Intact Smooth Muscle Strips Research Articles

Smooth Muscle Strips for Intestinal Tissue Engineering

Functionally contracting smooth muscle is an essential part of the engineered intestine that has not been replicated in vitro. The purpose of this study is to produce contracting smooth muscle in culture by maintaining the native smooth muscle organization. We employed intact smooth muscle strips and compared them to dissociated smooth muscle cells in culture for 14 days. Cells isolated by enzymatic digestion quickly lost maturity markers for smooth muscle cells and contained few enteric neural and glial cells. Cultured smooth muscle strips exhibited periodic contraction and maintained neural and glial markers. Smooth muscle strips cultured for 14 days also exhibited regular fluctuation of intracellular calcium, whereas cultured smooth muscle cells did not. After implantation in omentum for 14 days on polycaprolactone scaffolds, smooth muscle strip constructs expressed high levels of smooth muscle maturity markers as well as enteric neural and glial cells. Intact smooth muscle strips may be a useful component for engineered intestinal smooth muscle.

Aging-associated oxidative stress leads to decrease in IAS tone via RhoA/ROCK downregulation

Internal anal sphincter (IAS) tone plays an important role in rectoanal incontinence (RI). IAS tone may be compromised during aging, leading to RI in certain patients. We examined the influence of oxidative stress in the aging-associated decrease in IAS tone (AADI). Using adult (4-6 mo old) and aging (24-30 mo old) rats, we determined the effect of oxidative stress on IAS tone and the regulatory RhoA/ROCK signal transduction cascade. We determined the effect of the oxidative stress inducer LY83583, which produces superoxide anions (O2 (·-)), on basal and stimulated IAS tone before and after treatment of intact smooth muscle strips and smooth muscle cells with the O2 (·-) scavenger SOD. Our data showed that AADI was associated with a decrease in RhoA/ROCK expression at the transcriptional and translational levels. Oxidative stress with a LY83583-mediated decrease in IAS tone and relaxation of IAS smooth muscle cells was associated with a decrease in RhoA/ROCK signal transduction, which was reversible by SOD. In addition, LY83583 caused a significant decrease in IAS contraction produced by the RhoA activator and a known RhoA/ROCK agonist, U46619, that was also reversible by SOD. The inhibitory effects of LY83583 and the ROCK inhibitor Y27632 on the U46619-induced increase in IAS tone were similar. We conclude that an increase in oxidative stress plays an important role in AADI in the elderly and may be one of the underlying mechanisms of RI in certain aging patients.

Cyclic nucleotide-dependent phosphodiesterases (PDEI) inhibition by muscarinic antagonists in bovine tracheal smooth muscle

In bovine tracheal smooth muscle (TSM) strips, muscarinic antagonists (atropine, 4-DAMP, AFDX-116 and methoctramine) were able to increase simultaneously and a similar fashion the intracellular levels of cyclic nucleotides, with a cAMP/cGMP ratio higher than 2.0. These original pharmacological responses were time-and dose-dependent, exhibiting maximal values at 15 min, with a pEC 50 of 7.4 ± 0.2 for atropine and 4-DAMP. These effects on cAMP and cGMP levels were similar to the ones obtained with isobutyl-methylxantine (IBMX, 10 μM), a non-selective cyclic nucleotide phosphodiesterase (PDE) inhibitor, suggesting the involvement of PDEs in these muscarinic antagonist responses. Neither, rolipram (10 μM), a specific PDEIV inhibitor, nor zaprinast (10 μM), a PDEV inhibitor, exhibited this “atropine-like” responses. Instead, atropine enhanced the increments of cAMP levels induced by rolipram and cGMP levels by zaprinast. However, vinpocetine (20 μM), a non-calmodulin dependent PDEIC inhibitor was able to mimic these muscarinic antagonist responses in intact smooth muscle strips. In addition, in cell free systems, muscarinic antagonists inhibited the membrane-bound PDEIC activity whereas soluble (cytosol) PDEIC activity was not affected by these muscarinic drugs. These results indicate that muscarinic antagonists acting possibly as inverse agonists on M 2/M 3mAChRs anchored to sarcolemma membranes can initiate a new signal transducing cascade leading to the PDEIC inhibition, which produced a simultaneous rise in both cAMP and cGMP intracellular levels in tracheal smooth muscle.

Protein kinase C mediation of Ca(2+)-independent contractions of vascular smooth muscle.

