Fetal bovine bladder: physiology and pharmacology.
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Animals↗
Biomedical subjects
E J Macarak
Publications and source records attributed to E J Macarak.
1995 William J. Stickel Gold Award. High strain rate tissue deformation. A theory on the mechanical etiology of diabetic foot ulcerations.
Foot ulcerations are one of the most common and dangerous complications associated with chronic diabetes mellitus. Many studies have focused on neuropathy, in conjunction with elevated ground reactive forces, as the principal cause of these ulcerations. The authors discuss the mechanical cause of diabetic ulcerations at the cellular level. It is hypothesized that increased rate of tissue deformation associated with foot slap secondary to progressive motor neuropathy is the actual culprit, and not the magnitude of local pressure applied. The authors present a cellular model that shows that high rates of tissue deformation may result in elevated intracellular calcium concentrations, which may lead to cellular death, while comparable loads gradually applied do not. Furthermore, there is no significant difference in the response observed at 5 psi and 10 psi. Based on these findings, it is hypothesized that techniques such as ankle foot orthoses, which control the velocity of foot strike, may be useful in treating diabetic foot ulcerations.
Awards and Prizes↗
The ontogeny of bladder function in the fetal calf.
Although numerous experimental studies have addressed urinary bladder physiology and pharmacology, little information is available concerning the ontogeny of bladder function. The present in vitro study describes the developmental aspects of bladder compliance, pressure generation and emptying in bovine fetuses from early second trimester to term (280 days). The results can be summarized as follows: 1) bladder compliance increased 3-fold between early second trimester and term; 2) sustained contractile response to bethanechol was present in all bladders; 3) field stimulation produced a submaximal, nonsustained contraction and an active relaxation when the field stimulus was turned off; 4) bladder emptying in response to bethanechol was nearly 100% in mid- and late-gestational bladders but was only 50% at early gestation; 5) bladder emptying in response to field stimulation was 20% to 40% for all gestational age groups; 6) field stimulated relaxation was observed only in the fetal bladder. These studies demonstrate that bladder physiology in utero is different from postnatal bladder function. The presence of a relaxant response during the fetal period may reflect a unique and significant role of the in utero bladder in protecting the upper urinary tracts from sustained increased pressure.
Adenosine Triphosphate↗
Characterization of a fibroblast cell from the urinary bladder wall.
For the first time we report on the growth, culture, and matrix production characteristics of a cell type isolated from the lamina propria of the urinary bladder wall. A fibroblastlike cell was identified as distinct from bladder detrusor smooth muscle cells and urothelium based on morphology, growth characteristics, and immunohistochemical staining. Characterization of extracellular matrix synthesis by this cell type using 35S-methionine metabolic labeling demonstrated that these cells are capable of secreting components of the surrounding connective tissue, including several fibrillar collagens, a basement membrane collagen, and fibronectin.
Actins↗
Loss of elasticity in dysfunctional bladders: urodynamic and histochemical correlation.
