Force Development In Response Research Articles

Age-Related Decline in Vascular Responses to Phenylephrine Is Associated with Reduced Levels of HSP70.

Aging impairs the expression of HSP70, an emergent player in vascular biology. However, it is unknown if age-related alterations in HSP70 are linked to a decline in arterial function. In this study, we test the hypothesis that the contributions of HSP70 to vascular contraction are diminished in middle-aged animals. We determined the basal levels of HSP70 in the aorta of young and middle-aged Sprague Dawley male rats using Western blotting. Functional studies were performed in a wire myograph system. Force development in response to phenylephrine was assessed in the presence or absence of extracellular calcium (Ca2+), and in aortic rings treated or non-treated with an HSP70 inhibitor. Fluorescent probes were used to evaluate vascular oxidative stress and nitric oxide levels. We report that middle-aged rats have significantly lower levels of HSP70. Blockade of HSP70 attenuated vascular phasic and tonic contraction in isolated aortas. It appears that a functional HSP70 is required for proper Ca2+ handling as inhibition of this protein led to reduced force–displacement in response to Ca2+ dynamics. Furthermore, middle-aged aortic rings exposed to the HSP70 inhibitor display higher reactive oxygen species levels without changes in nitric oxide. In summary, we show that middle-aged animals have lower levels of HSP70 in aortas, which associates with an age-related decline in vascular responses to α-1 adrenergic stimulation.

Additional in-series compliance reduces muscle force summation and alters the time course of force relaxation during fixed-end contractions.

There are high mechanical demands placed on skeletal muscles in movements requiring rapid acceleration of the body or its limbs. Tendons are responsible for transmitting muscle forces, but, because of their elasticity, can manipulate the mechanics of the internal contractile apparatus. Shortening of the contractile apparatus against the stretch of tendon affects force generation according to known mechanical properties; however, the extent to which differences in tendon compliance alter force development in response to a burst of electrical impulses is unclear. To establish the influence of series compliance on force summation, we studied electrically evoked doublet contractions in the cane toad peroneus muscle in the presence and absence of a compliant artificial tendon. Additional series compliance reduced tetanic force by two-thirds, a finding predicted based on the force-length property of skeletal muscle. Doublet force and force-time integral expressed relative to the twitch were also reduced by additional series compliance. Active shortening over a larger range of the ascending limb of the force-length curve and at a higher velocity, leading to a progressive reduction in force-generating potential, could be responsible. Muscle-tendon interaction may also explain the accelerated time course of force relaxation in the presence of additional compliance. Our findings suggest that a compliant tendon limits force summation under constant-length conditions. However, high series compliance can be mechanically advantageous when a muscle-tendon unit is actively stretched, permitting muscle fibres to generate force almost isometrically, as shown during stretch-shorten cycles in locomotor activities. Restricting active shortening would likely favour rapid force development.

Histone deacetylase 8 regulates cortactin deacetylation and contraction in smooth muscle tissues.

Histone deacetylases (HDACs) are a family of enzymes that mediate nucleosomal histone deacetylation and gene expression. Some members of the HDAC family have also been implicated in nonhistone protein deacetylation, which modulates cell-cycle control, differentiation, and cell migration. However, the role of HDACs in smooth muscle contraction is largely unknown. Here, HDAC8 was localized both in the cytoplasm and the nucleus of mouse and human smooth muscle cells. Knockdown of HDAC8 by lentivirus-encoding HDAC8 shRNA inhibited force development in response to acetylcholine. Treatment of smooth muscle tissues with HDAC8 inhibitor XXIV (OSU-HDAC-44) induced relaxation of precontracted smooth muscle tissues. In addition, cortactin is an actin-regulatory protein that undergoes deacetylation during migration of NIH 3T3 cells. In this study, acetylcholine stimulation induced cortactin deacetylation in mouse and human smooth muscle tissues, as evidenced by immunoblot analysis using antibody against acetylated lysine. Knockdown of HDAC8 by RNAi or treatment with the inhibitor attenuated cortactin deacetylation and actin polymerization without affecting myosin activation. Furthermore, expression of a charge-neutralizing cortactin mutant inhibited contraction and actin dynamics during contractile activation. These results suggest a novel mechanism for the regulation of smooth muscle contraction. In response to contractile stimulation, HDAC8 may mediate cortactin deacetylation, which subsequently promotes actin filament polymerization and smooth muscle contraction.