Tumour-promoting phorbol esters induce slow, sustained contractions of vascular smooth muscle, suggesting that protein kinase C (PKC) may play a role in the regulation of smooth muscle contractility. In some cases, e.g., ferret aortic smooth muscle, phorbol ester induced contractions occur without a change in [Ca2+]i or myosin phosphorylation. Direct evidence for the involvement of PKC came from the use of single saponin-permeabilized ferret aortic cells. A constitutively active catalytic fragment of PKC induced a slow, sustained contraction similar to that triggered by phenylephrine. Both responses were abolished by a peptide inhibitor of PKC. Contractions of similar magnitude occurred even when the [Ca2+] was reduced to close to zero, implicating a Ca(2+)-independent isoenzyme of PKC. Of the two Ca(2+)-independent PKC isoenzymes, epsilon and zeta, identified in ferret aorta, PKC epsilon is more likely to mediate the contractile response because (i) PKC epsilon, but not PKC zeta, is responsive to phorbol esters; (ii) upon stimulation with phenylephrine, PKC epsilon translocates from the sarcoplasm to the sarcolemma, whereas PKC zeta, translocates from a perinuclear localization to the interior of the nucleus; and (iii) when added to permeabilized single cells of the ferret aorta at pCa 9, PKC epsilon, but not PKC zeta, induced a contractile response similar to that induced by phenylephrine. A possible substrate of PKC epsilon is the smooth muscle specific, thin filament associated protein, calponin. Calponin is phosphorylated in intact smooth muscle strips in response to carbachol, endothelin-1, phorbol esters, or okadaic acid. Phosphorylation of calponin in vitro by PKC (a mixture of alpha, beta, and gamma isoenzymes) dramatically reduces its affinity for F-actin and alleviates its inhibition of the cross-bridge cycling rate. Calponin is phosphorylated in vitro by PKC epsilon but is a very poor substrate of PKC zeta. A signal transduction pathway is proposed to explain Ca(2+)-independent contraction of ferret aorta whereby extracellular signals trigger diacylglycerol production without a Ca2+ transient. The consequent activation of PKC epsilon would result in calponin phosphorylation, its release from the thin filaments, and alleviation of inhibition of cross-bridge cycling. Slow, sustained contraction then results from a slow rate of cross-bridge cycling because of the basal level of myosin light chain phosphorylation (approximately 0.1 mol Pi/mol light chain). We also suggest that signal transduction through PKC epsilon is a component of contractile responses triggered by agonists that activate phosphoinositide turnover; this may explain why smooth muscles often develop more force in response, e.g., to alpha 1-adrenergic agonists than to K+.

Effects of a water-soluble forskolin derivative (NKH477) and a membrane-permeable cyclic AMP analogue on noradrenaline-induced Ca2+ mobilization in smooth muscle of rabbit mesenteric artery.

1. Effects were studied of 6-(3-dimethylaminopropionyl) forskolin (NKH477), a water-soluble forskolin derivative and of dibutyryl-cyclic AMP, a membrane-permeable cyclic AMP analogue on noradrenaline (NA)-induced Ca2+ mobilization in smooth muscle strips of the rabbit mesenteric artery. The intracellular concentration of Ca2+ ([Ca2+]i), isometric force and cellular concentration of inositol 1,4,5-trisphosphate (InsP3) were measured. 2. NA (10 microM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force in a solution containing 2.6 mM Ca2+. NKH477 (0.01-0.3 microM) attenuated the phasic and the tonic increases in both [Ca2+]i and force induced by 10 microM NA, in a concentration-dependent manner. 3. In Ca(2+)-free solution containing 2 mM EGTA with 5.9 mM K+, NA (10 microM) produced only phasic increases in [Ca2+]i and force. NKH477 (0.01 microM) and dibutyryl-cyclic AMP (0.1 mM) each greatly inhibited these increases. 4. NA (10 microM) led to the production of InsP3 in intact smooth muscle strips and InsP3 (10 microM) increased Ca2+ in Ca(2+)-free solution after a brief application of Ca2+ in beta-escin-skinned smooth muscle strips. NKH477 (0.01 microM) or dibutyryl-cyclic AMP (0.1 mM) modified neither the NA-induced synthesis of InsP3 in intact muscle strips nor the InsP3-induced Ca2+ release in skinned strips. 5. In Ca(2+)-free solution, high K+ (40 and 128 mM) itself failed to increase [Ca2+]i but concentration-dependently enhanced the amplitude of the increase in [Ca2+]i induced by 10 microM NA with a parallel enhancement of the maximum rate of rise. The extent of the inhibition induced by NKH477 (0.01 microM)or dibutyryl-cyclic AMP (0.1 mM) on the NA-induced [Ca2+] increase was inversely related to the maximum rate of rise of [Ca2+], induced by NA in Ca2+-free solution containing various concentrations of K+. These results suggest that the increase in the rate of Ca2+ release induced by NA can conceal the inhibitory action on NA-induced Ca2+ mobilization of agents that increase cyclic AMP.6. Repetitive application of 10 JAM NA in Ca2+-free solution led to a disappearance of the NA-induced increase in [Ca2+]j, but NA could again increase [Ca2+], in Ca2+-free solution after a brief application of Ca2+ with 40 mM K+ ('Ca2+-loading'). The magnitude of this NA-induced increase in [Ca2+]i depended on the duration of the Ca2+-loading. With application of dibutyryl-cyclic AMP (0.1 mM) during the Ca2+-loading period, the loading duration required for the restoration of the maximum NA-response was shortened.7. Cyclopiazonic acid (10 microM, an inhibitor of Ca2+-ATPase at intracellular storage sites) attenuated the inhibitory action of dibutyryl-cyclic AMP on the NA-induced increase in [Ca2+], in Ca2+-free solution.When NA (10 microM) was applied twice for 30 s with a 10 min interval in Ca2+-free solution, the amplitude of response to the second application was about one third of the first response. With application of 0.1 mM dibutyryl-cyclic AMP during the first application of NA, the increase in [Ca2+], induced by the first application of NA was inhibited, but the response induced by the second was enhanced. These results suggest that dibutyryl-cyclic AMP enhances Ca2+ uptake into the NA-sensitive storage sites.8. We conclude that, in smooth muscle of the rabbit mesenteric artery, agents that increase cyclic AMP inhibit the NA-induced increase in [Ca2+] through an activation of Ca2+ uptake into the cellular storage sites.

