Alteration of the responses of gastric smooth muscle to endothelin in streptozotocin-induced diabetic rats.

Diabetic gastropathy is suggested to be the result of not only an autonomic neuropathy but also to disorder of the spontaneous rhythmic motility of the gastric smooth muscle. Attempts were made to investigate the alteration of the effects of endothelin-1 (ET-1), which is known to enhance the spontaneous activity of gastrointestinal smooth muscle, on gastric activity in streptozotocin (STZ)-induced diabetic rats. STZ-induced diabetic rats were prepared by the injection of Sprague-Dawley (SD) rats with STZ (i.p.). Isometric mechanical responses were recorded in isolated circular smooth muscle strips of the stomach antrum, to measure changes in the rhythmicity of the smooth muscle. ET-1 (10 nM) significantly elevated the resting tension and the frequency of spontaneous contraction, but did not alter the amplitude of the spontaneous oscillatory contractions in normal rats. In diabetic rats, ET-1 elevated the resting tension, and spontaneous contractions were increased in frequency, however they were decreased in amplitude. In normal rats, sarafotoxin S6c (S6c, 10 nM), a selective ET(B) receptor agonist, elevated the resting tension slightly and increased both the frequency and amplitude of the spontaneous contractions. However, S6c significantly elevated the resting tension alone in STZ-induced diabetic rats. Selective stimulation of endothelin type A (ET(A)) receptors with ET-1, in the presence of a selective antagonist of ET(B) receptors, produced similar responses in the gastric muscle of both normal and diabetic rats. These results indicate that ET-1 elevates the resting tension and increases the frequency of the spontaneous oscillatory contractions in both normal and STZ-induced diabetic rats, to a similar extent. However, the specific actions on ET(B) receptors were quite different between the two: the elevating actions on the resting tension were much greater in STZ-diabetic rats than in normal rats. The results suggested the facilitation of ET(B) receptor signaling in the antrum during the pathogenesis of diabetic gastropathy.     

Regeneration of skeletal muscle in streptozotocin-induced diabetic rats

The present study analyzes the regeneration of skeletal muscle in diabetic rats. Intravenous injection of streptozotocin (STZ) was used to induce diabetes. Six weeks later the extensor digitorum longus (EDL) muscles from diabetic rats were either transplanted into diabetic or normal hosts to initiate regeneration. Normal EDL muscle transplants in normal and diabetic hosts were also performed for comparison. One, 2, 4, and 12 weeks after transplantation, the EDL regenerates were morphologically analyzed. Regeneration and formation of neuromuscular junctions were observed in all transplants, including diabetic regenerates in diabetic hosts. The overall mass and myofiber size of the diabetic EDL regenerate in the diabetic host was significantly reduced in spite of complete regeneration. Recovery of the diabetic muscle mass and the myofiber size was observed after transplantation into normal hosts. A reduction in mass and myofiber size was observed in normal EDL muscles transplanted into diabetic hosts. It is concluded that poor recovery of diabetic muscle is related to metabolic and structural alterations in the diabetic host, rather than to innate capacity of the muscle to per se undergo regeneration and reinnervation. The observed enhancement in recovery of diabetic muscle after transplantation in a normal host and deterioration of normal muscle after transplantation in a diabetic host shows that the host environment determines the success of muscle regeneration.     

TRPV1在链脲佐菌素诱导的糖尿病大鼠膀胱中的表达变化

目的本实验旨在研究瞬时感受器电位V型1(TRPV1)在链脲佐菌素(STZ)诱导的糖尿病大鼠的膀胱中的表达变化,探讨糖尿病膀胱功能障碍的机制。方法雄性Wistar大鼠随机分成STZ诱导的糖尿病组,5%蔗糖水诱导的多尿组和对照组,通过尿动力学检查对膀胱功能进行评价,应用免疫组织化学技术,逆转录-聚合酶链式反应(RT-PCR)和Western blot检测STZ诱导糖尿病后16周大鼠膀胱中TRPV1的表达变化。结果与同龄对照组和多尿对照组相比,16周糖尿病组大鼠的平均膀胱容量和平均膀胱内最大压力升高,排尿率降低,糖尿病组大鼠膀胱中TRPV1的mRNA表达水平和蛋白水平均降低。结论 TRPV1可能与糖尿病膀胱功能障碍有关。     

Series-elastic properties of strips of smooth muscle from pig urinary bladder

The series elasticity of strips of smooth muscle from pig urinary bladder was investigated by means of a series of computerised quick-release and quick-stretch measurements with and without stimulation of the muscle, and at different shortenings and force levels. The results cannot be interpreted in terms of a discrete passive series-elastic element. They can, however, be interpreted in terms of the sliding-filaments model for contracting muscle.     