To store adequate volumes of urine at low safe pressures an elastic bladder wall is required. We developed 2 new techniques to measure this ability in our urodynamic laboratory: pressure specific bladder volume, which measures the bladder capacity at a given pressure, and dynamic analysis of bladder compliance. Recently, morphometric and histochemical techniques have been used to determine the relative volume of connective tissue in the bladder wall and to measure the 2 major types (I and III) of collagen within the bladder wall. These methods quantitate 3 parameters of bladder ultrastructure: 1) relative volume of per cent connective tissue, 2) ratio of connective tissue to smooth muscle and 3) ratio of type III to type I collagen. These parameters have been shown to be abnormally elevated in patients with dysfunctional bladders compared to normals. The purpose of the study was to describe the ultrastructural changes that occur in the wall of dysfunctional bladders and to determine the ability of these new urodynamic techniques to detect reliably the clinical effect of these histological changes. The study included 29 consecutive patients with dysfunctional bladders necessitating bladder augmentation. All patients had upper tract changes and/or were incontinent despite treatment with clean intermittent catheterization and pharmacotherapy. Preoperative urodynamic evaluation included measurement of the total bladder capacity, pressure specific bladder volume and dynamic analysis of bladder compliance. Full thickness bladder biopsies were obtained from the dome of the bladders during augmentation. The per cent connective tissue and the ratio of connective tissue to smooth muscle were determined for all patients, and 4 unselected patients from this group had the ratio of type III to type I collagen determined. These histological results were compared to previously established normal values. All 29 patients had a decreased pressure specific bladder volume and dynamic analysis of bladder compliance, whereas 9 had a normal total bladder capacity. The per cent connective tissue was 35.19 +/- 2.84 and ratio of connective tissue to smooth muscle was 0.60 +/- 0.08 compared to normal values of 10.6 +/- 0.020 and 0.131 +/- 0.021, respectively (p < 0.05). Ratio of type III to type I collagen was also significantly elevated in the 4 samples analyzed (30.53 +/- 1.37 versus 24.00 +/- 2.50, p < 0.05). We conclude that poor storage function of poorly compliant bladders is secondary to an alteration in the connective tissue content of the bladder wall. Furthermore, these pathological ultrastructural changes are universally reflected by an abnormally low pressure specific bladder volume and dynamic analysis of bladder compliance. This strong association validates the use of these parameters and suggests that they are urodynamic indicators of a loss of elasticity in bladder wall.
Adolescent↗
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.
Animals↗
Protection of erythrocytes against hemolytic agents by cyclodextrin polysulfate.
Cyclodextrins generally exhibit hemolytic activity, some at concentrations as low as 1-10 mg/mL or lower. However, we found previously that a highly polysulfated cyclodextrin has no demonstrable hemolytic activity (Macarak et al., Biochem Pharmacol 42: 1502-1503, 1991). In the present study, we determined that, in fact, cyclodextrin polysulfate (CDS) actively protected erythrocytes against hemolysis induced by a wide spectrum of hemolytically active substances, ranging from pharmaceuticals, such as chlorpromazine, to solid suspensions of siliceous particles. The protective action was also effective against the hemolytic action of non-sulfated cyclodextrins. The similar kinetic responses of the erythrocytes to CDS protection against such chemically and structurally diverse hemolytic agents suggest a common mechanism involving the cell. Addition of the sulfated cyclodextrins to other cyclodextrin compounds used to solubilize poorly soluble pharmaceutical agents can extend the allowable maximum dosage without deleterious hemolytic action.
Cells, Cultured↗
Biochemical characterization and quantitation of the collagenous components of urethral stricture tissue.
The collagenous composition of normal and strictured human urethral tissue was analyzed qualitatively by immunohistochemistry and quantitatively by 2-dimensional gel electrophoresis of cyanogen bromide digested tissue. Histological comparison of the normal and strictured urethral tissue showed that the normal urethral spongiosum was replaced by densely packed connective tissue fibers interspersed with fibroblasts in the strictured tissue. The immunohistochemical analysis of urethral tissue identified the presence of types I and III collagen fibers in normal spongiosum and in the connective tissue scar of strictured tissue. Estimation of the collagen type III:I ratio using scanning densitometry revealed a CB5:CB8 peptide ratio of 0.357 +/- 0.058 in the normal tissue, while the urethral stricture tissue had a CB5:CB8 ratio of 0.203 +/- 0.079 (p = 0.010). Total collagen content, as determined by hydroxyproline analysis, revealed no statistically significant differences between control and strictured tissue. Therefore, the normal urethral spongiosum was comprised of 75.1% type I collagen and 24.9% type III collagen. In contrast, the type I collagen in urethral stricture tissue was increased (83.9%), with a corresponding decrease in type III collagen (16.1%). This alteration in the ratio of collagen type III:I may explain the fibrotic noncompliant nature of urethral stricture scar tissue.
Adolescent↗
Culture of urethral fibroblasts: cell morphology, proliferation and extracellular matrix synthesis.