ATP induced‐relaxation in the mouse bladder smooth muscle

1. The effect of adenosine 5'-triphosphate (ATP) on the free cytosolic Ca2+ concentration ([Ca2+]i) as measured with the fluorescent Ca(2+)-indicator fura-2, and on force was investigated in the intact smooth muscle strips of the mouse urinary bladder. 2. ATP elicited, when exogenously applied, a large increase of [Ca2+]i with limited force development resulting in a marked Ca(2+)-force dissociation. 3. Release of endogenous neurotransmitters by transmural electrical stimulation (TES) for 30 s induced a steady increase of [Ca2+]i and a peak contraction, followed within 15 s by a relaxation. 4. In carbachol-prestimulated preparations, ATP elicited an initial rise of [Ca2+]i followed by a return to the initial precontraction Ca(2+)-level. Force in contrast presented a biphasic pattern, i.e. an initial contraction was followed by a sustained relaxation. 5. In the K(+)-depolarized precontracted preparation, ATP elicited a slight initial rise of [Ca2+]i. The partial relaxation of the force during depolarization was not preceded by a transient contraction. 6. The ATP-induced relaxation of the K(+)-prestimulated preparations was not inhibited by 8-phenyltheophylline, a potent P1-purinoceptor antagonist. 7. The order of potency for relaxation of the ATP analogues was 2-MeSATP > ATP > beta gamma Me-ATP, which is characteristic for P2y-purinoceptors. 8. These results indicate that, besides its activating effect, ATP also relaxes the mouse urinary bladder. It is suggested that the relaxant effect, mediated through P2y-purinoceptors, is mainly responsible for the low contractile potency of ATP in the bladder.

Phosphorylation-contraction coupling in smooth muscle: role of caldesmon.

In intact smooth muscle strips from chicken gizzard, carbachol elicited brief, phasic contractions which were associated with a very rapid, transient phosphorylation of the 20 kDa myosin light chains. Phosphorylation was not significantly different from basal levels after 30 s while force still amounted to 50% of the peak value. The rate of tension decline could be increased by addition of atropine, even at apparently basal phosphorylation levels suggesting a phosphorylation independent regulation. The force, at a given level of phosphorylation, could also be modulated by addition of the actin binding, putative regulatory protein, caldesmon. Caldesmon, inhibits phosphorylation dependent force in skinned fiber bundles of chicken gizzard without affecting myosin light chain phosphorylation. This suggests that caldesmon might modulate contraction in smooth muscle. Moreover our results suggest that caldesmon does not function to maintain passive tension.

