Absence Of Stretch Research Articles

The effect of fiber-matrix interaction on the kinking instability arising in the torsion of stretched fibrous biofilaments

The response of fibrous soft tissues undergoing torsional deformations is a topic of current interest. Such deformations are common in ligaments and tendons and are also of particular interest in cardiac mechanics. The problem of torsion superimposed on extension of incompressible hyperelastic solid circular cylinders is a classic problem of nonlinear elasticity that has been considered by many authors in the context of rubber elasticity particularly for isotropic materials. A striking feature of such problems is the instability that arises with sufficiently large twist where a kink and then a knot suddenly appears. An energy approach to examining this instability when the extension and twist are prescribed was described by Gent and Hua (2004) and illustrated there for a neo-Hookean isotropic elastic material. The theoretical results were compared with experimental observations on natural rubber rods. Murphy (2015) has shown that the approach of Gent and Hua (2004) for isotropic materials can be simplified when the rods are assumed to be thin and this theory was applied to transversely isotropic materials by Horgan and Murphy (2016). In contrast with the case for isotropic materials, it was shown there that the kinking instability occurs even in the absence of stretch, i.e., for the case of pure torsion. Here we are concerned with the implications of this simplified thin rod instability theory for fiber-reinforced transversely isotropic materials that reflect fiber-matrix interaction. It is again shown that the kinking instability occurs even in the absence of stretch, i.e., for the case of pure torsion. The results are illustrated for a specific strain-energy density function that models fiber-matrix interaction. It is shown that the critical twist at which kinking occurs decreases as a measure of fiber-matrix interaction is increased so that the fiber-matrix interaction has a destabilizing effect. The results are illustrated using experimental data of other authors for skeletal muscles and for porcine brain white matter tissue.

As the endothelial cell reorients, its tensile forces stabilize

When adherent cells are subjected to uniaxial sinusoidal stretch at frequencies close to physiological, their body and their contractile stress fibers realign nearly perpendicularly to the stretch axis. A common explanation for this phenomenon is that stress fibers reorient along the direction where they are unaffected by the applied cyclic stretch and thus can maintain optimal (homeostatic) tensile force. The ability of cells to achieve tensional homeostasis in response to external disturbances is important for normal physiological functions of cells and tissues and it provides protection against diseases, including cancer and atherosclerosis. However, quantitative experimental data that support the idea that stretch-induced reorientation is associated with tensional homeostasis are lacking. We observed previously that in response to uniaxial cyclic stretch of 10% strain amplitudes, traction forces of single endothelial cells reorient in the direction perpendicular to the stretch axis. Here we carried out a secondary analysis of those data to investigate whether this reorientation of traction forces is associated with tensional homeostasis. Our analysis showed that stretch-induced reorientation of traction forces was accompanied by attenuation of temporal variability of the traction field to the level that was observed in the absence of stretch. These findings represent a quantitative experimental evidence that stretch-induced reorientation of cellular traction forces is associated with the cell’s tendency to achieve tensional homeostasis.

Kinking Instability in the Torsion of Stretched Anisotropic Elastomeric Filaments

The problem of torsion superimposed on extension of incompressible nonlinearly elastic solid circular cylinders is a classic problem of nonlinear elasticity that has been considered by many authors in the context of rubber elasticity particularly for isotropic materials. An interesting aspect of such problems is the instability that arises with sufficiently large twist where a kink or knot suddenly appears. An energy approach to examining this instability when the extension and twist are prescribed was given by Gent and Hua (Int. J. Nonlinear Mech. 39:483–489, 2004) and illustrated there for a neo-Hookean isotropic elastic material. The theoretical results were compared with experimental observations on natural rubber rods. In a recent paper Murphy (Int. J. Nonlinear Mech. 68:96–100, 2015) has shown that the approach in Gent and Hua for isotropic materials can be simplified when the rods are assumed to be thin. Motivated by potential applications to the biomechanics of soft tissues, we are concerned here with the implications of this simplified thin rod instability theory for transversely isotropic materials. The results are illustrated on using three specific strain-energy density functions. These models are quadratic in the anisotropic invariants, linear in the isotropic strain invariants and are consistent with the linear theory. In contrast with the case for isotropic materials, it is shown that the kinking instability arises even in the absence of stretch that is in the case of pure torsion. The results are illustrated on using elastic moduli for muscles based on experimental findings of other authors.