Mucosa of murine detrusor impairs β2 -adrenoceptor-mediated relaxation.

To investigate the role of the mucosa in (-)-isoprenaline-induced relaxation of mouse detrusor muscle and to characterize the β-adrenoceptor subtypes involved. Isolated intact and mucosa-denuded muscle strips from the urinary bladder of male C57BL6 mice were pre-contracted with KCl (40 mM) and were relaxed with increasing concentrations of the β-adrenoceptor (β-AR) agonist (-)-isoprenaline and forskolin in the presence and absence of the subtype-selective β-AR blockers CGP20712A (β1 -ARs), ICI118,551 (β2 -ARs), and L748,337 (β3 -ARs). Force development in response to KCl was larger in mucosa-denuded than in intact preparations and was almost completely relaxed with increasing concentrations of (-)-isoprenaline. Mucosa-denuded muscles were about 10-fold more sensitive to (-)-isoprenaline than intact muscles. CGP20712A did not affect the concentration-response curves (CRCs) to (-)-isoprenaline, ICI118,551 shifted the CRC further to the right in denuded than in intact strips so that the difference between them was abolished. Combined exposure to β1 -AR and β2 -AR blocker yielded the same result. L748,337 did not significantly affect the CRC to (-)-isoprenaline but caused additional blockade to ICI118,551 in the presence of intact mucosa. The mucosa of mouse detrusor strips impairs KCl-induced force development and reduces the sensitivity to β-AR-induced relaxation. The relaxing response to (-)-isoprenaline as well as the mucosa effect thereupon are mainly mediated by β2 -ARs. A minor involvement of β3 -ARs becomes apparent particularly at high (-)-isoprenaline concentrations.

Mucosa of human detrusor impairs contraction and β‐adrenoceptor‐mediated relaxation

To elucidate the impact of the mucosa on detrusor muscle function by investigating force of contraction under various stimulatory conditions and during subsequent relaxation using catecholamines. Detrusor tissue was obtained from patients who had undergone cystectomy for bladder cancer and strips of intact or mucosa-denuded muscle were set up for force measurement. Preparations were precontracted with KCl, carbachol or electric-field stimulation (EFS). Precontracted strips were relaxed using increasing concentrations of catecholamines in the absence and presence of the subtype-selective β-adrenoceptor (AR) blockers CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L-748,337 (β3-ARs). Force development in response to KCl (40 mM), carbachol (1 μM) or EFS was larger in the absence of mucosa than in intact muscle strips. The force of contraction of mucosa-denuded strips with detached urothelium incubated in the same chamber was as low as in intact strips. Noradrenaline relaxed precontracted detrusor strips to a significantly larger extent and at lower concentrations in denuded than in intact strips. CGP 20712A did not affect noradrenaline-induced relaxation of denuded and intact strips, and ICI 118,551 did not influence noradrenaline-induced relaxation in denuded strips, but abolished the difference between denuded and intact strips. The antagonism of the relaxant effects of noradrenaline by L-748,337 was slightly smaller in intact than denuded strips. The mucosa of human detrusor strips impairs force development when stimulated with KCl, carbachol or EFS. The mucosa also blunts the relaxing effects of catecholamines. The latter effect does not involve the activation of β1-ARs but only of β2-ARs, whereas β3-ARs mediate the relaxation of human detrusor.

