Stretch-induced Contraction Research Articles

Androgen-Induced Relaxation of Uterine Myocytes Is Mediated by Blockade of Both Ca(2+) Flux and MLC Phosphorylation.

Uterine quiescence must be maintained until pregnancy reaches term. Premature activation of myometrial contractility leads to preterm labor and delivery. To scrutinize the potential of androgens to relax the myometrium and the mechanism of their action. A pregnancy-derived myometrial smooth muscle cell line (PHM1-41) and myometrial strips prepared from tissues obtained from pregnant women (lean, n = 9; obese, n = 6) undergoing elective cesarean section at term and from nonpregnant C57BL/6 mice (n=5) were each utilized. The contraction of collagen-embedded PHM1-41s and the stretch-induced contraction of human and murine myometrial strips were assessed after incubation with Testosterone (T), dihydrotestosterone (DHT), and T conjugated to BSA. Intracellular calcium ([Ca(2+)]) and phosphorylated myosin light chain concentrations were quantified in PHM1-41s using a Fluo-4 Ca(2+) assay and in-cell Westerns, respectively. University research institute. DHT and T, but not T conjugated to BSA, impaired the contractile function of PHM1-41s and of human and murine myometrial strips. The response was rapid (observed within minutes), was sustainable for up to 48 hours, and was not abolished on knockdown of the androgen receptor. DHT (100 μm) reduced the amplitude of lean strip contraction to 2 ± 2% of the pretreatment value and T (100 μm) to 3.3 ± 1%. These values for obese strips were 15 ± 6.7% and 11 ± 6.7%, respectively. At the same doses, in murine strips, DHT reduced the amplitude to 4.8 ± 3% and T to 4.9 ± 3%. DHT (50 μm) pretreatment reduced the oxytocin-stimulated increase in [Ca(2+)] (P < .0001; n = 6) and phosphorylated myosin light chain (P < .05; n = 5) in PHM1-41s. Lipid-soluble androgens could be developed as tocolytic agents for the treatment of preterm labor.

PLD.12 Sex hormone cocktail drug - a possible therapeutic target for inhibition of preterm contractions?

Maintenance of uterine quiescence is critical for maintenance of pregnancy and prevention of preterm birth. Progesterone and other sex steroids appear to relax the myometrium but the mechanism of action...

Androgens in pregnancy: roles in parturition

BACKGROUNDUnderstanding the physiology of pregnancy enables effective management of pregnancy complications that could otherwise be life threatening for both mother and fetus. A functional uterus (i) retains the fetus in utero during pregnancy without initiating stretch-induced contractions and (ii) is able to dilate the cervix and contract the myometrium at term to deliver the fetus. The onset of labour is associated with successful cervical remodelling and contraction of myometrium, arising from concomitant activation of uterine immune and endocrine systems. A large body of evidence suggests that actions of local steroid hormones may drive changes occurring in the uterine microenvironment at term. Although there have been a number of studies considering the potential role(s) played by progesterone and estrogen at the time of parturition, the bio-availability and effects of androgens during pregnancy have received less scrutiny. The aim of this review is to highlight potential roles of androgens in the biology of pregnancy and parturition.METHODSA review of published literature was performed to address (i) androgen concentrations, including biosynthesis and clearance, in maternal and fetal compartments throughout gestation, (ii) associations of androgen concentrations with adverse pregnancy outcomes, (iii) the role of androgens in the physiology of cervical remodelling and finally (iv) the role of androgens in the physiology of myometrial function including any impact on contractility.RESULTSSome, but not all, androgens increase throughout gestation in maternal circulation. The effects of this increase are not fully understood; however, evidence suggests that increased androgens might regulate key processes during pregnancy and parturition. For example, androgens are believed to be critical for cervical remodelling at term, in particular cervical ripening, via regulation of cervical collagen fibril organization. Additionally, a number of studies highlight potential roles for androgens in myometrial relaxation via non-genomic, AR-independent pathways critical for the pregnancy reaching term. Understanding of the molecular events leading to myometrial relaxation is an important step towards development of novel targeted tocolytic drugs.CONCLUSIONSThe increase in androgen levels throughout gestation is likely to be important for establishment and maintenance of pregnancy and initiation of parturition. Further investigation of the underlying mechanisms of androgen action on cervical remodelling and myometrial contractility is needed. The insights gained may facilitate the development of new therapeutic approaches to manage pregnancy complications such as preterm birth.

