A role for Rho in smooth muscle phenotypic regulation

The role of the small GTP-binding protein Rho in the process of smooth muscle cell (SMC) phenotypic modulation was investigated using cultured rabbit aortic SMCs. Both Rho transcription and Rho protein expression were high for the first 3 days of culture (contractile state cells), with expression decreasing after change to the synthetic state and peaking upon return to the contractile phenotype. Activation of Rho (indicated by translocation to the membrane) also peaked upon return to the contractile state and was low in synthetic state SMCs. Transient transfection of synthetic state rabbit SMCs with constitutively active Rho (val14rho) caused a dramatic decrease in cell size and reorganization of cytoskeletal proteins to resemble those of the contractile phenotype; alpha-actin and myosin adopted a tightly packed, highly organized arrangement, whereas vimentin localized to the immediate perinuclear region and focal adhesions were enlarged. Conversely, specific inhibition of endogenous Rho, by expression of C3 transferase, resulted in the complete loss of actin and myosin filaments without affecting the distribution of vimentin. Focal adhesions were reduced in number. Thus, Rho plays a key role in regulating SMC phenotypic expression.

Rho and smooth muscle cell phenotype

Smooth muscle cell (SMC) phenotypic modulation from the mature ’contractile’ to a less differentiated ’synthetic’ phenotype involves not only altered expression but also a reorganisation of contractile and cytoskeletal proteins. Objective: To investigate the role of RhoA, a known regulator of the actin cytoskeleton, in SMC phenotypic regulation. Methods: Rho transcription (RT-PCR), expression (Western analysis) and activation (membrane translocation or Rho ’pull-down’ assay) was investigated in cultured rabbit aortic SMC during phenotypic modulation, and under the influence of known SM-regulatory proteins (thrombin, heparin and TGF- β). Rho’s effect on cell morphology was examined by transient transfection of ’synthetic’ state SMC with either constitutively active Rho (Val14RhoA) or its inhibitor, C3 transferase. Results: RhoA transcription was elevated in the first 3 days of primary culture, and protein expression peaked at 2 days post-confluence when SMC return to a more ’contractile’ state. However, RhoA showed augmented activation at three time-points in primary culture: the transition point when SMCs enter logarithmic growth and are highly motile, upon reaching quiescence, and when they return to a more ’contractile’ state. Thrombin, heparin and TGF-β activated RhoA in ’synthetic’ state SMCs. Transfection with Val14RhoA caused a dramatic decrease in SMC size and a reorganization of cytoskeletal proteins, reminiscent of the ’contractile’ phenotype. Specific inhibition of endogenous Rho by C3 transferase resulted in an almost complete loss of contractile proteins. Conclusion: These data indicate that Rho is an important determining factor of SMC functional state.

Protease-activated receptor-2 peptides activate neurokinin-1 receptors in the mouse isolated trachea

Protective roles for protease-activated receptor-2 (PAR2) in the airways including activation of epithelial chloride (Cl-) secretion are based on the use of presumably PAR(2)-selective peptide agonists. To determine whether PAR(2) peptide-activated Cl- secretion from mouse tracheal epithelium is dependent on PAR(2), changes in ion conductance across the epithelium [short-circuit current (I-SC)] to PAR(2) peptides were measured in Ussing chambers under voltage clamp. In addition, epithelium and endothelium-dependent relaxations to these peptides were measured in two established PAR(2) bioassays, isolated ring segments of mouse trachea and rat thoracic aorta, respectively. Apical application of the PAR(2) peptide SLIGRL caused increases in I-SC, which were inhibited by three structurally different neurokinin receptor-1 (NK1R) antagonists and inhibitors of Cl- channels but not by capsaicin, the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37), or the nonselective cyclooxygenase inhibitor indomethacin. Only high concentrations of trypsin caused an increase in I-SC but did not affect the responses to SLIGRL. Relaxations to SLIGRL in the trachea and aorta were unaffected by the NK1R antagonist nolpitantium (SR 140333) but were abolished by trypsin desensitization. The rank order of potency for a range of peptides in the trachea I-SC assay was 2-furoyl-LIGRL > SLCGRL > SLIGRL > SLIGRT > LSIGRL compared with 2-furoyl-LIGRL > SLIGRL > SLIGRT > SLCGRL (LSIGRL inactive) in the aorta relaxation assay. In the mouse trachea, PAR(2) peptides activate both epithelial NK1R coupled to Cl- secretion and PAR(2) coupled to prostaglandin E-2-mediated smooth muscle relaxation. Such a potential lack of specificity of these commonly used peptides needs to be considered when roles for PAR(2) in airway function in health and disease are determined.

