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Biomedical subjects

E Macarak

Publications and source records attributed to E Macarak.

15 recordsLinked to original sources
PubMed2001

Regulation of Cyr61 gene expression by mechanical stretch through multiple signaling pathways.

The cysteine-rich protein 61 (Cyr61) is a signaling molecule with functions in cell migration, adhesion, and proliferation. This protein is encoded by an immediate early gene whose expression is mainly induced by serum growth factors. Here we show that Cyr61 mRNA levels increase sharply in response to cyclic mechanical stretch applied to cultured bladder smooth muscle cells. Stretch-induced changes of Cyr61 transcripts were transient and accompanied by an increase of the encoded protein that localized mainly to the cytoplasm and nucleus of the cells. With the use of pharmacological agents that interfere with known signaling pathways, we show that transduction mechanisms involving protein kinase C and phosphatidylinositol 3-kinase activation partly blocked stretch-induced Cyr61 gene expression. Selective inhibition of Rho kinase pathways altered this stretch effect as well. Meanwhile, using inhibitors of the actin cytoskeleton, we show that Cyr61 gene expression is sensitive to mechanisms that sense actin dynamics. These results establish the regulation of Cyr61 gene by mechanical stretch and provide clues to the key signaling molecules involved in this process.

Animals↗
PubMed2001

The response of fetal sheep bladder tissue to partial outlet obstruction.

PURPOSE: We characterized the response of fetal ovine bladder strips to stimulated contraction and relaxation, and compared this response to that of strips from the pregnant mother and those obtained after a short duration of fetal bladder outlet obstruction. MATERIALS AND METHODS: Sham surgery or bladder obstruction was performed in fetal sheep at 90 days of gestation (term 147 days). Bladder tissue was obtained 3 and 5 days later. Isolated strips of full-thickness bladders from fetuses and pregnant females were mounted individually in Tyrode's solution containing glucose. The strips were subjected to electrical field stimulation. Alternate strips were stimulated by adding carbachol, adenosine triphosphate and KCl. Each strip stimulated by carbachol also underwent field stimulation in the presence of carbachol. Relaxation was also tested using isoproterenol and nitroprusside. RESULTS: The response of isolated strips to field stimulation showed phasic contraction or biphasic response, consisting of initial phasic contraction followed by phasic relaxation and a return to control tension after the end of stimulation. In fetal bladder strips field stimulation at all frequencies after carbachol stimulation produced phasic relaxation or a biphasic response with an initial relaxation phase followed by phasic contraction. This field stimulated relaxant response was not present in adult female bladder strips. In addition, field stimulation stimulated relaxation was completely eliminated by pretreatment with N-nitro-L-arginine-methyl ester, indicating that relaxation was nitric oxide mediated. The fetal responses to all forms of stimulation and relaxation were significantly greater than those of pregnant females. After 5 days or greater of obstruction the responses to field stimulation were reduced significantly. In contrast, there were no significant differences in contractile responses to adenosine triphosphate, carbachol or KCl, or the relaxant response to field stimulation after obstruction. However, there was a significant reduction in relaxant responses to isoproterenol and nitroprusside. CONCLUSIONS: In mid gestation sheep fetus contractile responses to field stimulation, adenosine triphosphate, carbachol and isoproterenol are well developed. The fetal ovine bladder shows a strong neuronal nitric oxide response that is not present in the pregnant mother and is maintained after short-term obstruction.

Animals↗
PubMed1997

A basic compositional requirement of agents having heparin-like cell-modulating activities.