UROTHELIAL EPAC: NOVEL MEDIATOR IN THE REGULATION OF BLADDER CAPACITY

Bladder filling elicits exocytosis and vesicle fusion increasing the apical surface area of epithelial cells (measured by membrane capacitance). One stimulus governing apical exocytosis is cAMP. Although initially the effects of cAMP were solely attributed to activation of protein kinase A (PKA), recent findings uncovered a novel c‐AMP sensor, EPAC (exchange protein directly activated by cAMP). This protein plays a critical role in calcium handling and ion transport, vesicle trafficking and secretion, and barrier function in various organs. Here we investigated the expression and role of EPAC in signaling apical membrane dynamics.Bladders from female Sprague Dawley rats and New Zealand white rabbits were used for western blot and immunohistochemistry or for culturing urothelial cells for fura‐2 calcium imaging and ATP release. Transepithelial capacitance was measured by placing bladder mucosa in modified Ussing chambers and increasing hydrostatic pressure to mimic bladder filling (in the presence or the absence of EPAC agonist‐ 8‐pCPT, or antagonist‐ ESI‐09).EPAC was expressed in the urothelium, however the agonist did not alter Ca2+ nor ATP release in cultured cells. Nevertheless, the EPAC agonist (used in rabbit UT) significantly increased transepithelial capacitance (30% vs 7% in control) in the absence of stretch; stretch‐induced capacitance was reduced by the EPAC antagonist.Our results indicate that EPAC plays an important role in augmenting bladder filling induced increases in urothelial surface area. The lack of ATP involvement suggests that EPAC mediated pathways could lead to increasing bladder capacity without affecting underlying bladder nerves.

TNF-α provokes electrical abnormalities in rat atrial myocardium via a NO-dependent mechanism

Stretch-induced depolarizations of cardiomyocytes, which are related to activity of mechano-gated cation channels (MGCs), can lead to serious arrhythmias. However, signaling pathways leading to activation of mechano-gated channels by stretch remain almost unexplored. Using standard sharp microelectrodes, the present study addresses the hypothesis that tumor necrosis factor-alpha (TNF-α) modulates stretch-induced electrophysiological abnormalities in rat atrial myocardium by a mechanism involving nitric oxide (NO)-dependent pathways. TNF-α (50 ng/ml) produced a marked prolongation of action potential, subsequently transforming into humplike depolarizations and, finally, leading to occurrence of arrhythmias. These effects developed slowly during 25 min of TNF-α application. Similar electrical effects were induced by stretching the preparations. A blocker of MGCs, Gd(3+) (40 μM), completely abolished action potential (AP) prolongations and electrical abnormalities caused by TNF-α or stretch. Further, a donor of exogenous NO, S-nitroso-N-acetylpenicillamine SNAP (300 μM), evoked the same electrical abnormalities as TNF-α and tissue stretch. Both TNF-α and stretch failed to produce their typical effects after pretreatment of the preparations with the NO-synthase inhibitor L-N(G)-nitroarginine methyl ester (L-NAME) (100 μM). Thus, the present study shows (i) that TNF-α and the NO-donor SNAP evoke MGC-mediated electrical abnormalities in rat atrial myocardium in the absence of stretch that is very similar to stretch-evoked electrical events and (ii) that the TNF-α-induced electrical abnormalities are mediated by NO synthase. In conclusion, our data suggest that NO is an endogenous modulator of MGCs and mediates proarrhythmic effects of TNF-α in mammalian organism.