Cardiomyocyte-driven gel network for bio mechano-informatic wet robotics

This paper reports on a cellular mechano-informatics network gel robot which was powered by culturing cardiomyocytes in the micro gel structure. Contraction activities propagated through the cardiomyocyte gel network will transmit a spatial mechanical wave as information about the chemical and mechanical responses to environmental changes. The cardiomyocyte gel network robot transmits electrically excited potential and mechanical stretch-induced contractions as information carried on the gel network. The cardiomyocyte gel network robot was fabricated from a mixture of primary cardiomyocytes and collagen gel and molded in a PDMS casting mold, which could produce serial, parallel lattice, or radial pattern networks. Fluorescent calcium imaging showed that the calcium activity of the cardiomyocytes in the gel network was segmented in small domains in the gel network; however, the local contraction that started on one branch of the gel network was propagated to a neighboring branch, and the propagation velocity was increased with increasing concentration of adrenaline. This increase was limited to ~20 mm/s. This proposed mechano-informatics kineticism will provide not only mechano-informatics for cardiomyocyte powered wet robotics but will also help show how cardiac disease occurs in activity propagation systems.

L-type Ca2+ channels, Ca2+-induced Ca2+ release, and BKCa channels in airway stretch-induced contraction

Resistance arteries constrict in response to mechanical stress. This response is myogenic in nature, and reliant on membrane depolarization, activation of L-type Ca2+ channels, Ca2+-induced Ca2+-release and large conductance Ca2+-dependent K+ channels (BKCa). Airway smooth muscle is also affected by mechanical stress: a deep inspiration produces a bronchodilation in healthy individuals, but bronchoconstriction in moderate to severe asthmatics. In this study, our objective was to investigate the regulation of this airway stretch-activated contractile response (Rstretch), and explore its similarities to the vascular myogenic response. Using a pharmacological approach in intact bovine bronchial segments cannulated horizontally in an organ bath, we showed the ability of carbachol (2-carbamoyloxyethyl-trimethyl-azanium), KCl, neurokinin-A, and U46619 (9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z, 13E-dien-1-oic acid) to generate Rstretch in a concentration-dependent manner. Rstretch was significantly reduced by nifedipine, ryanodine, and iberiotoxin, suggesting that it possesses characteristics similar to those of the vascular smooth muscle myogenic response, such as a role for membrane depolarization, L-type Ca2+ channel, ryanodine receptors and BKCa channel activation.This study demonstrates a novel role for the L-type Ca2+ channel in airway smooth muscle and provides new insights into possible mechanisms regulating the deep inspiration-induced bronchoconstriction seen in asthmatics.

Chronic hypoxia differentially modulates age‐dependent responses of ovine middle cerebral arteries to VEGF

To better understand how chronic hypoxia causes vascular remodeling, we hypothesized that hypoxic increases in VEGF alter cerebrovascular contraction through direct actions on both endothelium and arterial smooth muscle. Endothelium intact (E+) and denuded (E−) middle cerebral arteries (MCA) harvested from term fetuses and non‐pregnant adult sheep kept at sea level or 3820m for 110 days were serum starved 24h then cultured 24h with 0, 3, 10, or 30 ng/ml VEGF. All adult MCAs showed decreased K+ induced contractions with VEGF treatment, and E+ MCAs exhibited stronger K+ induced contractions than E− MCAs. VEGF produced a graded biphasic effect, negligible at 30 ng/ml in both E+ and E− normoxic adult MCAs. Fetal arteries exhibited little reactivity to VEGF. VEGF enhanced stretch‐induced contractions in E− normoxic adult MCAs, an effect absent at low concentrations in E+ normoxic and all hypoxic adult MCAs. Stretch‐induced contractions were generally greater in E− than in E+ arteries from both normoxic and hypoxic fetal MCAs. VEGF produced opposite biphasic effects on E+ and E− fetal arteries, of greater magnitude in normoxic than hypoxic arteries. These data support the hypothesis that VEGF can alter cerebrovascular contraction through direct actions on endothelium and arterial smooth muscle, thereby contributing to age‐dependent hypoxic cerebrovascular remodeling. Supported by P01‐HD31226.

