Local infusion of the nitric oxide donor Sin-1 after angioplasty. Effects on intimal hyperplasia in porcine coronary arteries.

PURPOSE: To investigate the development of intimal hyperplasia in response to percutaneous transluminal coronary angioplasty (PTCA) followed by local delivery of the nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1). MATERIAL AND METHODS: Overdilation PTCA was performed in coronary arteries in 20 healthy pigs. One of the dilated segments was additionally treated with local delivery of SIN-1 for 10 min. Segments distal to the treated part of the arteries served as controls. Arteries were radiographically depicted and analyzed after 1 and 8 weeks for actin, myosin and intermediate filaments (IF), nitric oxide synthetase (NOS) and histological evaluation. RESULTS: Segments treated with PTCA+SIN-1 showed a significantly (p=0.03) larger luminal diameter compared with PTCA only treated segments. The luminal loss after SIN-1 was not significant compared with the diameter prior to treatment. Endothelial NOS content was significantly lower in the PTCA+SIN-1 group compared with the PTCA group after 1 (p=0.03) and 8 weeks (p=0.013). IF/actin ratio after 1 week was significantly increased in PTCA-treated segments compared with untreated controls (p=0.004), and compared with PTCA+SIN-1-treated segments (p=0.004). CONCLUSION: PTCA-induced intimal hyperplasia was potently inhibited by local delivery of the NO donor SIN-1. Momentary events at the time of injury play a significant role in the development of intimal hyperplasia and long-lasting down-regulation of the endothelial NOS expression after SIN-1 exposure is suggested. The IF/actin ratio can be useful as an early marker of intimal hyperplasia.

Regeneration of detrusor muscle after subtotal cystectomy in the rat: effects on contractile proteins and bladder mechanics.

The aim of the present study was to determine to what extent adult rats can produce new contracting bladder muscle and to see if such newly formed bladder tissue possesses characteristic mechanical properties or whether the ability to recover mechanically is so pronounced that the prehistory of the bladder is unimportant. Subtotal cystectomy was performed in adult female rats, leading to a pronounced decrease in total bladder weight. At 10 weeks, bladder weight had normalized. The histological appearance of such bladders was similar to that of the controls. Active and passive length-tension relations for the detrusor muscle were determined in controls and up to 10 weeks after surgery. Immediately after surgery active and passive forces showed a leftward shift and maximum active force decreased markedly. With time the length-tension curves shifted back to normal, but a decreased active force still remained at 10 weeks. Detrusor actin concentration and detrusor myosin/actin ratio were unaffected by the subtotal cystectomy. Intermediate filament protein/actin ratio showed a significant but transitory increase. We conclude that there is a remarkable recovery of detrusor muscle function after subtotal cystectomy, leading to a normalization of optimum length for active force and a net synthesis of contractile and cytoskeletal proteins. The ability to produce active force does, however, not fully recover.

Increased expression of non-muscle myosin heavy chain-B in connective tissue cells of hypertrophic rat urinary bladder.

Expression of the non-muscle myosin heavy chain-B (NM-MHC-B, also denoted as the embryonic smooth muscle myosin heavy chain, SMemb) was examined in rat urinary bladder during growth in response to a partial urinary outflow obstruction. Following obstruction, the weight of the urinary bladder increased more than five-fold within 10 days. Immunohistochemistry with a polyclonal antiserum against the C-terminal sequence of NM-MHC-B revealed very few NM-MHC-B immunoreactive cells in the control urinary bladders. In hypertrophic bladders, the number of NM-MHC-B immunoreactive cells markedly increased. The majority of such cells were found in the interstitium surrounding smooth muscle bundles and also in the subserosal and submucosal layers. Western blot analysis showed that the NM-MHC-B expression was transient; the content of NM-MHC-B immunoreactive material had doubled 10 days after obstruction and then declined towards the control level after 6 weeks. Immunohistochemistry revealed co-localization of NM-MHC-B and vimentin within the same cells. NM-MHC-B did not co-localize with smooth muscle actin, suggesting that the source of NM-MHC-B is not a de-differentiated smooth muscle cell or myofibroblast but a non-muscle cell possibly reacting to tissue distension or stress. The NM-MHC-B-positive cells could have a role in the production of extracellular matrix and growth factors or be involved in modulation of spontaneous contractile activity.

