remodeling of the Bladder Wall in Growing Rabbits After Partial Outlet Obstruction

We asked whether a urethral constriction gradually developed during growth would give rise to a structural remodeling of the bladder wall distinct from that of the mature rabbits in terms of cellular response. We examined the serosa and detrusor muscle in immature rabbits whose urethra was obstructed at 30 days postnatal and studied 7 to 30 days after partial outlet obstruction. Morphometry, bromo-deoxyuridine (BrdU) incorporation, Western blotting and immunocytochemical staining with a panel of monoclonal antibodies specific to selected cytoskeletal, cytocontractile and membrane-related proteins unique to non-muscle and smooth muscle cells (SMC) were used to analyze the effects of obstruction on the differentiation pattern. In comparison with results in adult obstructed bladders, we have found that in growing rabbits: (1) the cell conversion from fibroblasts to SMC, occurring within the 'extrinsic' region of serosal thickening, takes place earlier; (2) newly formed SMC are localized exclusively to the thickened serosa, and can group in bundles depending on the density of the regional innervation; (3) the peak level of BrdU incorporation is more elevated than in the adult bladder wall; and (4) change in the phenotypic profile of SMC of detrusor muscle is delayed. These data indicate that the basic features of structural remodeling in the two models are similar, though partial outlet obstruction produced in growing animals accelerates the fibroblast conversion to SMC and their spatial, differentiation-specific arrangement in the serosa. The late phenotypic changes in obstructed detrusor muscle correlate with the decline of the DNA synthesis level after an initial burst and strongly suggest that newly formed SMC in the serosa do not derive from pre-existing SMC.

Comparative analysis of colchicine induced micronuclei in different cell types in vitro

In the present study we analyzed the induction of micronuclei (MN) by colchicine, at different treatment times, in four histogenetically different cell cultures: human skin fibroblasts (HSF), bovine skin fibroblasts (BSF), bovine bladder fibroblasts (BBF) and human skin epithelial cells (HK), developed and characterized in our laboratory. The frequencies of dead cells, nuclear budding and mitotic index were also evaluated. The HSF and BSF cell lines showed similar frequencies of micronucleated cells (4,7% and 4,9%, respectively) at 96 h of treatment time. The BBF cell line showed the lowest frequency of micronucleated cells (2,6%) and HK did not show any MN. The studied cell lines differed in their responses to colchicine. The data revealed the relevance of utilization of other end-points as growth curves, dead cells, mitotic index and other nuclear alterations for accurate analysis of the effect of agents that disturb cell cycle or are cytotoxic.

Cultured bladder cells and their response to mechanical strain.

The mechanism responsible for the transformation of a normal bladder into a poorly compliant storage vesicle is not well understood. However, a common feature of many poorly compliant bladders is an abnormal accumulation and distribution of matrix proteins within the bladder wall1–4. Histological examination of tissues from bladders with outlet obstruction is frequently associated with infiltration of connective tissue2. This abnormal accumulation is uniquely associated with the muscle bundles of the detrusor layer and has been shown by immunohistochemistry to be largely composed of type III collagen4. Biochemical studies of myelodysplastic bladders show this characteristic fibrosis to result from a quantitative increase in type III collagen5. Since these same bladders have altered compliance, we believe that the loss of elasticity is related to the changed collagen composition of the bladder wall. A common link in pathological conditions with poor bladder compliance is premorbid exposure of the bladder wall and its constituent cells to an abnormal physical environment, e.g., uncontrolled intermittent contractions. The impact of this abnormal environment upon bladder function is difficult to assess in whole bladder studies. It is likewise difficult to assess the relative contributions of the various tissue components within the bladder wall to the overall loss of compliance. We have previously isolated and maintained and characterized fetal bovine bladder urothelial, fibroblastic and smooth muscle cells in culture6–8. To begin to understand the role of mechanical forces in altering synthesis of connective tissue proteins and assess the relative responses of the individual cell types found in the bladder wall, we have applied equibiaxial deformations to the three major cell types found in the bladder wall9.