The Actin Associated Protein Palladin Is Important for the Early Smooth Muscle Cell Differentiation

Palladin, an actin associated protein, plays a significant role in regulating cell adhesion and cell motility. Palladin is important for development, as knockdown in mice is embryonic lethal, yet its role in the development of the vasculature is unknown. We have shown that palladin is essential for the expression of smooth muscle cells (SMC) marker genes and force development in response to agonist stimulation in palladin deficient SMCs. The goal of the study was to determine the molecular mechanisms underlying palladin's ability to regulate the expression of SMC marker genes. Results showed that palladin expression was rapidly induced in an A404 cell line upon retinoic acid (RA) induced differentiation. Suppression of palladin expression with siRNAs inhibited the expression of RA induced SMC differentiation genes, SM α-actin (SMA) and SM22, whereas over-expression of palladin induced SMC gene expression. Chromatin immunoprecipitation assays provided evidence that palladin bound to SMC genes, whereas co-immunoprecipitation assays also showed binding of palladin to myocardin related transcription factors (MRTFs). Endogenous palladin was imaged in the nucleus, increased with leptomycin treatment and the carboxyl-termini of palladin co-localized with MRTFs in the nucleus. Results support a model wherein palladin contributes to SMC differentiation through regulation of CArG-SRF-MRTF dependent transcription of SMC marker genes and as previously published, also through actin dynamics. Finally, in E11.5 palladin null mouse embryos, the expression of SMA and SM22 mRNA and protein is decreased in the vessel wall. Taken together, our findings suggest that palladin plays a key role in the differentiation of SMCs in the developing vasculature.

Smoothelin-like 1 Protein Regulates Myosin Phosphatase-targeting Subunit 1 Expression during Sexual Development and Pregnancy*[S

Pregnancy coordinately alters the contractile properties of both vascular and uterine smooth muscles reducing systemic blood pressure and maintaining uterine relaxation. The precise molecular mechanisms underlying these pregnancy-induced adaptations have yet to be fully defined but are likely to involve changes in the expression of proteins regulating myosin phosphorylation. Here we show that smoothelin like protein 1 (SMTNL1) is a key factor governing sexual development and pregnancy induced adaptations in smooth and striated muscle. A primary target gene of SMTNL1 in these muscles is myosin phosphatase-targeting subunit 1 (MYPT1). Deletion of SMTNL1 increases expression of MYPT1 30-40-fold in neonates and during development expression of both SMTNL1 and MYPT1 increases over 20-fold. Pregnancy also regulates SMTNL1 and MYPT1 expression, and deletion SMTNL1 greatly exaggerates expression of MYPT1 in vascular smooth muscle, producing a profound reduction in force development in response to phenylephrine as well as sensitizing the muscle to acetylcholine. We also show that MYPT1 is expressed in Type2a muscle fibers in mice and humans and its expression is regulated during pregnancy, suggesting unrecognized roles in mediating skeletal muscle plasticity in both species. Our findings define a new conserved pathway in which sexual development and pregnancy mediate smooth and striated muscle adaptations through SMTNL1 and MYPT1.

Cyclooxygenase-2 contributes to functional changes seen on experimental hemorrhagic cystitis induced by ifosfamide in rat urinary bladder

Ifosfamide (IFS) is often involved in the occurrence of hemorrhagic cystitis due to direct contact of its metabolite acrolein with uroepithelium. It has been shown that COX-2 is involved in this pathogenesis. Thus, we aimed to study the functional changes on the urinary bladder in the putative modifications induced by IFS, as well as the COX-2 role in this process. IFS-treated rats were evaluated by cystometrography in absence or presence of COX inhibitors indomethacin or etoricoxib or in the presence of mesna. Experiments with isolated strips of urinary bladder obtained from animals with IFS-induced cystitis, either treated or not treated with COX inhibitors or mesna, were performed. Histological analyses, immunohistochemistry for COX-2, and measurement of plasma PGE(2) were also performed. IFS treatment caused severe inflammation of the bladder tissue. Cystometrography recordings of IFS-treated rats revealed bladder with increased micturition frequency and enhanced filling intravesical pressure. Contractility of the isolated smooth muscle from the rat's bladder with IFS-induced cystitis showed decreased force development in response to KCl and CCh. Almost all effects induced by IFS were ameliorated by the use of COX inhibitors or mesna. Enzyme expression in the urinary bladder tissue was positive, and plasma concentration of PGE(2) was increased in IFS-treated animals and decreased significantly in etoricoxib-treated animals. IFS causes important changes in the micturition physiology in rats, and the inhibition of the isoenzyme COX-2 could be an important event that could prevent the detrimental effects elicited by IFS-induced hemorrhagic cystitis.