Stretch induced endothelin-1 secretion by adult rat astrocytes involves calcium influx via stretch-activated ion channels (SACs)

The expression of endothelins (ETs) and ET-receptors is often upregulated in brain pathology. ET-1, a potent vasoconstrictor, also inhibits the expression of astrocyte glutamate transporters and is mitogenic for astrocytes, glioma cells, neurons, and brain capillary endothelia. We have previously shown that mechanical stress stimulates ET-1 production by adult rat astrocytes. We now show in adult astrocytes that ET-1 production is driven by calcium influx through stretch-activated ion channels (SACs) and the ET-1 production correlates with cell proliferation. Mechanical stimulation using biaxial stretch (<20%) of a rubber substrate increased ET-1 secretion, and 4 μM GsMTx-4 (a specific inhibitor of SACs) inhibited secretion by 30%. GsMTx-4 did not alter basal ET-1 levels in the absence of stretch. Decreasing the calcium influx by lowering extracellular calcium also inhibited stretch-induced ET-1 secretion without effecting ET-1 secretion in unstretched controls. Furthermore, inhibiting SACs with the less specific inhibitor streptomycin also inhibited stretch-induced ET-1 secretion. The data can be explained with a simple model in which ET-1 secretion depends on an internal Ca 2+ threshold. This coupling of mechanical stress to the astrocyte endothelin system through SACs has treatment implications, since all pathology deforms the surrounding parenchyma.

Rab11a-dependent exocytosis of discoidal/fusiform vesicles in bladder umbrella cells

The discoidal/fusiform vesicles (DFV) of bladder umbrella cells undergo regulated exocytosis in response to stretch, but little is known about their biogenesis or the molecular machinery that modulates this process. We observed that Rab11a was expressed in umbrella cells (but not Rab11b or Rab25) and was associated with DFV. Using adenovirus-mediated delivery we transduced umbrella cells in situ with either dominant active (DA) or dominant negative (DN) mutants of Rab11a. DA-Rab11a stimulated an increase in apical surface area in the absence of stretch, whereas DN-Rab11a inhibited stretch-induced changes. Endocytosed fluid and membrane markers had little access to Rab11a-positive DFV, but virally expressed human growth hormone (hGH), a secretory protein, was packaged into DFV. Whereas expression of DA-Rab11a stimulated release of hGH into the bladder lumen, expression of DN-Rab11a had the opposite effect. Our results indicate that DFV may be biosynthetic in nature and that their exocytosis depends on the activity of the Rab11a GTPase.

Stretch-induced regulation of angiotensinogen gene expression in cardiac myocytes and fibroblasts: Opposing roles of JNK1/2 and p38α MAP kinases

The cardiac renin–angiotensin system (RAS) has been implicated in mediating myocyte hypertrophy, remodeling, and fibroblast proliferation in the hemodynamically overloaded heart. However, the intracellular signaling mechanisms responsible for regulation of angiotensinogen (Ao), a substrate of the RAS system, are largely unknown. Here we report the identification of JNK1/2 as a negative, and p38α as a major positive regulator of Ao gene expression. Isolated neonatal rat ventricular myocytes (NRVM) and fibroblasts (NRFB) plated on deformable membranes coated with collagen IV, were exposed to 20% equiaxial static-stretch (0–24 h). Mechanical stretch initially depressed Ao gene expression (4 h), whereas after 8 h, Ao gene expression increased in a time-dependent manner. Blockade of JNK1/2 with SP600125 increased basal Ao gene expression in NRVM (10.52 ± 1.98 fold, P < 0.001) and NRFB (13.32 ± 2.07 fold, P < 0.001). Adenovirus-mediated expression of wild-type JNK1 significantly inhibited, whereas expression of dominant-negative JNK1 and JNK2 increased basal and stretch-mediated (24 h) Ao gene expression, showing both JNK1 and JNK2 to be negative regulators of Ao gene expression in NRVM and NRFB. Blockade of p38α/β by SB202190 or p38α by SB203580 significantly inhibited stretch-induced (24 h) Ao gene expression, whereas expression of wild-type p38α increased stretch-induced Ao gene expression in both NRVM (8.41 ± 1.50 fold, P < 0.001) and NRFB (3.39 ± 0.74 fold, P < 0.001). Conversely, expression of dominant-negative p38α significantly inhibited stretch response. Moreover, expression of constitutively active MKK6b (E) significantly stimulated Ao gene expression in the absence of stretch, indicating that p38 activation alone is sufficient to induce Ao gene expression. Taken together p38α was demonstrated to be a positive regulator, whereas JNK1/2 was found to be a negative regulator of Ao gene expression. Prolonged stretch diminished JNK1/2 activation, which was accompanied by a reciprocal increase in p38 activation and Ao gene expression. This suggests that a balance in JNK1/2 and p38α activation determines the level of Ao gene expression in myocardial cells.