Bovine Bladder Compliance Increases with Normal Fetal Development

In this study we characterized the elastic properties of the normal bovine bladder throughout fetal life, the newborn period and into adulthood. The elasticity of the bladder was measured with a novel circularly clamped bladder testing system. Pressurization of a circularly clamped bladder tissue sample caused the tissue to deflect upward repetitively in the shape of a spherical cap. If the centerline deflection is much larger than the tissue thickness, the elastic modulus, considered the inverse of compliance, can be determined using linear regression techniques.The results of our analyses showed that the elastic modulus decreases in direct proportion with increasing gestational age. These data suggest that during normal development of the bovine fetal bladder there is a progressive change from a rather stiff noncompliant bladder characterized by a high elastic modulus to a compliant bladder with a lower elastic modulus. Moreover, the increase in compliance appears in the developmental period when urine production first occurs. These observations suggest that volume work may be a significant event in the normal development process of the bovine bladder and results in an increase in bladder compliance. Conversely, the poorly compliant fetal bladder may explain some of the transient dilatations of the upper urinary tract which have been documented in utero. Finally, from the newborn period to the mature adult bovine we documented a relatively modest increase in the elastic modulus or decrease in bladder compliance which may reflect the normal aging process.

Type III Collagen Decreases in Normal Fetal Bovine Bladder Development

In normal fetal bovine bladder development we have shown that compliance increases at approximately the same time that urine production first occurs. The late first trimester fetal bladders are relatively stiff with a progressive increase in bladder compliance peaking in the newborn period. From the newborn period through adulthood, we documented a relatively modest decrease in bladder compliance, which may result from the normal aging process. To account for these changes, we have used the bovine model to perform biochemical analyses of the major structural collagens that are found in the bladder (types I and III). These results show that the per cent of type III collagen decreases in the developing bladder from the end of the first trimester until the newborn period. Comparing the newborn bladder to that of a mature adult, we documented a relatively modest increase in the amount of type III collagen. We demonstrated that the ratio of type III-to-type I collagen parallels the normal compliance changes in the developing fetal and mature bovine bladder.

Developmental Changes in Normal Fetal Bovine Whole Bladder Physiology

In the present study, normal and experimentally altered whole fetal bovine bladders have been used in vitro to study developmental changes in compliance and capacity. For the first time, we have attempted to define the relative contributions of the detrusor and mucosal layers to bladder compliance. Fetal bovine bladder compliance increases with fetal development. Elimination of the active component of smooth muscle tension improves compliance and increases capacity more than 60% in younger fetuses but only 35% in older fetuses. Active smooth muscle tension as evaluated by whole bladder cystometry is highest in the youngest fetuses and decreases with fetal age. Surgical removal of the detrusor layer (smooth muscle and connective tissue) also increases compliance and capacity substantially in all fetal bladders. These observations show that both smooth muscle and connective tissue are important in the function of the developing fetal bladder. Changes in both of these bladder wall components probably occur during development and are responsible for the physiologic changes observed.

Comparative Studies on the Ontogeny and Autonomic Responses of the Fetal Calf Bladder at Different Stages of Development: Involvement of Nitric Oxide on Field Stimulated Relaxation

This initial study correlates the passive length-tension relationship, contractile and relaxant responses to field stimulation and contractile responses to specific autonomic agonists and antagonists with gestational age. Fetal bovine bladders were separated into three groups based on the head-rump length (FL): 30 to 45cm. (early gestation), 50 to 65cm. (middle gestation) and 70 to 85cm. (late gestation). Each bladder was separated into upper and lower bladder segments; longitudinal strips of smooth muscle were isolated and placed in individual muscle baths.Passive length-tension studies demonstrated that compliance was greatest in the bladder of late gestation and lowest in the bladder of early gestation period. Field stimulation (FS) elicited frequency-dependent contractile responses in all strips. In the upper bladder, the maximal response and maximal rate of tension generation to FS was lowest in the youngest fetuses and increased in proportion to the gestational age. In the lower bladders, there were no gestational age-related differences in the maximal response or maximal rate of tension generation in response to field stimulation.The maximal response of the upper bladder to bethanechol increased significantly from the youngest gestational age to mid-gestation, with no further changes between mid- and late gestation. The maximal response to field stimulation and bethanechol were equal between upper and lower bladder segments for the youngest gestational bladders, whereas for the oldest gestational ages, the maximal response of the upper bladder to FS and bethanechol were significantly greater than the responses of the lower bladder.In the presence of maximal precontraction with bethanechol, FS induced a rapid and marked decrease in tension. The magnitude of the relaxation was substantially greater for the strips of lower bladder than for the strips of upper bladder at late gestation. In lower bladders, the magnitude of the field stimulated relaxation was greater in the strips from the older fetuses than in the strips from younger fetuses. In all strips, field stimulated relaxations were completely inhibited by pretreatment with L-NAME (an inhibitor of nitric oxide synthesis), indicating that the FS-induced relaxation was due to nitric oxide. In addition to nitric oxide-induced relaxation, beta adrenergic stimulation also induced a significant relaxation of the isolated strips.In summary, these data suggest that, in the tubular shaped fetal bovine bladder, there were distinct differences in the autonomic responses between the upper bladder segment and the lower bladder segment in the late gestation period.