The in vitro morphology and proliferative activity of urethral fibroblasts were characterized from primary isolates of fetal bovine urethral spongiosum. Cells were identified as fibroblastic based on their spindle-shaped morphology and lack of expression of phenotypic markers characteristic of either urethral smooth muscle or epithelial cells. Urethral fibroblasts proliferated at a steady rate and did not exhibit contact-inhibition of growth in a culture medium of Medium 199 supplemented with 10% newborn calf serum. Both immunohistochemistry and metabolic labeling analyses of extracellular matrix synthesis demonstrated the presence of collagen types I and III, and fibronectin. Quantitation of the collagenous components of the cell medium revealed that urethral fibroblasts synthesize 85% type I collagen, 14% type III collagen and < 1% type V collagen. In contrast, the urethral fibroblast cell layer matrix consisted of 98% type I collagen, < 1% type III and < 1% type V collagen.
Animals↗
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 alpha-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 alpha 1[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.
Animals↗
An analysis of the time-dependent changes in intracellular calcium concentration in endothelial cells in culture induced by mechanical stimulation.
When bovine pulmonary artery endothelial cells in culture are subjected to mechanical strain, their physiology is altered. Experimentally, this mechanical strain is generated by increased tension in the substrate to which the cells are attached and results in altered levels of fibronectin. Studies of the structural response of the endothelial cell suggest that this stimulus is transmitted to the cell membrane, organelles, and cytoskeleton by natural cell attachments in a quantifiable and predictable manner. This report examines altered intracellular calcium homeostasis as a possible messenger for the observed strain-induced physiologic response. In particular, using the intracellular trapped calcium indicator dyes, Quin2 and Fura2, we observed changes in cytosolic free calcium ion concentration in response to biaxial strain of bovine pulmonary artery endothelial cells in culture. The magnitude and time course of this calcium transient resemble that produced by treatment with the calcium ionophore, Ionomycin, indicating that mechanical stimulation may alter cell membrane permeability to calcium. Additional experiments in the presence of EDTA indicated that calcium was also released from intracellular stores in response to strain. In order to explain the stretch-induced calcium transients, a first-order species conservation model is presented that takes into account both the cell's structural response and the calcium homeostatic mechanisms of the cell. It is hypothesized that the cell's calcium sequestering and pumping capabilities balanced with its mechanically induced changes in calcium ion permeability will determine the level and time course of calcium accumulation in the cytosol.
Animals↗
Angiogenesis and heparin mimics.
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Animals↗
Is lamina propria matrix responsible for normal bladder compliance?
Immunohistochemistry using monoclonal and polyclonal antibodies to extracellular matrix proteins is a highly sensitive tool for the characterization of matrix components. For the first time in the normal and noncompliant human bladder we have used antibodies to collagen types I, III and IV, and elastin to provide morphological correlation with mechanical properties noted clinically. In the normal bladder elastin and collagen types I and III showed intense localization in the lamina propria with modest localization in the detrusor layer. In contrast, lamina propria staining in the noncompliant bladder was essentially unchanged, while there was intense localization within the detrusor layer. Significantly, this intense localization consisted of collagen type III and elastin with little increase in type I. Type IV collagen is associated with basement membranes and individual smooth muscle cells, and shows commensurate increase in specimens with muscle hypertrophy and/or hyperplasia. These observations suggest that in the normal bladder the lamina propria may be a major structural capacitance layer with the smooth muscle covering it. The collagen fibers of the lamina propria may gradually unfold during filling, thus, accounting for normal compliance while in the noncompliant bladder the capacitance layer shifts outward to the infiltrated smooth muscle, thus, preventing the normal expansion of the lamina propria. The smooth muscle infiltration consists of a deposition of collagen type III and elastin with little increase of collagen type I, and it results in a loss of compliance. The pattern of localization would suggest that the smooth muscle is responsible for this accumulation.
Adolescent↗
Sulfation and hemolytic activity of cyclodextrin.
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Cyclodextrins↗
Progressive modulation of endothelial phenotype during in vitro blood vessel formation.