Up-regulation of bradykinin response in rat and human bladder smooth muscle.

PURPOSE: Responses to bradykinin were investigated in vitro in isolated control and hypertrophic smooth muscle strips from rat bladder. MATERIALS AND METHODS: Bladder hypertrophy was induced by a 10-day period of partial urinary outflow obstruction. In addition, human bladder strips were also investigated. RESULTS: Bradykinin (1 nM. to 1 microM.) caused contractions in all tissues studied. In the freshly isolated rat bladder preparations bradykinin induced contractions were similar and of small amplitude in control and hypertrophic tissues. After a 4-hour equilibratory period contractile responses to bradykinin and the B1 specific bradykinin receptor agonist desArg9 bradykinin were slightly increased in the controls but there was approximately a 6-fold increase in the hypertrophic muscle strips. After 4 hours of equilibration the human bladder strips showed a smaller but still significant increase in contractile response to bradykinin. Indomethacin, a cyclooxygenase inhibitor, almost abolished the increased response, which suggests that prostanoids are involved in the up-regulated response. The protein synthesis inhibitor cycloheximide inhibited up-regulation by approximately 50% in hypertrophic and control muscle strips from rat bladder and normal muscle from human bladder. CONCLUSIONS: These results demonstrate that bradykinin receptor responses are present in rat and human detrusor muscle and they can be up-regulated in vitro. Experiments on hypertrophic rat bladder revealed that this process is enhanced in hypertrophy.

Enteric neuronal plasticity and a reduced number of interstitial cells of Cajal in hypertrophic rat ileum.

BACKGROUND: Partial obstruction of the ileum causes a notable hypertrophy of smooth muscle cells and enteric neurones in the proximally located intestine. AIMS: To study the expression of neuromessengers in the hypertrophic ileum of rat as little is known about neuromessenger plasticity under these conditions. To investigate the presence of interstitial cells of Cajal (ICC) in hypertrophic ileum. METHODS: Ileal hypertrophy was induced by circumferential application of a strip of plastic film for 18-24 days. Immunocytochemistry, in situ hybridisation, nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry, and ethidium bromide staining were used to investigate the number of enteric neurones expressing neuropeptides and nitric oxide synthase, and the frequency of ICC. RESULTS: In the hypertrophic ileum several neuronal populations showed changes in their expression of neuromessengers. Myenteric neurones expressing vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide, and galanin were notably increased in number. In submucous ganglia the number of VIP immunoreactive neurones decreased while those expressing VIP mRNA increased. NADPH diaphorase positive submucous neurones increased dramatically while the number of neuronal type nitric oxide synthase expressing ones was unchanged. The number of ICC decreased notably in hypertrophic ileum. CONCLUSION: Enteric neurones change their levels of expression of neuromessengers in hypertrophic ileum. ICC are also affected. The changes are presumably part of an adaptive response to the increased work load.

Mechanical alterations in smooth muscle from mice lacking desmin.