Role of Secretory Phospholipase A2 in Rhythmic Contraction of Pulmonary Arteries of Rats With Monocrotaline-Induced Pulmonary Arterial Hypertension

Excessive stretching of the vascular wall in accordance with pulmonary arterial hypertension (PAH) induces a variety of pathogenic cellular events in the pulmonary arteries. We previously reported that indoxam, a selective inhibitor for secretory phospholipase A2 (sPLA2), blocked the stretch-induced contraction of rabbit pulmonary arteries by inhibition of untransformed prostaglandin H2 (PGH2) production. The present study was undertaken to investigate involvement of sPLA2 and untransformed PGH2 in the enhanced contractility of pulmonary arteries of experimental PAH in rats. Among all the known isoforms of sPLA2, sPLA2-X transcript was most significantly augmented in the pulmonary arteries of rats with monocrotaline-induced pulmonary hypertension (MCT-PHR). The pulmonary arteries of MCT-PHR frequently showed two types of spontaneous contraction in response to stretch; 27% showed rhythmic contraction, which was sensitive to indoxam and SC-560 (selective COX-1 inhibitor), but less sensitive to NS-398 (selective COX-2 inhibitor); and 47% showed sustained incremental tension (tonic contraction), which was insensitive to indoxam and SC-560, but sensitive to NS-398 and was attenuated to 45% of the control. Only the rhythmically contracting pulmonary arteries of MCT-PHR produced a substantial amount of untransformed PGH2, which was abolished by indoxam. These results suggest that sPLA2-mediated PGH2 synthesis plays an important role in the rhythmic contraction of pulmonary arteries of MCT-PHR.

Superoxide modulates myogenic contractions of mouse afferent arterioles

Reactive oxygen species are reported to enhance or impair renal autoregulation. Since both superoxide and hydrogen peroxide can be vasoconstrictors, we tested the hypothesis that stretch generates superoxide and/or hydrogen peroxide that mediate the myogenic response. Increasing perfusion pressure of isolated renal afferent arteriole in mouse from 40 to 80 mmHg reduced the diameter by 13.3 ± 1.8 % (p<0.001) and increased reactive oxygen species, indexed by ethidium: dihydroethidium fluorescence, by 7.3 ± 2.1% (p<0.05). Stretch-induced fluorescence was prevented by incubation of vessels with tempol or pegylated superoxide dismutase (SOD) but not with pegylated catalase (CAT), relating it to vascular superoxide. Compared to vehicle, Tempol or SOD both prevented stretch-induced contractions (Tempol: 4.1 ± 1.0 %, p<0.001, and SOD 5.4 ± 0.8 %, p<0.05) whereas contractions persisted with catalase (10.1 ± 1.6 %, NS). Bath addition of tempol had no further effect after SOD but remained fully effective after CAT. Hydrogen peroxide added to vessels with myogenic tone caused constraction only at concentrations of 50 μM (contracted by 8.6 ± 2.9 %) and above. In conclusion, stretch increased afferent arteriolar superoxide which enhanced myogenic contraction of normal mouse perfused renal afferent arterioles.

Integrin‐dependent and ‐independent potentiation of L‐type Calcium Current (Cav1.2) by cell stretch