Heparin has been recognized as possessing a large variety of cell-modulating activities. Using compositionally simple, structurally rigid, and low molecular weight saccharide molecules (cyclodextrins), we demonstrated that these activities depend primarily on a single, gross compositional parameter: a minimum intramolecular density of neighboring anionic (sulfate) groups. This same critical parameter is shown to be involved in achieving cell-modulating behavior as diverse as angiogenesis, endothelial proliferation, inhibition of smooth muscle cell growth, and cell protection against virus invasion. Physical chemical evidence is presented that associates this property with multi-ionic complex formation between the clusters of anionic and cationic sites on the complexing partners. These observations revive early suggestions of the decisive role of electrostatic complexation capabilities of glycosaminoglycans like heparin; taken together with numerous observations on heparinoids and other agents reported in diverse specialized fields of cell biology and medicine, they provide evidence that molecular agents of critical anionic (sulfate) density (MACADs) represent a broad class of molecules that, in contrast to proteins, do not rely on structural detail for their cell biological activities, but function by ionic complexation with proteinic agents (e.g. growth factors), thereby modifying their structure-specific activities.

Alcian Blue↗
PubMed1997

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.

Animals↗
PubMed1997

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 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. 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.

Animals↗
PubMed1994

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.

Aging↗
PubMed1994

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.

Animals↗
PubMed1994

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 45 cm. (early gestation), 50 to 65 cm. (middle gestation) and 70 to 85 cm. (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.

Adenosine Triphosphate↗
PubMed1993

Distribution and function of the adrenergic and cholinergic receptors in the fetal calf bladder during mid-gestational age.

Previous studies on the developing fetal bovine bladder demonstrate that compliance is low during early stages of fetal development and increases with fetal age. In addition, the pharmacological response of isolated fetal bovine bladder smooth muscle strips to field stimulation and bethanechol increased in proportion with the gestational age. In the adult bladder (rabbit), the contractile response to autonomic receptor stimulation and the autonomic receptor density showed a parallel gradation in the the bladder between bladder dome and the urethra. The present studies were designed to determine the distribution of the cholinergic and adrenergic receptors in the urinary bladder of the fetal calf at mid-gestational age, and to correlate the receptor density with the magnitude of the response to receptor stimulation. Each bladder body was separated into upper, middle, and lower segments. For the functional studies, circular and longitudinal strips were cut from each bladder segment and stimulated with field stimulation (FS), bethanechol, methoxamine, isoproterenol, and KCl. Autonomic receptor assays were performed using 3H-QNB (muscarinic cholinergic), 3H-DHE (alpha adrenergic), and 3H-DHA (beta adrenergic) as ligands. In general, there were no significant differences in receptor density among the three bladder segments. However, the density of muscarinic receptors was significantly greater than the density of alpha- or beta-adrenergic receptors for all bladder segments. The receptor densities correlated very well with the functional response of isolated bladder strips to the specific autonomic agonists. The contractile response of isolated bladder strips to the specific autonomic agonists. The contractile responses of the strips to FS, bethanechol, and methoxamine were not significantly different among the three different bladder segments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

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.

Animals↗
PubMed1993

Effect of mechanical forces on extracellular matrix synthesis by bovine urethral fibroblasts in vitro.

The role of mechanical forces in normal physiological processes is just beginning to be elucidated. Using a system developed in our laboratory, we can apply precise and reproducible mechanical deformations (biaxial strain) to cells. These deformations alter cell activities in a reproducible fashion and may mimic the physical environment found in portions of the urinary tract. At a low strain of 1.8% no change in the synthesis of types I and III collagen by urethral fibroblasts was found. However, at a high strain (4.9%) types I and III collagen showed a significant increase in synthesis compared to controls (type I, 1.4 +/- 0.25 microgram. versus 0.9 +/- 0.27 microgram., p = 0.053; type III, 110 +/- 7 ng. versus 88 +/- 10 ng., p = 0.036). In addition, fibronectin synthesis was increased at low and high strains when compared to controls (low strain 3.20 +/- 1.03 micrograms. versus 1.46 +/- 0.15 microgram., p = 0.042; high strain 8.90 +/- 1.09 micrograms. versus 3.12 +/- 0.69 microgram., p = 0.001). We have shown at the cellular level that mechanical force applied to fetal bovine urethral fibroblasts results in an increase in the amount of collagen synthesis and fibronectin synthesis. These findings suggest that alterations in the physical environment of cells found in the urethral wall can affect biochemical processes including those that govern the synthesis of structural macromolecules such as collagen.