Reduced urinary bladder afferent conduction velocities in streptozocin diabetic rats.

Previous experiments in our laboratory have described the method used to measure the conduction velocity distribution of a selected group of fibers (Brain Res., 520 (1990) 83-89). We have applied this technique to the 2 month streptozotocin-diabetic rat. Glycosylated hemoglobin values measured at the time of death were 17.19 +/- 4.74% (diabetic, n = 8) and 4.07 +/- 0.74% (controls, n = 6). Diabetic bladders were thicker and heavier. The wet weights were 0.50 +/- 0.11 g (diabetic, n = 7) and 0.16 +/- 0.01 g (controls, n = 6). The conduction velocities of a total of 151 and 86 single afferent fibers were measured in the diabetic and control animals respectively. The conduction velocity distribution of the diabetics showed a shift towards slower speeds when compared to controls. The mean conduction velocities were 1.70 m/s for diabetics and 2.84 m/s for controls. The percent of units with conduction velocities greater than 2.5 m/s was 17.2 for diabetics and 36.0 for controls. This experiment demonstrates, for the first time, that diabetes causes a significant reduction of afferent conduction velocities in a functionally well-defined system.     

Active mechanical properties of the smooth muscle of the urinary bladder

Strips of pig bladder have been maximally stimulatedin vitro at 37°C via electrodes placed in the muscle, in order, particularly, to measure the dependence of the resulting active force on the velocity of shortening and on length changes. The active isometric force and the passive viscoelastic force are approximately, but not precisely, additive. The active isometric force, like the steady (equilibrium) passive force, is a function of the extension of the strip above its rest length, which is increased after subjection to a high passive force. The steady passive force increases quasiexponentially with this extension, of which it is therefore a measure. The active isometric force F_iso increases approximately linearly with the extension until it approaches a maximum in the region where it and the steady passive force are comparable in size. The maximum is partly obscured by rest-length changes. The dependence of the active force F on the speed of shortening of the strip has been measured in a new way, with a correction for passive viscoelastic effects. For a given strip the ratio F/F_iso is, approximately, a function of the contraction velocity only. The function is similar to that of the classical Hill equation but not identical, possibly for geometrical reasons. The results imply that a velocity parameter v^*, analogous to Hill’s parameter b, is approximately constant for each strip, independent of changes of length and rest length.     

Induction of smooth muscle cell-like phenotype in marrow-derived cells among regenerating urinary bladder smooth muscle cells

Tissue regeneration on acellular matrix grafts has great potential for therapeutic organ reconstruction. However, hollow organs such as the bladder require smooth muscle cell regeneration, the mechanisms of which are not well defined. We investigated the mechanisms by which bone marrow cells participate in smooth muscle formation during urinary bladder regeneration, using in vivo and in vitro model systems. In vivo bone marrow cells expressing green fluorescent protein were transplanted into lethally irradiated rats. Eight weeks following transplantation, bladder domes of the rats were replaced with bladder acellular matrix grafts. Two weeks after operation transplanted marrow cells repopulated the graft, as evidenced by detection of fluorescent staining. By 12 weeks they reconstituted the smooth muscle layer, with native smooth muscle cells (SMC) infiltrating the graft. In vitro, the differential effects of distinct growth factor environments created by either bladder urothelial cells or bladder SMC on phenotypic changes of marrow cells were examined. First, supernatants of cultured bladder cells were used as conditioned media for marrow cells. Second, these conditions were reconstituted with exogenous growth factors. In each case, a growth factor milieu characteristic of SMC induced an SMC-like phenotype in marrow cells, whereas that of urothelial cells failed. These findings suggest that marrow cells differentiate into smooth muscle on acellular matrix grafts in response to the environment created by SMC.     