Stretch-induced contraction of VSM requires Ca influx through L-type Ca channels (Cav1.2). Integrins play a role in this process because alpha5 beta1 and alphaVbeta3 integrin blocking antibodies prevent myogenic constriction. Recent studies in our lab indicate that Cav1.2 current is potentiated by alpha5beta1 integrin activation and requires phosphorylation by PKA and c-Src of Cav1.2 C-terminal residues. To test whether Cav1.2 channels are mechanosensitive and the possible role of integrins in this process, patch clamp methods were used to investigate the properties of either native or heterologously expressed Cav1.2 cannels by stretch of single cells plated onto a flexible substrate. Thin silicone membranes were coated with either fibronectin (FN) or poly-l-lysine (PLL) to assess integrin-dependent and -independent responses, respectively, and stretched using two blunt micropipettes driven in equal and opposite directions by piezoelectric translators. Graded stretch to 130% of resting cell length induced graded increases in Cav1.2 current (up to 63%) in HEK 293 cells expressing the neuronal channel isoform (Cav1.2c). The increase in current was ~2-fold greater for cells adhering to FN than for cells on PLL. On FN, 130% longitudinal stretch of primary VSM cells induced ~50% increases in Cav1.2 current. However, the magnitude of stretch-activated Cav1.2 current was the same on FN or PLL for cells expressing a Cav1.2 construct containing two C-terminal mutations (Y2122F/S1901A) to prevent phosphorylation by PKA and c-Src, or for cells expressing a Cav1.2 construct with the C-terminus truncated. Our results suggest that the Cav1.2 channel can be potentiated by membrane stretch, with one component due to intrinsic mechanosensitivity of the channel and a second component due to signaling through an integrin-dependent process.

Inhibition of Untransformed Prostaglandin H2 Production and Stretch-Induced Contraction of Rabbit Pulmonary Arteries by Indoxam, a Selective Secretory Phospholipase A2 Inhibitor

Involvement of secretory phospholipase A2 (sPLA2) in the stretch-induced production of untransformed prostaglandin H2 (PGH2) in the endothelium of rabbit pulmonary arteries was investigated. The stretch-induced contraction was significantly inhibited by indoxam, a selective inhibitor for sPLA2, and NS-398, a selective inhibitor for cyclooxygenase-2 (COX-2). Indoxam inhibited the RGD-sensitive-integrin–independent production of untransformed PGH2, but did not affect the RGD-sensitive-integrin–dependent production of thromboxane A2 (TXA2). These results suggest that the stretch-induced contraction and untransformed PGH2 production was mediated by sPLA2–COX-2 pathway, making it a new possible target for pharmacological intervention of pulmonary artery contractility.

Integrin-Dependent and -Independent Potentiation of L-type Calcium Current (Cav1.2) by Cell Stretch

Stretch-induced (myogenic) contraction of vascular smooth muscle (VSM) requires calcium influx through L-type calcium channels (Cav1.2). Integrins play a role in this process because alpha5 beta1 and alphaVbeta3 integrin blocking antibodies prevent myogenic constriction. Recent studies in our lab indicate that Cav1.2 current is potentiated by alpha5beta1 integrin activation and requires phosphorylation by PKA and c-Src of Cav1.2 C-terminal residues. To test whether Cav1.2 channels are mechanosensitive and the possible role of integrins in this process, patch clamp methods were used to investigate the properties of either native or heterologously expressed Cav1.2 cannels by stretch of single cells plated onto a flexible substrate. Thin silicone membranes were coated with either fibronectin (FN) or poly-l-lysine (PLL) to assess integrin-dependent and -independent responses, respectively, and stretched using two blunt micropipettes driven in equal and opposite directions by piezoelectric translators. Graded stretch to 130% of resting cell length induced graded increases in Cav1.2 current (up to 63%) in HEK 293 cells expressing the neuronal channel isoform (Cav1.2c). The increase in current was ∼2-fold greater for cells adhering to FN than for cells on PLL. On FN, 130% longitudinal stretch of primary VSM cells induced ∼50% increases in Cav1.2 current. However, the magnitude of stretch-activated Cav1.2 current was the same on FN or PLL for cells expressing a Cav1.2 construct containing two C-terminal mutations (Y2122F/S1901A) to prevent phosphorylation by PKA and c-Src, or for cells expressing a Cav1.2 construct with the C-terminus truncated. Our results suggest that the Cav1.2 channel can be potentiated by membrane stretch, with one component due to intrinsic mechanosensitivity of the channel and a second component due to signaling through an integrin-dependent process.