Contribution of beta-adrenoceptor subtypes to relaxation of colon and oesophagus and pacemaker activity of ureter in wild-type and beta(3)-adrenoceptor knockout mice

1 The smooth muscle relaxant responses to the mixed beta(3)-, putative beta(4)-adrenoceptor agonist, (-)-CGP 12177 in rat colon are partially resistant to blockade by the beta(3)-adrenoceptor antagonist SR59230A suggesting involvement of beta(3)- and putative beta(4)-adrenoceptors. We now investigated the function of the putative beta(4)-adrenoceptor and other beta-adrenoceptor subtypes in the colon, oesophagus and ureter of wild-type (WT) and beta(3)-adrenoceptor knockout (beta(3)KO) mice. 2 (-)-Noradrenaline and (-)-adrenaline relaxed KCl (30 mM)-precontracted colon mostly through beta(1)-and beta(3)-adrenoceptors to a similar extent and to a minor extent through beta(2)-adrenoceptors. In colon from beta(3)KO mice, (-)-noradrenaline was as potent as in WT mice but the effects were mediated entirely through beta(1)-adrenoceptors. (-)-CGP 12177 relaxed colon from beta(3)KO mice with 2 fold greater potency than in WT mice. The maintenance of potency for (-)-noradrenaline and increase for (-)-CGP 12177 indicate compensatory increases in beta(1)- and putative beta(4)-adrenoceptor function in beta(3)KO mice. 3 In oesophagi precontracted with 1 mu M carbachol, (-)-noradrenaline caused relaxation mainly through beta(1)-and beta(3)-adrenoceptors. (-)-CGP 12177 (2 mu M) relaxed oesophagi from WT by 61.4+/-5.1% and beta(3)KO by 67.3+/-10.1% of the (-)-isoprenaline-evoked relaxation, consistent with mediation through putative beta(4)-adrenoceptors. 4 In ureter, (-)-CGP 12177 (2 mu M) reduced pacemaker activity by 31.1+/-2.3% in WT and 31.3+/-7.5% in beta(3)KO, consistent with mediation through putative beta(4)-adrenoceptors. 5 Relaxation of mouse colon and oesophagus by catecholamines are mediated through beta(1)- and beta(3)- adrenoceptors in WT. The putative beta(4)-adrenoceptor, which presumably is an atypical state of the beta(1)-adrenoceptor, mediates the effects of(-)-CGP 12177 in colon, oesophagus and ureter.

Peptidergic control of gastrointestinal blood flow in the estuarine crocodile, Crocodylus porosus

Peptidergic mechanisms influencing the resistance of the gastrointestinal vascular bed of the estuarine crocodile, Crocodylus porosus, were investigated. The gut was perfused in situ via the mesenteric and the celiac arteries, and the effects of different neuropeptides were tested using bolus injections. Effects on vascular resistance were recorded as changes in inflow pressures. Peptides found in sensory neurons [substance P, neurokinin A, and calcitonin gene-related peptide (CGRP)] all caused significant relaxation of the celiac vascular bed, as did vasoactive intestinal polypeptide (VIP), another well-known vasodilator. Except for VIP, the peptides also induced transitory gut contractions. Somatostatin and neuropeptide Y (NPY), which coexist in adrenergic neurons of the C. porosus, induced vasoconstriction in the celiac vascular bed without affecting the gut motility. Galanin caused vasoconstriction and occasionally activated the gut wall. To elucidate direct effects on individual vessels, the different peptides were tested on isolated ring preparations of the mesenteric and celiac arteries. Only CGRP and VIP relaxed the epinephrine-precontracted celiac artery, whereas the effects on the mesenteric artery were variable. Somatostatin and NPY did not affect the resting tonus of these vessels, but somatostatin potentiated the epinephrine-induced contraction of the celiac artery. Immunohistochemistry revealed the existence and localization of the above-mentioned peptides in nerve fibers innervating vessels of different sizes in the gut region. These data support the hypothesis of an important role for neuropeptides in the control of the vascular bed of the gastrointestinal tract in C. porosus.