Energy turnover and lactate dehydrogenase activity in detrusor smooth muscle from rats with streptozotocin-induced diabetes

Force generation and tissue glucose metabolism were measured in the urinary bladder smooth muscle from rats with streptozotocin-induced diabetes (7–8 wk duration). Bladder wet wt was almost 4–fold higher in the diabetic animals compared with the untreated controls. Morphological analysis showed that the growth was associated with hypertrophy of the smooth muscle component in the bladder wall. Force generation of isolated bladder strip preparations was measured in vitro at different ambient oxygen tensions. Activation of intramural nerves, with electrical field stimulation, induced contractions that were unaffected by reduction of oxygen tension down to Po₂ 100 mmHg for both control and diabetic muscle strips. At zero Po₂ force was reduced by approximately 10–20% in both groups. High-K⁺ solution induced ‘tonic’ contractions that were slightly more inhibited by lowering Po₂. At intermediate Po₂ (between 100 and 20 mmHg) the diabetic muscle gave slightly higher force. At zero Po₂ no significant difference could be detected between strips from control and diabetic animals. Oxygen consumption and lactate production in the preparations were determined at a Po₂ of 290 mmHg and related to the volume of smooth muscle. At zero Po₂ lactate formation increased 3- to 4-fold. The metabolic tension cost was lower at zero Po₂ No differences in basal and contraction related metabolic rates could be detected between the two groups under normoxic and anoxic conditions. The maximal activity of lactate dehydrogenase (LDH) determined in tissue sampIes was about 2-fold higher in the diabetic bladder muscle. This increased enzymatic activity could thus not be correlated with any altered metabolic properties of the smooth muscle in the urinary bladder from diabetic rats.     

Analysis of the length response to a force step in smooth muscle from rabbit urinary bladder

Responses to isotonic quick release of AC-stimulated smooth muscle strips from rabbit urinary bladder were analysed. Releases were performed at the peak of contraction and at a preset tension level in the contraction and relaxation phase. In other expts. responses at 37 degrees C and 27 degrees C were compared. The length response always consisted of 3 parts: (1) elastic recoil, (2) rapid length change (isotonic transient), (3) steady length change. Qualitatively, phases (1)-(3) could be distinguished also in responses to isotonic quick stretch. The immediate elastic recoils, phase (1), were described by exponential stress-strain relations. Stiffness was found to be somewhat lower during relaxation than during contraction. No effect of temperature on the elastic recoil was seen. The initial velocity in phase (2) was 2-3 times greater than the velocity 100 ms after release. By means of computer analysis of the length records during phases (2) and (3) two decaying exponential processes with widely different time constants could be separated. The time constant of the faster process was of the order of 15-30 ms at 37 degrees C. It decreased with increasing force steps and with increasing temperature. The amount of shortening associated with this process was correlated with the size of the force step, reaching a maximum of about 1.2% of the muscle length. The shortening velocities in phase (3), measured 100 ms after release, were described by Hill's equation. Vmax in the rising part and at the peak of contraction were 0.7 and 0.6 L/s respectively at 37 degrees C. Lower values were found during relaxation and at 27 degrees C. We suggest that part of the elastic recoil in phase (1) occurs in structures associated with the individual cross-bridges, that phase (2) is dominated by a change in the distribution of conformations of bridges in the attached position and that the shortening rate in phase (3) is determined by the entire cycle of events during turnover of bridges after the muscle has adapted to the new load. Observations on the force response to length steps and on shifts from isometric to afterloaded isotonic contraction and vice versa are consistent with this interpretation.     

Influence of muscle length on the force-velocity relation of K+-contractures in smooth muscle from rabbit urinary bladder

Force-velocity relations of K+-contractures of longitudinal smooth muscle from rabbit urinary bladder were studied by isotonic quick release at 37 degrees C. In order to minimize the influence of parallel elasticity the study was limited to the rising part of the length-tension curve. The force-velocity data fitted well with Hill's equation. The in situ length of the strip at a bladder volume of 10 ml is called L10. This length is 50% of that at which maximum active tension is developed. At L10 Vmax was 0.29 muscle lengths per second and it was estimated to be 0.36 lengths/s at optimum length. Constant b in Hill's equation had a value of 0.052 L10/s and it was unaffected by length changes over the interval 0.69 L10-1.44L10. At L10 a/Po was 0.17. In the interval given above, a/Po decreased with increasing length in proportion to the increase in Po, indicating that a was also length independent. According to Hill's equation [V = b(Po - P)/(P + a)], V should increase in proportion to (Po - P) when the muscle length is increased if a and b are constants. Such a linear relation was found at shorter lengths but at lengths close to or at the length for maximum active tension, V increased more than (Po - P). Two possible explanations were considered; firstly that b/(P + a) increased, and secondly that the load on the contractile element could be less that P due to an influence of the considerable tension in the parallel elastic element at these lengths. The series elastic recoil of the active muscle amounted to 3-4% of the muscle length when released to zero tension.     