Interactive Role of Protein Phosphatase 2A and Protein Kinase Cα in the Stretch-Induced Triphosphorylation of Myosin Light Chain in Canine Cerebral Artery

The interactive role of protein kinase C (PKC) isoforms and protein phosphatase 2A (PP2A) in the mechanisms underlying the gradual reduction in stretch-induced contraction through triphosphorylation of 20-kDa myosin light chain (MLC₂₀) was investigated in the canine basilar artery. In the presence of 5 mM tetraethylammonium, stretching at a rate of 1 mm/s from the initial length (L_i) to 1.5 L_i produced a contraction. Maintaining the stretched state for 15 min (15-min stretch) produced triphosphorylation of MLC₂₀at Ser-19, Thr-18 and Thr-9, and a gradual reduction in the contraction, both of which were reversed by Gö6976 (1 µM), an inhibitor of conventional PKC. The 15-min stretch increased PKCα activity whereas it decreased PP2A activity, both of which were blocked by Y-27632, an inhibitor of rho kinase. Okadaic acid (OA; 1 µM), a PP2A inhibitor, also produced triphosphorylation of MLC₂₀ at the same amino acid residues and activated PKCα, which was inhibited by Gö6976. Stretching and OA increased phosphorylation of 17-kDa PKC-potentiated inhibitory phosphoprotein (CPI-17), and this phosphorylation was inhibited by Gö6976. The present results suggest that activation of PKCα mediated by an inhibitor of PP2A is involved in the stretch-induced triphosphorylation of MLC₂₀, and that this triphosphorylation counteracts the stretch-induced contraction.

The stiffness and phase frequency response of taenia coli smooth muscle: Comparison of the step and sinusoidal perturbation analysis

The present study aimed to show that the stiffness and phase frequency responses obtained from sinusoidal and step length changes have similar form in guinea pig taenia coli smooth muscles. Sinusoidal length changes at 20 discrete frequencies in the range of 0.0007–1 Hz or step length changes with a rise time of 60 ms were applied during plateau level of the tetanic contraction (60 mM KCl) and the force responses were recorded. Similar experiments were performed on the passive muscle with 0.1 mg/mL verapamil. The stiffness and phase responses for tetanic contraction and passive muscle were obtained by using Fourier transformed results of force and length. Then the frequency response of the passive muscle was subtracted from that obtained for tetanic contraction. The general profiles of the stiffness and phase responses were similar for the two types of perturbations. The minimum stiffness occurred at 0.0012 ± 0.0005 Hz for sinusoidal perturbation and at 0.0022 ± 0.0009 Hz for step length perturbation. The minimum stiffness value was 3.79 ± 0.59 mN/mm ( n = 10) for sinusoidal perturbation and 4.02 ± 1.18 mN/mm ( n = 10) for step perturbation. The minimum phase angle was −30.2 ± 5.5° for sinusoidal perturbation and −28.2 ± 11.6° for step perturbation. In conclusion, results showed that step length perturbations instead of sinusoidal length perturbations could be used to find the frequency characteristics of smooth muscles. The results also indicate that 0.001–0.002 Hz corresponds to the cycling rate of overall processes for the stretch-induced contraction mechanism in taenia coli smooth muscle contracted with high K, including the depolarization of the plasma membrane, calcium influx, phosphorylation of light chain, and cross-bridge action.

Coupling a change in intraluminal pressure to vascular smooth muscle depolarization: still stretching for an explanation

pressure-induced (myogenic) constriction of arterioles is believed to be a fundamental contributor to the local regulation of microvascular blood flow and pressure. Myogenic constriction provides a level of tone upon which vasodilators can act to lower resistance and match blood flow to metabolic

Roles of stretch-activated cation channel and Rho-kinase in the spontaneous contraction of airway smooth muscle