Actin Depolymerization Factor/Cofilin Activation Regulates Actin Polymerization and Tension Development in Canine Tracheal Smooth Muscle

The contractile activation of airway smooth muscle tissues stimulates actin polymerization, and the inhibition of actin polymerization inhibits tension development. Actin-depolymerizing factor (ADF) and cofilin are members of a family of actin-binding proteins that mediate the severing of F-actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of smooth muscle was evaluated in intact canine tracheal smooth muscle tissues. Two-dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues, and that approximately 40% of the total ADF/cofilin in unstimulated tissues is phosphorylated. Phospho-ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho-cofilin mimetic (cofilin S3E) but not wild type cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh-induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh-induced dephosphorylation of ADF/cofilin required the Ca2+-dependent activation of calcineurin (PP2B). The results indicate that the activation of ADF/cofilin is regulated by contractile stimulation in tracheal smooth muscle and that cofilin activation is required for actin polymerization and tension development in response to contractile stimulation.

Skeletonization with an ultrasonic scalpel is as safe as a non-skeletonized dissection in preserving the endothelial function of the human gastroepiploic artery

The right gastroepiploic artery (GEA) is frequently used as another in situ artery, other than the internal thoracic artery (ITA) in coronary artery bypass grafting (CABG). Skeletonizing the graft with an ultrasonic scalpel is now regarded as a useful technique; however, this technique may damage the endothelial function during harvesting the graft resulting in postoperative graft stenosis or occlusion. In the present study, GEA segments from nine patients were excised in both a skeletonized and non-skeletonized manner with an ultrasonic scalpel, and then were transported to the laboratory. The vessels were trimmed as rings, and were allotted to the group of skeletonized or non-skeletonized, accordingly. The force development in response to 1 mumol/l norepinephrine did not differ between the skeletonized and non-skeletonized groups. Endothelium-dependent relaxation induced by either acetylcholine or bradykinin was not impaired in the skeletonized group in comparison to the non-skeletonized group. No significant difference was observed in endothelium-independent relaxation elicited by sodium nitroprusside. Therefore, the skeletonization of the GEA with an ultrasonic scalpel was thus found to be as safe as a non-skeletonized dissection in preserving the vascular contractile ability or endothelium-dependent and -independent relaxation of the graft.

Role of vimentin in smooth muscle force development

Vimentin intermediate filaments undergo spatial reorganization in cultured smooth muscle cells in response to contractile activation; however, the role of vimentin in the physiological properties of smooth muscle has not been well elucidated. Tracheal smooth muscle strips were loaded with antisense oligonucleotides (ODNs) against vimentin and then cultured for 2 days to allow for protein degradation. Treatment with vimentin antisense, but not sense, ODNs suppressed vimentin protein expression; neither vimentin antisense nor sense ODNs affected protein levels of desmin and actin. Force development in response to ACh stimulation or KCl depolarization was lower in vimentin-deficient tissues than in vimentin sense ODN- or non-ODN-treated muscle strips. Passive tension was also depressed in vimentin-depleted muscle tissues. Vimentin downregulation did not attenuate increases in myosin light chain (MLC) phosphorylation in response to contractile stimulation or basal MLC phosphorylation. In vimentin sense ODN-treated or non-ODN-treated smooth muscle strips, the desmosomal protein plakoglobin was primarily localized in the cell periphery. The membrane-associated localization of plakoglobin was reduced in vimentin-depleted muscle tissues. These studies suggest that vimentin filaments play an important role in mediating active force development and passive tension, which are not regulated by MLC phosphorylation. Vimentin downregulation impairs the structural organization of desmosomes, which may be associated with the decrease in force development.