Relation between cell length and force production in urinary bladder smooth muscle

Guinea-pig and rabbit urinary bladders were fixed in glutaraldehyde at different volumes. Strips were dissected out, embedded and cut for phase contrast and electron microscopy. Muscle wall thickness decreased with increased bladder volume as did the radial number of muscle cells. Cell length, measured by a morphometric method increased linearly with bladder radius, indicating that no slippage between the muscle cells occurred. Number of cells per mm² cross sectional area increased linearly with bladder radius. Volume-active force relations were obtained by pelvic nerve stimulations of guinea pig bladders filled to different volumes. Maximum pressure was obtained at 0.15 ml bladder volume, and maximum wall tension at a volume of 2.5 ml which corresponds to a cell length of 400 μm and a cell packing density of about 107000×mm^-2. Estimation of the length-active tension curve for the average muscle cell in the guinea-pig bladder indicated a maximum active force of 5.5 μN/cell. Maximum active force per cm² muscle bundle was calculated to be about 59 N. No compensation for extracellular space and nonmuscular tissue within the muscle bundle was made.     

Inhibition of EGFR signaling abrogates smooth muscle proliferation resulting from sustained distension of the urinary bladder

Urinary bladder outlet obstruction results in sustained stretch of the detrusor muscle and can lead to pathological smooth muscle hyperplasia and hypertrophy. The epidermal growth factor receptor (EGFR) is a cognate receptor for mitogens implicated in bladder hyperplasia/hypertrophy. Here, we investigated the potential for modulation of this pathway by pharmacologic targeting with a clinically available EGFR antagonist using an organ culture model of bladder stretch injury as a test system. Urinary bladders from adult female rats were distended in vivo with medium containing the EGFR inhibitor ZD1839 (gefitinib, Iressa). The bladders were excised and incubated in ex vivo organ culture for 4-24 h. EGFR phosphorylation, DNA proliferation, and the extent of apoptosis in the cultured tissues were assessed. To verify that the smooth muscle cells (SMC) are a target of the EGFR inhibitor, primary culture human and rat bladder SMC were subjected to cyclic mechanical stretch in vitro in the presence of ZD1839. Levels of phosphorylated EGFR were significantly increased in the detrusor muscle with 12 h of stretch in the organ cultures. This activation coincided with a subsequent 23-fold increase in DNA synthesis and a 30-fold decrease in apoptosis in the muscle compartment at 24 h. In the presence of ZD1839, DNA synthesis was reduced to basal levels without an increase in the rate of apoptosis under ex vivo conditions. Mechanical stretch of bladder SMC in vitro resulted in a significant increase in DNA synthesis, which was completely abrogated by treatment with ZD1839 but not by AG825, an inhibitor of the related receptor, ErbB2. Our results indicate that the EGFR pathway is a physiologically relevant signaling mechanism in hypertrophic bladder disease resulting from mechanical distension and may be amenable to pharmacologic intervention.     

Effects of neonatal capsaicin treatment and streptozotocin-induced diabetes on urinary bladder function in rats

A significant increase in the size and weight of the urinary bladder was observed 2 weeks after streptozotocin treatment and 2 months after neonatal capsaicin treatment. Both treatments induced a significant increase in the level of [³H]quinuclidinyl benzilate binding to muscarinic cholinergic receptors in the urinary bladder membranes. However, contractile responses of urinary bladder muscle strips to carbachol (0.3–20 μM) were not significantly affected by either treatment. On the other hand, neonatal capsaicin treatment, but not streptozotocin treatment, significantly enhanced contractile responses of bladder strips to electric field stimulation.     