"Sprouting" vascular endothelial cells were used as an in vitro model system to study the progressive morphologic and biosynthetic changes associated with the formation of tubular structures. In vitro, sprouting endothelial cells formed spontaneously without the addition of any exogenous factors from cultures of cloned endothelium exhibiting a polygonal/cobblestone phenotype. These phenotypically variant endothelial cells differentiated to form associated cell networks or nodules which gradually reorganized into tubular structures. Concomitant with these morphologic changes, the biosynthesis of extracellular matrix proteins was modulated, as determined by Northern blot analysis, metabolic labeling, and immunocytochemistry. The initial sprouting phase was characterized by the induction of type I collagen synthesis and the appearance of fibronectin containing the ED-A domain, in comparison to their absence in cloned cultures displaying a stable polygonal/cobblestone phenotype. The organizational stage, where the sprouting endothelial cells assembled into tubular structures, was additionally characterized by the expression of type IV collagen. These studies demonstrate that the progression from polygonal/cobblestone to sprouting cultures, and subsequent tubular organization, involves major alterations in extracellular matrix protein expression. This developmental phenomenon, although not completely analogous to blood vessel formation in vivo, nevertheless may be helpful in understanding the role of matrix macromolecules in the angiogenic process.
Animals↗
Contraction of vascular smooth muscle in cell culture.
The use of cultured vascular smooth muscle cells for the study of events related to excitation and contraction of smooth muscle has been limited by the inability to reliably induce contractile responses after subculturing of the cells. This limitation has been overcome by the cell culture preparation described herein. We demonstrate that appropriate responses to both smooth muscle agonists and vasodilators were preserved in cells that were serially subcultured. Fetal bovine pulmonary artery and aortic cell cultures were established following enzymatic dispersion of the medial portion of freshly harvested vessels. At various times after isolation, cells were transferred to microscope coverslips coated with a polymerized silicone preparation (polydimethyl siloxane). Tension forces generated by the cells were manifested as wrinkles and distortions of this flexible growth surface. Visual evidence of cell contraction in the form of increased wrinkling was documented for cells exposed to angiotensin II, carbachol, and KCl. Decreases in cell tension occurred following treatment with isoproterenol, and those relaxing effects were overcome by subsequent treatment with the agonist carbachol. The contractile responses did not diminish with prolonged maintenance in culture or repeated subculturing. Phosphorylation of the light chains on the contractile protein myosin was also measured as a biochemical index of agonist-induced contraction. Cells depolarized with KCl or exposed to carbachol showed increased myosin phosphorylation when analyzed by 2-dimensional gel electrophoresis. The responses remained intact through 7 passages and 9 weeks in culture. These results show that cultured vascular smooth muscle cells do not necessarily undergo a phenotypic modulation with loss of contractility under prolonged maintenance in culture.
Angiotensin II↗
Collagen and fibronectin gene expression in cultured endothelial cells.
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Animals↗
Cyclic biaxial strain of pulmonary artery endothelial cells causes an increase in cell layer-associated fibronectin.
Bovine pulmonary artery endothelial (PAE) cells were cultured on an artificial compliant substrate (Mitrathane) and were strained biaxially at a frequency of 1/s for 2, 4, 6, 7, or 24 h. Total protein synthesis, determined by estimating the incorporation of radiolabeled precursors into nondialyzable protein, was increased in cultures that had been biaxially strained for 6, 7, or 24 h, with differences more apparent in the cell layer fraction than in the medium fraction. Medium and cell layer-associated fibronectin were quantitated by enzyme-linked immunosorbent assay and by densitometric analysis of the autoradiograms of electrophoresed protein. Fibronectin levels in the medium of biaxially strained cells were initially depressed in comparison to nonstrained controls but, with time, began to approach control values. Cell layer-associated fibronectin of biaxially strained cultures was significantly elevated at 24 h, whereas DNA synthesis was not altered. Immunohistochemical localization of fibronectin and factor VIII-von Willebrand antigen revealed a more intense staining pattern in strained cultures. Distribution of stress fibers containing fibrous actin was visualized by staining with rhodamine-phalloidin and was altered in strained cultures. These observations indicate that cells respond to cyclic biaxial strain by selectively enhancing structural components associated with cell adhesion.
Animals↗