Mechanical deformation of ventricular myocytes modulates both TRPC6 and Kir2.3 channels

Cardiomyocytes respond to mechanical stretch with an increase [Ca2+]i. Here, we analyzed which ion channels could mediate this effect. Murine ventricular myocytes were attached to a glass coverslip and a cell-attached glass stylus sheared the upper cell part versus the attached cell bottom. At negative clamp potentials, stretch induced inward currents that increased with the extent of stretch and reversed within 2 min after relaxation from stretch. Stretch activated a nearly voltage-independent GsMTx-4-sensitive non-selective cation conductance Gns, antibodies against TRPC6 prevented Gns activation. In addition, stretch deactivated a Cs+-sensitive inwardly rectifying potassium conductance GK1, antibodies against Kir2.3 inhibited this effect. Immunolabeling localized TRPC6 and Kir2.3 in T-tubular membranes, and stretch-induced changes in membrane currents were absent in cells whose T-tubules had been removed. In absence of stretch, we could activate Gns and deactivate GK1 by 1-oleoyl-2-acetyl-sn-glycerol (OAG) and other amphipaths. We interpret that the function of TRPC6 and Kir2.3 channels is controlled by both tension and curvature of the surrounding lipid bilayer that are changed by incorporation of amphipaths. Stretch-activation of TRPC6 channels may increase Ca2+ influx directly and indirectly, by membrane depolarization (activation of voltage-gated Ca2+ channels) and by elevated [Na+]i (augmented Na+,Ca2+-exchange).

Tissue stretch decreases soluble TGF‐β1 and type‐1 procollagen in mouse subcutaneous connective tissue: Evidence from ex vivo and in vivo models

Transforming growth factor beta 1 (TGF-beta1) plays a key role in connective tissue remodeling, scarring, and fibrosis. The effects of mechanical forces on TGF-beta1 and collagen deposition are not well understood. We tested the hypothesis that brief (10 min) static tissue stretch attenuates TGF-beta1-mediated new collagen deposition in response to injury. We used two different models: (1) an ex vivo model in which excised mouse subcutaneous tissue (N = 44 animals) was kept in organ culture for 4 days and either stretched (20% strain for 10 min 1 day after excision) or not stretched; culture media was assayed by ELISA for TGF-beta1; (2) an in vivo model in which mice (N = 22 animals) underwent unilateral subcutaneous microsurgical injury on the back, then were randomized to stretch (20-30% strain for 10 min twice a day for 7 days) or no stretch; subcutaneous tissues of the back were immunohistochemically stained for Type-1 procollagen. In the ex vivo model, TGF-beta1 protein was lower in stretched versus non-stretched tissue (repeated measures ANOVA, P < 0.01). In the in vivo model, microinjury resulted in a significant increase in Type-1 procollagen in the absence of stretch (P < 0.001), but not in the presence of stretch (P = 0.21). Thus, brief tissue stretch attenuated the increase in both soluble TGF-beta1 (ex vivo) and Type-1 procollagen (in vivo) following tissue injury. These results have potential relevance to the mechanisms of treatments applying brief mechanical stretch to tissues (e.g., physical therapy, respiratory therapy, mechanical ventilation, massage, yoga, acupuncture).

Stretch‐induced TRPC4 downregulation in RASM cells may be due to changes in intracellular calcium