ROLE OF GLUTAMATE RECEPTORS IN THE DEVELOPMENT AND MAINTENANCE OF BLADDER OVERACTIVITY AFTER CEREBRAL INFARCTION IN THE RAT

To investigate the role of glutamate receptors in overactive bladder (OAB) caused by cerebral infarction (CI) we examined the effects of 2 different types of receptors antagonists on OAB induced by left middle cerebral artery (MCA) occlusion. Female rats were intravenously injected with dizocilpine, an NMDA (N-methyl-D-aspartate) receptor antagonist, or NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7-sulfonamide), an AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor antagonist, before or after MCA occlusion. Awake rats were cystometrically examined for 8 hours. Detrusor strips were evaluated for force development in response to dizocilpine and NBQX. In CI rats without pretreatment bladder capacity (BC) was significantly decreased after MCA occlusion and remained consistently below half that of pre-occlusion capacity. Dizocilpine (0.5 mg/kg intravenously) administered before MCA occlusion blocked the decrease in BC in awake rats 5 to 8 hours after MCA occlusion. In CI rats pretreated with NBQX (10 or 30 mg/kg intravenously) BC was not different from that in rats without pretreatment. Increasing doses of dizocilpine (0.01 to 10 mg/kg) or NBQX (0.1 to 30 mg/kg) increased rat BC 2 hours after MCA occlusion. NBQX did not change the BC of sham operated rats. No differences in the contractile response to dizocilpine or NBQX of detrusor strips from sham operated and CI rats were observed. These results indicate that NMDA receptor has an essential role in the development of OAB after CI. AMPA receptor antagonist cannot block the development of OAB. However, AMPA receptor antagonist temporally inhibits OAB after it is established by CI.

Differential effects of catecholamines on vascular rings from ductus venosus and intrahepatic veins of fetal sheep

Ductus venosus (DV) sparing means the maintenance of blood flow through the DV following reduction of liver venous blood supply during fetal hypoxia. The present study compared the reactions of the isthmic portion of the DV and intrahepatic veins (IHVs) to catecholamines in vitro. Vessel rings of 1 mm width and 3 mm diameter were obtained from 17 fetal sheep (88-136 days gestational age, median 120 days). The immunohistochemical examination of the DV and IHV was performed in eight cases using an antibody against alpha-smooth muscle actin and an antibody against alpha-adrenergic receptors. Five vessel rings of the DV in early gestation (median 95 days) did not respond to KCl-induced depolarisation. Force development in response to KCl of both vessel types increased with gestational age (P < 0.05). The IHV required 4.1 +/- 0.8 min (mean +/- S.E.M.) and the DV 14.5 +/- 4.0 min to reach the maximum tension in response to KCl, which was 5.0 +/- 4.0 mN in the IHV and 2.2 +/- 1.9 mN in the DV (n = 12, P < 0.05). The maximum forces developed in response to noradrenaline (norepinephrine; 42 microM, n = 9) and adrenaline (epinephrine; 100 microM, n = 12) were about sixfold higher in the IHV rings than in the DV rings (P < 0.05). The EC50 values of the DV and the IHV rings to noradrenaline were 5.9 +/- 1.3 microM and 5.0 +/- 1.3 microM, respectively (P = 0.03). The EC50 values of the adrenaline responses were 2.5 +/- 0.5 microM for the DV and 2.2 +/- 0.7 microM for the IHV (not significant). The alpha-adrenergic receptors were present in the well-structured media of IHVs, but were less distinctive in the wall of the DV. DV sparing can be attributed to an increased resistance of IHVs to catecholamines compared with the DV. The different responses can be explained by different anatomical and functional properties of the two vessel types.

Differential effects of catecholamines on vascular rings from ductus venosus and intrahepatic veins of fetal sheep.