In guinea pigs, it is well-known that mechanical stretch of airway smooth muscle exhibits spontaneous tone which is mediated by cyclooxygenase (COX) activation. We tested the hypothesis that this spontaneous contraction of airway smooth muscle is mediated by stretch-activated non-selective cation channels and the Rho/Rho-kinase pathway, as well as COX-2 using a pharmacological approach. Isometric force and intracellular Ca 2+ concentrations ([Ca 2+] i) were assessed in isolated guinea pig tracheal smooth muscle tissues. The samples were stretched to a given level and the muscle behavior was monitored under isometric conditions. We observed an increase in [Ca 2+] i and subsequent force generation over a 15-min period. The augmented [Ca 2+] i and spontaneous contraction due to the stretch were markedly attenuated by application of Gd 3+, an inhibitor of stretch-activated channels, and removal of extracellular Ca 2+. In contrast, nifedipine only had a mild inhibitory effect on the contraction. (R)-(+)- trans- N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide (Y-27632; a Rho-kinase inhibitor) abolished the spontaneous contraction with no changes in [Ca 2+] i. Simvastatin, which down-regulates Rho activity, also significantly inhibited the contraction. Moreover, indomethacin, an inhibitor of COX-1 and -2, and N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398; a COX-2 inhibitor) abolished the stretch-induced contraction without affecting [Ca 2+] i, whereas the inhibitory effect of 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC560; a COX-1 inhibitor) on the contraction was much less. These findings demonstrated that Ca 2+ entry via stretch-activated channels, the Rho/Rho-kinase pathway, and COX-2 are involved in the mechanotransduction in guinea pig tracheal smooth muscle. Additionally, while the Rho/Rho-kinase pathway and COX-2 regulate the spontaneous contraction independently of [Ca 2+] i, COX-1 is not involved in the stretch-induced force generation.

Age‐dependent stretch‐induced myofilament calcium sensitization in ovine cerebral arteries

Myogenic tone is a well-established characteristic of cerebral arteries, but the mechanisms of this response are not fully understood, particularly in immature cerebral arteries. Given that myofilament calcium sensitivity is typically greater in fetal than adult cerebral arteries, we explored the hypothesis that the characteristics of myogenic tone should differ in fetal and adult arteries. To test this idea, we measured intracellular calcium via Fura-2 in intact ovine posterior communicating arteries and found that stretch induced a graded increase in calcium (max ?ratio = .292), but only in adult arteries even though the magnitudes of stretch-induced contraction were similar in adult (265% Kmax) and fetal (247% Kmax) arteries. Maximum levels of stretch-induced myosin light chain (MLC) phosphorylation were also similar (?41–44%) in fetal and adult arteries. However, when length-tension measurements were performed in permeabilized arteries held at pCa 7, graded stretch produced graded increases in force that were greater in adult (207% Kmax) than fetal (110% Kmax) arteries, indicating stretch-induced calcium sensitization. Similar results were obtained at pCa 5.5. Together, these results indicate that myogenic tone is of greater magnitude in adult than fetal posterior communicating arteries, and that this difference is due to a greater calcium transient and a greater calcium sensitization response in adult compared to fetal arteries. Supported by NIH HD31226 and HL54120.

Mechanisms of mechanotransduction by specialized low-threshold mechanoreceptors in the guinea pig rectum

The guinea pig rectum, but not the colon, is innervated by a specialized class of distension-sensitive mechanoreceptors that have transduction sites corresponding to rectal intraganglionic laminar endings (rIGLEs). Rectal mechanoreceptors recorded in vitro had low threshold to circumferential stretch, adapted slowly, and could respond within 2 ms to mechanical stimulation by a piezo-electric probe. Antagonists to ionotropic N-methyl-D-aspartate (NMDA; CGS 19755, memantine) and non-NMDA (6,7-dinitroquinoxaline-2,3-dione) glutamate receptors did not affect mechanotransduction. In normal Krebs solution, the P2X purinoreceptor agonist alpha,beta-methylene ATP reduced mechanoreceptor firing evoked by distension but simultaneously relaxed circular smooth muscle and inhibited stretch-induced contractions. Neither ATP nor alpha,beta-methylene ATP affected mechanotransduction when transduction sites were directly compressed with von Frey hairs. The P2 purinoreceptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid did not affect stretch-induced firing but reduced the inhibitory effect of alpha,beta-methylene ATP on stretch-induced firing. Under isometric conditions, blocking synaptic transmission in Ca2+-free solution reduced stretch-evoked firing but not when basal tension was restored to control levels. Under isotonic condition, Ca2+-free solution did not significantly affect load-evoked firing. The blockers of mechanogated and/or transient receptor potential channels, benzamil, Gd3+, SKF 96365, and ruthenium red inhibited stretch-induced firing but, in parallel, significantly reduced stretch-induced contractions. Benzamil and SKF 96365 were able to inhibit mechanotransduction when transduction sites were compressed with von Frey hairs. The results show that mechanotransduction is rapid but does not depend on fast exocytotic release of mediators. It is likely that stretch-activated ion channels on rIGLEs are involved in direct, physical mechanotransduction by rectal low-threshold mechanoreceptors.