Comparison of indoleamine 2,3-dioxygenase activity in ciliary body, retina, and aqueous humor of bovine eyes

In bovine tracheal smooth muscle (TSM) strips, muscarinic antagonists (atropine, 4-DAMP, AFDX-116 and methoctramine) were able to increase simultaneously and a similar fashion the intracellular levels of cyclic nucleotides, with a cAMP/cGMP ratio higher than 2.0. These original pharmacological responses were time-and dose-dependent, exhibiting maximal values at 15 min, with a pEC50 of 7.4 ± 0.2 for atropine and 4-DAMP. These effects on cAMP and cGMP levels were similar to the ones obtained with isobutyl-methylxantine (IBMX, 10 μM), a non-selective cyclic nucleotide phosphodiesterase (PDE) inhibitor, suggesting the involvement of PDEs in these muscarinic antagonist responses. Neither, rolipram (10 μM), a specific PDEIV inhibitor, nor zaprinast (10 μM), a PDEV inhibitor, exhibited this “atropine-like” responses. Instead, atropine enhanced the increments of cAMP levels induced by rolipram and cGMP levels by zaprinast. However, vinpocetine (20 μM), a non-calmodulin dependent PDEIC inhibitor was able to mimic these muscarinic antagonist responses in intact smooth muscle strips. In addition, in cell free systems, muscarinic antagonists inhibited the membrane-bound PDEIC activity whereas soluble (cytosol) PDEIC activity was not affected by these muscarinic drugs. These results indicate that muscarinic antagonists acting possibly as inverse agonists on M2/M3mAChRs anchored to sarcolemma membranes can initiate a new signal transducing cascade leading to the PDEIC inhibition, which produced a simultaneous rise in both cAMP and cGMP intracellular levels in tracheal smooth muscle.

Effects of the calmodulin antagonist W-7 on isometric tension development and myosin light chain phosphorylation in bovine tracheal smooth muscle.

Effects of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on isometric tension development and myosin light chain (P-LC) phosphorylation were determined in intact smooth muscle strips of bovine trachea. Addition of 10(-5) M carbachol resulted in a marked increase in the P-LC phosphorylation within 30 sec of stimulation. Subsequently, there was a gradual decrease in the P-LC phosphorylation. In contrast, steady-state contraction was not attained until 10 min of stimulation. In these strips, W-7 itself caused a contraction, probably due to the release of endogenous histamine that was blocked by pyrilamine. In the presence of W-7 and pyrilamine, the isometric tension development and P-LC phosphorylation during the initial 5-min stimulation period were inhibited. Concentrations of W-7 that caused a 50% inhibition were 145 and 125 microM, respectively, for tension and phosphorylation. N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), a weak calmodulin antagonist, produced no significant inhibition on the carbachol-induced isometric tension development and P-LC phosphorylation. After 10-min exposure to carbachol, W-7 inhibited the steady-state contraction without inhibition of the P-LC phosphorylation. These results suggest that 1) W-7 inhibits the smooth muscle contraction through the inhibition of the initial increase in the P-LC phosphorylation and 2) the maintenance of isometric tension (after 10 min exposure to carbachol) in unlikely to depend on the P-LC phosphorylation.

Effects of the Calmodulin Antagonist W-7 on Isometric Tension Development and Myosin Light Chain Phosphorylation in Bovine Tracheal Smooth Muscle

Effects of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on isometric tension development and myosin light chain (P-LC) phosphorylation were determined in intact smooth muscle strips of bovine trachea. Addition of 10-5 M carbachol resulted in a marked increase in the P-LC phosphorylation within 30 sec of stimulation. Subsequently, there was a gradual decrease in the P-LC phosphorylation. In contrast, steady-state contraction was not attained untillOminofstimulation. In these strips, W-7 itself caused a contraction, probably due to the release of endogenous histamine that was blocked by pyrilamine. In the presence of W-7 and pyrilamine, the isometric tension development and P-LC phosphorylation during the initial 5-min stimulation period were inhibited. Concentrations of W-7 that caused a 50% inhibition were 145 and 125 μM, respectively, for tension and phosphorylation. N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), a weak calmodulin antagonist, produced no significant inhibition on the carbachol-induced isometric tension development and P-LC phosphorylation. After 10-min exposure to carbachol, W-7 inhibited the steady-state contraction without inhibition of the P-LC phosphorylation. These results suggest that 1) W-7 inhibits the smooth muscle contraction through the inhibition of the initial increase in the P-LC phosphorylation and 2) the maintenance of isometric tension (after 10 min exposure to carbachol) in unlikely to depend on the P-LC phosphorylation.

Rapid myosin phosphorylation transients in phasic contractions in chicken gizzard smooth muscle

In intact smooth muscle strips from chicken gizzard, electrical stimulation and carbachol elicited brief, phasic contractions which were associated with a very rapid, transient phosphorylation of the 20 kDa myosin light chains. The phosphorylation transients reached their peak after 3 s and 6 s and preceded that of force. Phosphorylation was not significantly different from basal levels after 10 s and 30 s while force still amounted to 50% of the peak value. The rate of tension decline could be increased by cessation of stimulation or by addition of atropine, even at apparently basal phosphorylation levels suggesting a phosphorylation independent regulation.