Ductus venosus (DV) sparing means the maintenance of blood flow through the DV following reduction of liver venous blood supply during fetal hypoxia. The present study compared the reactions of the isthmic portion of the DV and intrahepatic veins (IHVs) to catecholamines in vitro. Vessel rings of 1 mm width and 3 mm diameter were obtained from 17 fetal sheep (88-136 days gestational age, median 120 days). The immunohistochemical examination of the DV and IHV was performed in eight cases using an antibody against alpha-smooth muscle actin and an antibody against alpha-adrenergic receptors. Five vessel rings of the DV in early gestation (median 95 days) did not respond to KCl-induced depolarisation. Force development in response to KCl of both vessel types increased with gestational age (P < 0.05). The IHV required 4.1 +/- 0.8 min (mean +/- S.E.M.) and the DV 14.5 +/- 4.0 min to reach the maximum tension in response to KCl, which was 5.0 +/- 4.0 mN in the IHV and 2.2 +/- 1.9 mN in the DV (n = 12, P < 0.05). The maximum forces developed in response to noradrenaline (norepinephrine; 42 microM, n = 9) and adrenaline (epinephrine; 100 microM, n = 12) were about sixfold higher in the IHV rings than in the DV rings (P < 0.05). The EC50 values of the DV and the IHV rings to noradrenaline were 5.9 +/- 1.3 microM and 5.0 +/- 1.3 microM, respectively (P = 0.03). The EC50 values of the adrenaline responses were 2.5 +/- 0.5 microM for the DV and 2.2 +/- 0.7 microM for the IHV (not significant). The alpha-adrenergic receptors were present in the well-structured media of IHVs, but were less distinctive in the wall of the DV. DV sparing can be attributed to an increased resistance of IHVs to catecholamines compared with the DV. The different responses can be explained by different anatomical and functional properties of the two vessel types.

Calcium exerts a larger regulatory effect on potassium+ channels in small mesenteric artery mycoytes from spontaneously hypertensive rats compared to Wistar-Kyoto rats

Hypertension is associated with a remodeling of arterial smooth muscle K+ channels with Ca2+-gated K+ channel (BKCa) activity being enhanced and voltage-gated K+ channel (Kv) activity depressed. Because both of these channel types are modulated by intracellular Ca2+, we tested the hypothesis that Ca2+ had a larger effect on both BKCa and Kv channels in arterial myocytes from hypertensive animals. Myoctyes were enzymatically dispersed from small mesenteric arteries (SMA) of 12-week-old Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Using whole cell patch clamp methods, BKCa and Kv current components were determined as iberiotoxin-sensitive and -insensitive currents, respectively. The effects of Ca2+ on these K+ current components were determined from measurements made with 0.2 and 2 mmol/L external Ca2+. Increasing external Ca2+ from 0.2 to 2 mmol/L Ca2+ increased BKCa currents recorded using myocytes from both WKY rats and SHR with a larger effect in SHR. Increasing external Ca2+ decreased Kv currents recorded using myocytes from both WKY and SHR also with a larger effect in SHR. In other experiments, currents through voltage-gated Ca2+ channels (Cav) measured at 0.2 mmol/L external Ca2+ were 12 ± 2% (n = 12) of those recorded at 2 mmol/L Ca2+ with no differences in percent effect between WKY and SHR. In isolated SMA segments, isometric force development in response to 140 mmol/L KCl at 0.2 mmol/L external Ca2+ was about 23 ± 6% (n = 8) of that measured at 2 mmol/L external Ca2+. These results suggest that an increase in Ca2+ influx through Cav or in intracellular Ca2+ secondary to an increase in external Ca2+ augments BKCa currents and inhibits Kv currents in SMA myocytes with a larger effect in SHR compared to WKY. This mechanism may contribute to the functional remodeling of K+ currents of arterial myocytes in hypertensive animals.

Candesartan, an Insurmountable Antagonist of Angiotensin II-mediated Contractile Effects in Isolated Vascular Preparations: Comparison with Irbesartan, Losartan and its Active Metabolite (EXP-3174)