Our previous work showed that cyclic stretch significantly decreases TRPC4 expression in rat aortic smooth muscle (RASM) cells and is prevented by CPA. TRPC4 downregulation may be a compensatory mechanism to reduce increased [Ca2+]SR resulting from entry through stretch-activated channels. However, we do not yet know the role of cytosolic Ca2+. We hypothesized that TRPC4 downregulation may also serve to reduce stretch-induced increases in cytosolic [Ca2+] and outlined the following aims: (1) demonstrate that increased cytosolic [Ca2+] in the absence of stretch can decrease TRPC4 protein expression, and (2) establish whether blocking Ca2+ entry into the cytosol can prevent stretch-induced TRPC4 downregulation. RASM cells were subjected to cyclic stretch for 6h using the Flexercell Strain Unit FX-4000. Control cells were not stretched. To address Aim (1), the [Ca2+] of the cell culture medium was increased to 10 mM in both stretch and non-stretch conditions, and TRPC4 expression was measured by Western blot. 10 mM Ca2+ decreased TRPC4 protein expression in the absence of stretch (p<0.05). However, high Ca2+ conditions in the presence of stretch did not show any additional downregulation. To address Aim (2), gadolinium was added before mechanical strain to block stretch-activated channels. Nifedipine was used to assess the contribution of L-type channels. Addition of either drug in the absence of stretch increased TRPC4 expression. However, stretch-induced downregulation was still present within each drug condition. These results suggest that TRPC4 proteins may play an important role in stabilizing Ca2+ levels in RASM cells during mechanical or pharmacological changes. Support: COBRE P20 RR18766 “Mentoring in Cardiovascular Biology”

Cyclic stretch-induced reorganization of the cytoskeleton and its role in enhanced gene transfer

Cyclic stretch is known to alter a number of cellular and subcellular processes, including those involved in nonviral gene delivery. We have previously shown that moderate equibiaxial cyclic stretch (10% change in basement membrane area, 0.5 Hz, 50% duty cycle) of human pulmonary A549 cells enhances gene transfer and expression of reporter plasmid DNA in vitro, and that this phenomena may be due to alterations in cytoplasmic trafficking. Although the path by which plasmid DNA travels through the cytoplasm toward the nucleus is not well understood, the cytoskeleton and the constituents of the cytoplasm are known to significantly hinder macromolecular diffusion. Using biochemical techniques and immunofluorescence microscopy, we show that both the microfilament and microtubule networks are significantly reorganized by equibiaxial cyclic stretch. Prevention of this reorganization through the use of cytoskeletal stabilizing compounds mitigates the stretch-induced increase in gene expression, however, depolymerization in the absence of stretch is not sufficient to increase gene expression. These results suggest that cytoskeletal reorganization plays an important role in stretch-induced gene transfer and expression.

TIPping Toward Muscle or Fat

Stretch plays an important role in differentiation in developing embryos, and in adult tissues, stretch triggers various cellular responses. Jakkaraju et al. identified a new protein family of chromatin remodeling enzymes called TIP, for tension-induced or -inhibited proteins. The abundance of TIP-1 was increased [detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting] when undifferentiated mouse lung mesenchymal cells were subjected to stretch and persisted when these cells differentiated into smooth muscle myoblasts in response to stretch. TIP-3 was present, but unaffected by stretch, in the undifferentiated progenitor cells and was absent from the smooth muscle myoblasts. Although both TIP-1 and TIP-3 were present in the cytoplasm and nucleus of the progenitor cells, stretch promoted the accumulation of TIP-1 in the nuclei of smooth muscle myoblasts. Overexpression of TIP-1 or TIP-3 stimulated myogenic or adipogenic gene expression, respectively, in lung progenitor cells. Inhibition of TIP-1 using RNA interference converted smooth muscle myoblasts into lipoblasts (an adipose-related cell type). Chromatin immunoprecipitation (ChIP) experiments indicated that TIP-1 was recruited to the serum response factor (SRF) promoter (a gene involved in myogenesis) and that TIP-3 was recruited to the PPARγ2 promoter (a gene involved in adipogenesis). Furthermore, the promoters also possessed acetylated histone H4, and in vitro assays with immunopurified TIP-1 or TIP-3 showed that these proteins have histone acetyltransferase (HAT) activity. Recruitment to the promoters required the nuclear receptor binding boxes (NRBs) of TIP-1 and TIP-3, which is consistent with a requirement for interaction with other transcription factors for promoter recruitment (neither TIP-1 nor TIP-3 directly bound DNA based on electrophoretic mobility shift assay). These results suggest that in response to stretch, amounts of TIP-1 are increased, allowing it to serve as a coregulator and HAT for transcriptional activation of myogenic genes. In the absence of stretch, these same progenitor cells possess TIP-3, which allows an adipogenic differentiation pathway to proceed. S. Jakkaraju, X. Zhe, D. Pan, R. Choudhury, L. Schuger, TIPs are tension-responsive proteins involved in myogenic versus adipogenic differentiation. Dev. Cell 9 , 39-49 (2005). [PubMed]

Angiotensin II (AT1) receptors and NADPH oxidase regulate Cl- current elicited by beta1 integrin stretch in rabbit ventricular myocytes.