Vascular ENaC proteins are required for renal myogenic constriction

The myogenic response is an essential component of renal blood flow autoregulation and is the inherent ability of vascular smooth muscle cells (VSMCs) to contract in response to increases in intraluminal pressure. Although mechanosensitive ion channels are thought to initiate VSMC stretch-induced contraction, their molecular identity is unknown. Recent reports suggest degenerin/epithelial Na(+) channels (DEG/ENaC) may form mechanotransducers in sensory neurons and VSMCs; however, the role of DEG/ENaC proteins in myogenic constriction of mouse renal arteries has not been established. To test the hypothesis that DEG/ENaC proteins are required for myogenic constriction in renal vessels, we first determined expression of ENaC transcripts and proteins in mouse renal VSMCs. Then, we determined pressure- and agonist-induced constriction and changes in vascular smooth muscle cytosolic Ca(2+) and Na(+) in isolated mouse renal interlobar arteries following DEG/ENaC inhibition with amiloride and benzamil. We detect alpha-, beta-, and gammaENaC transcript and protein expression in cultured mouse renal VSMC. In contrast, we detect only beta- and gamma- but not alphaENaC protein in freshly dispersed mrVMSC. Selective DEG/ENaC inhibition, with low doses of amiloride and benzamil, abolishes pressure-induced constriction and increases in cytosolic Ca(2+) and Na(+) without diminishing agonist-induced responses in isolated mouse interlobar arteries. Our findings indicate that DEG/ENaC proteins are required for myogenic constriction in mouse interlobar arteries and are consistent with our hypothesis that DEG/ENaC proteins may be components of mechanosensitive ion channel complexes required for myogenic vasoconstriction.

Regional difference of endothelium-dependent contractility in human umbilical artery

To investigate whether vascular reactivity of the umbilical artery influences differences in Doppler waveform depending on the sampling site along the cord, we studied a regional difference of endothelium-dependent vascular contractility in human umbilical artery using mechanical responses to the quick stretch of vascular smooth muscle strips. The umbilical artery resistance index (RI) values for the fetal end were significantly higher than those for the placental end. In endothelium-intact strips, stretch-induced contraction of the umbilical artery appears to decrease from the fetal to the placental end of the umbilical artery. No significant differences were noted between strips from the two locations when endothelial cells were rubbed, indicating that this regional difference is related to endothelial cell function. Indo-methacin decreased the stretch-induced contraction of endothelium-intact strips, but tetrodotoxin did not affect this association, suggesting that arachidonic acid metabolism via the cyclooxygenase pathway contributes to modulation of human umbilical artery responsiveness to stretch. These findings suggest that the difference in Doppler waveform of the umbilical artery depending on the sampling site is related to differences in vascular resistance that result from endothelium-related function.

Degenerin/epithelial Na+ channel proteins: components of a vascular mechanosensor.

Mechanosensitive ion channels are thought to mediate stretch-induced contraction in vascular smooth muscle cells (VSMCs); however, the molecular identity of the mechanosensitive ion channel complex is unknown. Although recent reports suggest degenerin/epithelial Na+ channel (DEG/ENaC) proteins may be mechanosensors in sensory neurons, their role as mechanosensors in vascular tissue has not been examined. We first tested whether DEG/ENaC subunits are expressed in cerebral blood vessels and VSMCs and then examined their role as mechanosensors in mediating the myogenic response in intact blood vessels. Using RT-PCR, we found ENaC transcripts expressed in rat cerebral arteries and freshly dissociated rat cerebral VSMCs. We also detected ENaC expression in isolated blood vessels and VSMCs by immunoblotting and immunolocalization. Moreover, inhibition of ENaC with amiloride (1 micromol/L) and benzamil (30 nmol/L, 1 micromol), an amiloride analog, blocked myogenic constriction in isolated rat cerebral arteries. These data suggest that DEG/ENaC proteins are required for vessel responses to pressure and are consistent with the evolutionary conservation of mechanosensory function of DEG/ENaC proteins.