Primary culture of bovine gall bladder epithelial cells.

Gall bladder epithelial cells serve numerous biological functions and abnormalities in their function are important in the pathogenesis of several gall bladder diseases. Direct studies on cell function are rare due to lack of reliable methods to culture this epithelium. This study reports a reliable and reproducible method of harvesting and culturing gall bladder epithelial cells. Normal bovine gall bladder epithelium, obtained within 20 minutes of slaughter, was rinsed with modified Hanks's balanced salt solution, the mucosa separated and incubated in trypsin--EDTA solution at 37 degrees C. The cells were isolated and resuspended in Dulbeco's modification of Eagles' medium containing 10% fetal calf serum and, after filtration and centrifugation, were plated under aseptic conditions. The growth rate was established by flow cytometry and the morphological characteristics of the growing cells by electron microscopy. Gall bladder epithelial cells grew successfully and visible clusters of cells were present by day two, confluency being reached at 8 to 10 days in collagen coated plates and 12 to 14 days in uncoated plates. Electron microscopy showed typical gall bladder epithelia with microvilli, tight junctions, and mucus droplets. This method proved reliable and reproducible for the culture of gall bladder epithelial cells and should allow direct studies of the biological properties of these cells in human tissue.

Effect of Physical Forces on Bladder Smooth Muscle and Urothelium

Abnormalities in bladder physiology may be due to obstruction (pressure) and/or neurological impairment. Clinically they can result in an increase in connective tissue and a decrease in bladder compliance. To study the effects of physical forces on the bladder without the influence of the nerves we developed a cellular model system by isolating the 2 major cell types in the bladder: smooth muscle and urothelial cells. Extracellular matrix protein biosynthesis by these 2 cell types in vitro has been characterized by metabolic labeling of proteins with [14C] radiolabeled proline and analysis by gel electrophoresis. These studies demonstrate that fetal bovine bladder smooth muscle and urothelial cells synthesize fibronectin and types I and III interstitial collagen.Since bladder cells exist in an active physical environment, we have attempted to simulate this at the cellular level. Using a device developed in our laboratory, we applied a precise and reproducible mechanical strain (physical force) to these 2 cell types. By enzyme linked immunosorbent assay we quantitated collagen types I and III and fibronectin synthesized by fetal bovine bladder smooth muscle and urothelial cells undergoing mechanical strain (4.9%). These cells were compared to unstrained control cells that were exposed to the same experimental conditions. For bladder smooth muscle cells we found a significant increase in collagen type III and fibronectin synthesis when compared to unstrained cells. In contrast, collagen type I synthesis decreased with mechanical strain. For bladder urothelial cells we found an increase in collagen type I and fibronectin while collagen type III remained unchanged.These studies demonstrate that extracellular matrix synthesis by urothelial and smooth muscle cells can be modulated by stretch (strain) in the absence of neurological input. It is likely that bladder function may be impaired as a result of abnormal synthesis of connective tissue.

Bladder Smooth Muscle Cells in Culture: I. Identification and Characterization

This report documents the growth and culture characteristics of human and fetal bovine bladder smooth muscle cells in vitro. Bladder smooth muscle cell strains have been identified by their spindle shaped morphology, noncontact inhibited growth characteristics and the expression of smooth muscle cell specific a-actin. Extracellular matrix protein biosynthesis by these cells in vitro has been characterized by metabolic labeling of proteins with [14C] radiolabeled proline and analysis by SDS gel electrophoresis. These studies demonstrate that bladder smooth muscle cells synthesize predominantly types I and III collagen, and fibronectin. In addition type III collagen exists in both a partially processed (pN α1[III]) form and processed form.Complementary immunohistochemical studies show localization of type I, III, and IV collagens, and fibronectin to bladder smooth muscle cell extracellular matrix. We conclude that both fetal bovine and human smooth muscle bladder cells are capable of secreting the classic components of the surrounding connective tissue.