Direct stretch of β1 integrin activates an outwardly rectifying, tamoxifen-sensitive Cl− current (Cl− SAC) via focal adhesion kinase (FAK) and/or Src. The characteristics of Cl− SAC resemble those of the volume-sensitive Cl− current, ICl,swell. Because myocyte stretch releases angiotensin II (AngII), which binds AT1 receptors (AT1R) and stimulates FAK and Src in an autocrine-paracrine loop, we tested whether AT1R and their downstream signaling cascade participate in mechanotransduction. Paramagnetic beads coated with mAb for β1-integrin were applied to myocytes and pulled upward with an electromagnet while recording whole-cell anion current. Losartan (5 μM), an AT1R competitive antagonist, blocked Cl− SAC but did not significantly alter the background Cl− current in the absence of integrin stretch. AT1R signaling is mediated largely by H2O2 produced from superoxide generated by sarcolemmal NADPH oxidase. Diphenyleneiodonium (DPI, 60 μM), a potent NADPH oxidase inhibitor, rapidly and completely blocked both Cl− SAC elicited by stretch and the background Cl− current. A structurally unrelated NADPH oxidase inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF, 0.5 and 2 mM), also rapidly and completely blocked Cl− SAC as well as a large fraction of the background Cl− current. With continuing integrin stretch, Cl− SAC recovered upon washout of AEBSF (2 mM). In the absence of stretch, exogenous AngII (5 nM) activated an outwardly rectifying Cl− current that was rapidly and completely blocked by DPI (60 μM). Moreover, exogenous H2O2 (10, 100, and 500 μM), the eventual product of NADPH oxidase activity, also activated Cl− SAC in the absence of stretch, whereas catalase (1,000 U/ml), an H2O2 scavenger, attenuated the response to stretch. Application of H2O2 during NADPH oxidase inhibition by either DPI (60 μM) or AEBSF (0.5 mM) did not fully reactivate Cl− SAC, however. These results suggest that stretch of β1-integrin in cardiac myocytes elicits Cl− SAC by activating AT1R and NADPH oxidase and, thereby, producing reactive oxygen species. In addition, NADPH oxidase may be intimately coupled to the channel responsible for Cl− SAC, providing a second regulatory pathway.

C-Met expression and mechanical activation of satellite cells on cultured muscle fibers.

Single-fiber cultures can be used to model satellite cell activation in vivo. Although technical deficiencies previously prevented study of stretch-induced events, here we describe a method developed to study satellite cell gene expression by in situ hybridization (ISH) using protocol modifications for fiber adhesion and fixation. The hypothesis that mechanical stretching activates satellite cells was tested. Fiber cultures were established from normal flexor digitorum brevis muscles and plated on FlexCell dishes with a layer of Vitrogen. After 2 hr of stretch in the presence of BrdU, satellite cells on fibers attached to Vitrogen were activated above control levels. In the absence of activating treatments or mechanical stretch, ISH studies showed 0-6 c-Met+ satellite cells per fiber. Time course experiments demonstrated stable quiescence in the absence of stretch and significant peaks in activation after 30 min and 2 hr of stretch. Frequency distributions for unstretched fiber cultures showed a significantly greater number of quiescent c-Met+ satellite cells than were activated by stretching, suggesting that typical activation stimuli did not trigger cycling in the entire c-Met+ population of satellite cells. These methods have a strong potential to further dissect the nature of stretch-induced activation and gene expression among characterized populations of individual quiescent and activated satellite cells.