Beta1-adrenoceptor-mediated relaxation with isoprenaline and the role of MaxiK channels in guinea-pig esophageal smooth muscle.

The possible functional coupling between beta1-adrenoceptor and MaxiK channels which results in smooth muscle relaxation was examined in the guinea-pig esophageal muscularis mucosae. Isoprenaline-elicited relaxation of esophageal smooth muscle was confirmed to be mediated through beta1-adrenoceptors as the response was competitively antagonized by a beta1-selective antagonist atenolol with a pA2 value of 7.01. Iberiotoxin (IbTx, 10(-7) M), a selective MaxiK channel inhibitor, substantially diminished the relaxant response to isoprenaline. The extent of the MaxiK channel contribution to the relaxant response was 15-40% of the control response when estimated as the E50%-Emax responses to isoprenaline. The relaxation to isoprenaline was also attenuated by high-KCl (80 mM) to the same degree as the relaxant response generated in the presence of IbTx, and thus the estimated extent of the K+ channel contribution was 10-40%. These findings indicate that beta1-adrenoceptors are substantially coupled with MaxiK channels to produce relaxation of esophageal smooth muscle in the guinea-pig. Although MaxiK channels account for the contribution of K+ channels to the beta1-adrenoceptor-mediated relaxation in this smooth muscle preparation, their contribution seems to be less when compared to the beta2-adrenoceptor-mediated relaxation of tracheal smooth muscle.     

Ultrastructural changes in Leydig cells of streptozotocin-induced diabetic rats.

Hyperglycemia (experimental diabetes) was induced in adult male rats by destruction of the pancreatic beta cells with a single intravenous injection of streptozotocin (STZ). Testes from diabetic, from insulin-treated diabetic, and from sham-injected normal rats were fxed by vascular perfusion. The fine structure of Leydig cells was examined at two, three, and four weeks after the STZ injection in the untreated diabetic animals, and at four weeks in the controls and insulin-treated diabetic rats. A number of morphological changes was observed in Leydig cells of untreated diabetic animals. Most obvious of these was an accumulation of lipid droplets, not normally present in Leydig cells in adults of this species. Smooth endoplasmic reticulum (SER) was markedly reduced in Leydig cells of the hyperglycemic rats. Several types of intracellular bodies were seen exclusively in Leydig cells of the untreated diabetic animals. Many resembled secondary lysosomes or dense bodies, while others appeared to be autophagic vacuoles. In addition, a small, granule-containing lamellar structure was seen either within a typical dense body or free in the cytoplasm. Myelin-like structures were commonly observed within the cytoplasm of the Leydig cell or within mitochondria. The appearance of the mitochondria in diabetic rats was otherwise normal. The extracellular spaces surrounding Leydig cells from untreated hyperglycemic rats also contained large accumulations of myelin-like material. These structural changes appear to be direct consequences of the diabetic state of the animals, since the ultrastructure of insulin-treated diabetic rats did not differ from that of the controls. These findings may reflect an alteration or breakdown of Leydig cell components normally involved in the synthesis of androgen, and correlate with previous reports of lowered circulating levels of testosterone in diabetic rats.     

Changes in length and volume of smooth muscle cells of the hypertrophied rat urinary bladder

Rat urinary bladders were denervated by bilateral excision of the pelvic ganglion. After 10 days the hypertrophied bladders were removed, filled with 0.75 ml Krebs solution, fixated and embedded for electron microscopy. Normal bladders filled with the same volume were used as controls. Tangential and transverse sections of longitudinal muscle bundles were cut both for phase contrast and electron microscopy. The profiles of cross-sectioned cells from control bladders had regular, often almost circular contours, and were rather uniform in size. The cell contours from the denervated bladders were irregular with wrinkled surfaces and the cells varied considerably in size. In tangential sections the cells from denervated bladders had corrugated surfaces in contrast to the control cells. Mean length and volume of the muscle cells were determined morphometrically. Whereas cell volume was larger in denervated (3 800 μm³) than in control bladders (2 200 μ³), cell length was less (226 versus 335 μm). The finding of shorter cells in denervated bladders favour the opinion, brought forward previously (Ekström & Uvelius 1981), that the rightward shift of the active length-tension curve in denervated bladders is due to an increase in the number of muscle cells that are coupled in series around the bladder circumference.