Effects of beta-escin and saponin on the transverse-tubular system and sarcoplasmic reticulum membranes of rat and toad skeletal muscle

Mechanically skinned skeletal muscle fibres from rat and toad were exposed to the permeabilizing agents beta-escin and saponin. The effects of these agents on the sealed transverse tubular system (t-system) and sarcoplasmic reticulum (SR) were examined by looking at changes in the magnitude of the force responses to t-system depolarization, the time course of the fluorescence of fura-2 trapped in the sealed t-system, and changes in the magnitude of caffeine-induced contractures following SR loading with Ca2+ under defined conditions. In the presence of 2 mu g ml(-1) beta-escin and saponin, the response to t-system depolarization was not completely abolished, decreasing to a plateau, and a large proportion of fura-2 remained in the sealed t-system. At 10 mu g ml(-1), both agents abolished the ability of both rat and toad preparations to respond to t-system depolarization after 3 min of exposure, but a significant amount of fura-2 remained in sealed t-tubules even after exposure to 100 mu g ml(-1) beta-escin and saponin for 10 min. beta-Escin took longer than saponin to reduce the t-system depolarizations and fura-2 content of the sealed t-system to a similar level. The ability of the SR to load Ca2+ was reduced to a lower level after treatment with beta-escin than saponin. This direct effect on the SR occurred at much lower concentrations for rat (2 mu g ml(-1) beta-escin and 10 mu g ml(-1) saponin) than toad (10 mu g ml(-1) beta-escin and 150 mu g ml(-1) saponin). The reverse order in sensitivities to beta-escin and saponin of t-system and SR membranes indicates that the mechanisms of action of beta-escin and saponin are different in the two types of membrane. In conclusion, this study shows that: (1) beta-escin has a milder action on the surface membrane than saponin; (2) beta-escin is a more potent modifier of SR function; (3) simple permeabilization of membranes is not sufficient to explain the effects of beta-escin and saponin on muscle membranes; and (4) the t-system network within muscle fibres is not a homogeneous compartment.

Comparison of pulmonary vascular function and structure in early and established hypoxic pulmonary hypertension in rats

In pulmonary hypertension, changes in pulmonary vascular structure and function contribute to the elevation in pulmonary artery pressure. The time-courses for changes in function, unlike structure, are not well characterised. Medial hypertrophy and neomuscularisation and reactivity to vasoactive agents were examined in parallel in main and intralobar pulmonary arteries and salt-perfused lungs from rats exposed to hypoxia (10% O-2) for 1 and 4 weeks (early and established pulmonary hypertension, respectively). After 1 week of hypoxia, in isolated main and intralobar arteries, contractions to 5-hydroxytryptamine and U46619 (thromboxane-mimetic) were increased whereas contractions to angiotensins I and II and relaxations to acetylcholine were reduced. These alterations varied quantitatively between main and intralobar arteries and, in many instances, regressed between 1 and 4 weeks. The alterations in reactivity did not necessarily link chronologically with alterations in structure. In perfused lungs, constrictor responses to acute alveolar hypoxia were unchanged after 1 week but were increased after 4 weeks, in conjunction with the neomuscularisation of distal alveolar arteries. The data suggest that in hypoxic pulmonary hypertension, the contribution of altered pulmonary vascular reactivity to the increase in pulmonary artery pressure may be particularly important in the early stages of the disease.