Couple stress based strain gradient theory for elasticity

The deformation behavior of materials in the micron scale has been experimentally shown to be size dependent. In the absence of stretch and dilatation gradients, the size dependence can be explained using classical couple stress theory in which the full curvature tensor is used as deformation measures in addition to the conventional strain measures. In the couple stress theory formulation, only conventional equilibrium relations of forces and moments of forces are used. The couple's association with position is arbitrary. In this paper, an additional equilibrium relation is developed to govern the behavior of the couples. The relation constrained the couple stress tensor to be symmetric, and the symmetric curvature tensor became the only properly conjugated high order strain measures in the theory to have a real contribution to the total strain energy of the system. On the basis of this modification, a linear elastic model for isotropic materials is developed. The torsion of a cylindrical bar and the pure bending of a flat plate of infinite width are analyzed to illustrate the effect of the modification.

Sulfonylurea receptor ligands modulate stretch-induced ANF secretion in rat atrial myocyte culture.

Stretch-induced atrial natriuretic factor (ANF) secretion was studied in cultures of neonate atrial appendage myocytes. Stretch, applied for 40 min by hypotonic swelling, increased the mean area of 44 individually imaged myocytes by 4.8-8.8% (P < 0.0001) at 6 min and by 2.3-6.2% (P < 0.05) at 35 min. Stretch increased immunoreactive ANF release by 42% (P < 0.05) from a baseline of 315 pg/ml. The ATP-sensitive K(+) (K(ATP))-channel blocker tolbutamide (100 micromol/l) increased the stretch-stimulated release to 84% (P < 0.01) over baseline, whereas lower concentrations (1, 10, and 30 micromol/l) had no stimulatory effect. The K(ATP)-channel opener diazoxide (0.1, 1, 10, 30, and 100 micromol/l) inhibited stretch- plus tolbutamide-stimulated ANF release in a concentration-dependent manner, with IC(50) = 2.2 micromol/l, although 100 micromol/l diazoxide did not reduce the increase in mean cell area. The stretch-stimulated K(ATP) current, monitored in 82 whole cell recordings with sulfonylurea receptor (SUR) ligands, was inversely correlated with the stretch-induced ANF release (r(2) = 0.79, P < 0. 0001). In the absence of stretch, the K(ATP) current had no relationship with baseline ANF release, and baseline ANF release was not affected by the K(ATP)-channel modulators. The results show that SUR ligands that open K(ATP) channels inhibit stretch-induced ANF release in atrial myocytes, in correlation with the stretch-activated K(ATP) current. The subcellular site of action of the SUR ligands-plasmalemma or intracellular organelles-remains to be determined.

Muscle shortening induced by tenotomy does not reduce activity levels in rat soleus.

1. A slow postural muscle was tenotomized to determine the role of muscle stretch on chronic recruitment patterns in freely moving animals. 2. Different amounts of muscle shortening were induced in the soleus muscles of ten rats by severing the tendon of insertion (n = 3), the whole Achilles' tendon (n = 4) or the origins and insertions (n = 3). 3. Bipolar wire electrodes were implanted on each muscle to record the electromyographic activity (EMG) under control and tenotomized conditions. The complex interference pattern was continuously analysed to determine the number and amplitude of peak potentials (called turns). The numbers of these 'turns' and their amplitudes were determined during 4 control and at least 5 experimental days. Sham-operated controls and groups matched according to the type of tenotomy were analysed for length changes and pathological changes 5 and 10 days post-tenotomy. 4. The total activity levels in all three tenotomy conditions were not significantly changed when compared with their own control levels. No differences in total activity level were found between the three tenotomized conditions. 5. The normal diurnal patterns of muscle recruitment were preserved during the tenotomized conditions, with the highest levels consistently occurring during the first 3 h of the dark cycle. 6. Tenotomy of the soleus, whether induced by distal (ST), distal and proximal (DT) or Achilles' tenotomy (AT) resulted in muscle shortening (9-26 %). No muscle pathology was found in the ST or AT groups. Degeneration was found in the DT group after 5 days, with further increases at 10 days. 7. These data suggest that the absence of stretch had no discernible influence on the aggregate activity levels in the slow postural soleus muscle. Whether tenotomy caused changes in recruitment within individual step cycles was not evaluated.

Voltage-induced slow activation and deactivation of mechanosensitive channels in Xenopus oocytes.