Animals↗
PubMed1984

Serum fibronectin is elevated during normobaric and hyperbaric oxygen exposure in rats.

Serum fibronectin concentration was measured by immunoelectrophoresis in rats exposed to 1 atmosphere absolute (ata) of oxygen for 24 to 72 h and at 30-min intervals during 1 to 5 h of exposure to 4 ata O2. At 1 ata O2, there was a progressive increase in serum fibronectin, which became significant at 48 h and was 2.5 times the control value by 72 h (p less than 0.01). At 4 ata O2, the rise in serum fibronectin was significant at 2 h, and was followed by an accelerated rate of increase to 5 h, at which time serum fibronectin concentration was 1.75 times the control value (p less than 0.05). By SDS-polyacrylamide gel electrophoresis and crossed immunoelectrophoresis, there was no evidence of fragmentation of fibronectin. This study demonstrated a progressive increase in serum fibronectin in the oxygen-exposed rat. The magnitude and course of the increase suggest increased synthesis and secretion of intact fibronectin molecules.

Animals↗
PubMed1981

Bovine endothelial cells in culture produce thromboxane as well as prostacyclin.

Bovine aortic endothelial and smooth muscle cells in culture were incubated with arachidonic acid or prostaglandin H2. The amount of prostacyclin nd thromboxane A2 synthesized ws then determined by specific radioimmunoassay for 6-keto-prostaglandin F1 alpha and thromboxane B2. Although smooth muscle cells produced only 6-keto-prostaglandin F1 alpha and thromboxane B2 in a ratio of 5:1 to 10:1. The same ratio of these metabolites of arachidonic acid ws also found when prostaglandin production from endogenous arachidonic acid was stimulated in endothelial cells by the ionophore A23187. Cyclooxygenase inhibitors inhibited the production of both metabolites equally, whereas thromboxane synthetase inhibitors selectively inhibited the production of thromboxane B2. Cells in culture were also incubated with [1-14C]arachidonic acid and the extracted products were identified by two-dimensional thin-layer chromatography. 6-Keto-prostaglandin F1 alpha was the only metabolite produced by smooth muscle cells, but endothelial cells synthesized 6-keto prostaglandin F1 alpha, thromboxane B2, prostaglandin E2, and prostaglandin F2 alpha.

Animals↗
PubMed1977

Production of 5-hydroxyindoleacetic acid from serotonin by cultured endothelial cells.

Endothelial cells cells from bovine aorta and human umbilical vein and fibroblasts from human foreskin were cultured and subsequently evaluated for ability to metabolize serotonin (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA). Cells were incubated for three hours with 4 X 10(-6) M [14C] 5-HT creatinine sulfate. [14C] 5-HIAA was separated from labeled 5-HT by column chromatography and measured for scintillation counting. Production of 5-HIAA by bovine aorta cells was 39.0+/-7.5 (S.E.M., n=6) nmoles per 10(9) cells per hour. Production of 5-HIAA was markedly inhibited by the presence of 10(-4) M iproniazid (an inhibitor of monoamine oxidase) or 10(-4) M imipramine (an inhibitor of amine transport). 5-HIAA was the only product of 5-HT metabolism detected by thin layer chromatography. Production of 5-HIAA by human umbilical vein endothelial cells was 5.4+/-2.0 nmoles per 10(9) cells per hour (n=5) and by human foreskin fibroblasts was 3.9+/-1.4 nmoles per 10(9) cells per hour (n=5). The results obtained during incubation in the presence and absence of inhibitors indicate that bovine aorta endothelial cells maintained in tissue culture are able to transport serotonin with subsequent production of 5-HIAA. By contrast, human umbilical vein endothelial cells and fibroblasts exhibited relatively low rates of 5-HT uptake and metabolism.

Animals↗