Mice with a null mutation introduced in the desmin gene were used to study the mechanical role of intermediate filaments in smooth muscle cells. Vas deferens (VD), urinary bladder (UB) and portal vein (PV) preparations were obtained from adult animals lacking desmin (Des -/-) and from age- and weight-matched wild-type animals (Des +/+). Active force per cross-sectional area was decreased in the smooth muscle of the Des -/- compared with Des +/+ mice (VD to 42%; UB to 34%). Quantitative gel electrophoresis suggests a marginally lower cellular content of myosin, but the organization of the contractile apparatus appeared unchanged by electron microscopy. A similar reduction in stress was measured in Des -/- skinned fibres showing that altered activation mechanisms were not involved. The results indicate that the reduced active force is caused by low intrinsic force generation of the contractile filaments or subtle modifications in the coupling between the contractile elements and the cytoskeleton. The relationship between length and passive stress was less steep in the Des -/- samples and a second length force curve after maximal extension revealed a loss of passive stress. The maximal shortening velocity was reduced in Des -/- skinned VD and UB preparations by approximately 25-40%. This was associated with an increased relative content of the basic essential myosin light chain, suggesting that alterations in the contractile system towards a slower, more economical muscle had occurred. PV preparations showed no difference in mechanical properties in Des +/+ and Des -/- animals, a result that was consistent with the predominance of vimentin instead of desmin in this vascular tissue. In conclusion, the results show that, although intermediate filaments in smooth muscle are not required for force generation or maintenance of passive tension, they have a role in cellular transmission of both active and passive force.

Old age does not affect shortening velocity or content of contractile and cytoskeletal proteins in the rat detrusor smooth muscle.

The influence of old age on mechanical properties of the urinary bladder was investigated using smooth muscle strips from urinary bladders of control (14-16 weeks) and old-age (104 weeks) female Sprague-Dawley rats. Bladder weight of the aged rats had increased by about 30%. The maximal shortening velocity and stiffness in skinned activated urinary bladder fibers from old animals were unchanged compared to controls. The relative content of intermediate filament proteins to actin and the relative content of myosin to actin was unchanged. The concentration of myosin was unchanged (about 6.5 microg/mg wet weight). The results suggest that old age is not associated with pronounced changes in the cellular contractile and cytoskeletal proteins or in the mechanical properties of the contractile machinery. The age-related changes in mechanical properties previously reported for intact smooth muscle from urinary bladder are most likely due to alterations in the activation systems.

Contraction kinetics and myosin isoform composition in smooth muscle from hypertrophied rat urinary bladder.

Mechanical properties and isoform composition of myosin heavy and light chains were studied in hypertrophying rat urinary bladders. Growth of the bladder was induced by partial ligation of the urethra. Preparations were obtained after 10 days. In maximally activated skinned preparations from the hypertrophying tissue, the maximal shortening velocity and the rate of force development following photolytic release of ATP were reduced by about 20 and 25%, respectively. Stiffness was unchanged. The relative content of the basic isoform of the essential 17 kDa myosin light chain was doubled in the hypertrophied tissue. The expression of myosin heavy chain with a 7 amino acid insert at the 25K/50K region was determined using a peptide-derived antibody against the insert sequence. The relative amount of heavy chain with insert was decreased to 50% in the hypertrophic tissue. The kinetics of the cross-bridge turn-over in the newly formed myosin in the hypertrophic smooth muscle is reduced, which might be related to altered expression of myosin heavy or light chain isoforms.

Effects of purinoceptor agonists on smooth muscle from hypertrophied rat urinary bladder.

Tension responses induced by the purinoceptor agonists ATP and the stable ATP analogue alpha, beta-methylene ATP were investigated in isolated muscle strips from normal and hypertrophic urinary bladders from the rat. Hypertrophy was induced by a partial ligation of the urethra giving an increase in mean bladder weight from 65 mg to 300 mg. Activation with ATP and alpha, beta-methylene ATP caused phasic, concentration-dependent, contractions. The sensitivity to ATP was about 100-fold lower than that for alpha, beta-methylene ATP. The force of the contractions induced by the purinoceptor agonists was significantly lower in the hypertrophied bladder compared to the controls. The kinetics of the ATP-induced responses was studied by photolytic release of ATP from caged-ATP in intact fibre bundles. The rate of contraction following photolytic release of ATP was slower, and the force amplitude lower, in the hypertrophic preparations compared to the controls. The results suggest changes in the purinoceptor function or in the responses of the contractile system to transient increases in intracellular Ca2+ in the hypertrophic bladder.