1. The relationship between stretch and voltage activation of mechanosensitive (MS) channels from Xenopus oocytes was studied in excised patches of membrane using the patch clamp technique. 2. As is characteristic of MS channels to oocytes, stretching the membrane by applying negative pressure to the patch pipette at -50 mV activated the MS channels rapidly. The channels then deactivated rapidly when the stretch was removed. The stretch-activated MS channels entered a main conductance level (45 pS) and one or more subconductance levels in the range of about 75-90% of the main conductance level. 3. In the absence of stretch, a depolarizing step from -50 to +50 mV activated apparent MS channels after long delays of typically 1-20 s (range, 100 ms to 6 min). Upon repolarization, the channels deactivated slowly with a single exponential (mean time constant of 4 s) or double exponential (mean time constants of 0.8 and 3 s) time course. 4. Delayed activation with depolarization and slow deactivation upon repolarization were also observed for apparent MS channels in on-cell patches. 5. The voltage-activated channels were cation selective and had the same selectivity and conductance levels as the stretch activated MS channels. Applying stretch during voltage-induced channel activity did not activate any additional channels, and the same maximal number of channels were typically activated by either stretch or by voltage. These observations suggest that voltage activates the same MS channels that are activated by stretch. 6. The opening of MS channels following steps to +50 mV occurred in an apparently co-operative manner in 70% of the excised patches containing multiple MS channels. 7. In the absence of stretch, the opening frequency and open probability of MS channels increased with depolarization in the examined voltage range of -60 to -20 mV. 8. Applying a brief stretch during the delay to activation at +50 mV activated the MS channels rapidly, which then remained active when the stretch was removed. In contrast, applying a brief stretch during the slow deactivation induced by stepping from +50 to -50 mV abruptly terminated the voltage induced channel activity upon release of the stretch and inhibited subsequent depolarization-induced activity. 9. Depolarizing steps from -50 to +50 mV inhibited any spontaneous channel activity that was present before the depolarizing step. If the potential was stepped back to -50 mV before the channels activated at +50 mV, a delayed activation could occur at -50 mV, followed by normal deactivation, indicating that the depolarizing step initiated activation processes that were initially masked by inhibition. 10. These observations suggest that voltage and stretch can induce different functional gating configurations of MS channels with associated structures, and that these different gating configurations can interconvert.

Expression of connexin-43 and connexin-26 in the rat myometrium during pregnancy and labor is differentially regulated by mechanical and hormonal signals.

We investigated the effects of uterine stretch on the levels of messenger RNA (mRNA) encoding the gap junction proteins connexin-43 (Cx-43) and connexin-26 (Cx-26) as well as the presence of gap junction plaques formed by Cx-43 within the myometrium. In nonpregnant ovariectomized rats, stretch of one uterine horn with a polyvinyl tube induced a significant increase in myometrial Cx-43 mRNA levels, an effect that was blocked by progesterone; no expression of Cx-26 was detected in the presence or absence of stretch. To investigate whether pregnancy and parturition modified the response to stretch, unilaterally pregnant rats underwent either sham operation or placement of a tube in the nongravid uterine horns. On day 20 of pregnancy, expression of Cx-43 mRNA in gravid horns was low, and stretch did not increase this level. Cx-26 mRNA expression was elevated at this time, but only in the gravid horns. Cx-43 mRNA was highly expressed in the myometrium of gravid horns during labor, but Cx-43 expression in sham-operated, nongravid horns remained low. In contrast, nongravid horns stretched with tubes expressed Cx-43 mRNA at levels similar to those in gravid horns. Levels of Cx-26 mRNA in gravid horns fell between days 20 and 23, and this was not altered by stretch. Punctate Cx-43 immunofluorescence (indicative of gap junction formation) also increased in the myometrium after uterine stretch and in gravid horns during labor. Our data demonstrate that differential mechanisms regulate the expression of Cx-43 and Cx-26 in the pregnant myometrium. Cx-43 expression during labor is dependent upon myometrial stretch under conditions of low progesterone. In contrast, Cx-26 expression during late pregnancy, although requiring the presence of the fetal/placental unit, does not require stretch of the myometrium.