Candesartan is a new angiotensin II type 1 (AT 1 ) receptor blocker that produces more effective 24-h blood pressure lowering than losartan in hypertensive patients. In in vitro tissue preparations, candesartan displays insurmountable antagonism of the responses to angiotensin II. Both irbesartan and EXP-3174, the active metabolite of losartan, have also previously been described in some studies as insurmountable AT 1 -receptor blockers, whereas losartan exhibits surmountable blockade of the AT 1 -receptor. We compared the properties of candesartan, irbesartan, losartan and EXP-3174 in isolated vascular preparations of rat portal vein and rabbit aortic strips. The concentrations of the different AT 1 -receptor antagonists that were effective in these in vitro preparations were also correlated to the non-protein bound plasma concentrations obtained in clinical use. Preparations of the rabbit aorta and the rat portal vein were dissected, mounted on a force-displacement transducer and submerged in oxygenated Krebs' buffer at 37°C. The vessel strips were pre-stretched to a passive force of 5 and 20 mN for portal vein and aorta, respectively. The response to angiotensin II, measured as the mean force development in response to increasing concentrations of angiotensin II, was recorded in the absence and presence of candesartan, 0.003-10 nmol/l, irbesartan, 1-100 nmol/l, losartan, 1-100 nmol/l, and EXP-3174, 0.01-10 nmol/l, for a period of 90 min. In rabbit aortic strips, candesartan caused a non-parallel shift and suppression of the angiotensin II concentration-response curve, with complete suppression of the response to angiotensin II at a dose of candesartan of 1 nmol/l. In contrast, irbesartan, losartan and EXP-3174 all caused a parallel shift of the concentration-response curve. No suppression of the angiotensin II response was seen with losartan, while its active metabolite caused saturable suppression of the maximal response at higher concentrations. For irbesartan, some degree of suppression of the maximal response could not be excluded at the highest concentration studied. Similar concentration-response curves were obtained in rat portal vein. Data on protein binding for the different AT 1 -receptor blockers are variable in the literature. Plasma protein binding for the different AT 1 -receptor blockers was determined (in triplicate) by liquid chromatography with fluorescence detection after equilibrium dialysis (6 h) of cold drug at a concentration of 1500 nmol/l. Protein binding was high (see Table) and, for candesartan, losartan and its active metabolite EXP-3174, in accordance with previously reported levels. For irbesartan, a large discrepancy in protein binding between previously reported and the present experimental data, obtained from two different non-associated laboratories, was found. The higher plasma protein binding for irbesartan found in the present study may explain why high doses of irbesartan seem to be needed for clinical efficacy. It appears unlikely that losartan exerts any significant inhibitory effect at therapeutic plasma levels, and the main AT 1 -blocking effect observed after oral losartan is probably exerted by EXP-3174. It is concluded that AT 1 -receptor blockers differ in their ability to inhibit angiotensin II-mediated vascular contraction, and that the antagonistic characteristics are similar in vessel preparations of different origins and with different degrees of AT 1 -receptor reserve.

Actin polymerization stimulated by contractile activation regulates force development in canine tracheal smooth muscle.

1. The role of actin polymerization in the regulation of smooth muscle contractility was investigated in canine trachealis muscle strips. The effect of contractile activation on the content of monomeric globular (G)-actin was estimated by the method of DNase I inhibition. The G-actin content was 30 % lower in extracts of muscle strips activated with 10-4 M acetylcholine (ACh) than in extracts from unstimulated muscle strips. The decrease in G-actin in response to contractile stimulation was prevented by latrunculin-A, an agent that prevents actin polymerization by binding to G-actin monomers. 2. The inhibition of actin polymerization by latrunculin-A markedly depressed force development in response to ACh but had no effect on ACh-induced myosin light chain (MLC) phosphorylation. Latrunculin also suppressed the length sensitivity of force during ACh-induced isometric contractions. The actin-capping agent cytochalasin-D also markedly inhibited force and caused only a slight decrease in MLC phosphorylation. Cytochalasin-D also inhibited force in alpha-toxin-permeabilized muscle strips that were activated either by Ca2+ or by ACh at constant pCa. No disorganization of smooth muscle cell ultrastructure was detected by electron microscopy or by immunofluorescence microscopy of muscles treated with either agent. 3. The results suggest that the polymerization of actin is stimulated by the contractile activation of tracheal smooth muscle and that this actin polymerization contributes directly to force development. In addition, actin filament remodelling contributes to the length sensitivity of tracheal smooth muscle contractility.

Effect of milrinone on small mesenteric artery vasoconstriction: role of K(+) channels.

We examined whether milrinone-mediated attenuation of small mesenteric artery vasoconstriction results predominantly from the activation of vascular smooth muscle K(+) channels. Resistance arteries (approximately 150 micrometers) were dissected from rat mesentery and were mounted on a wire myograph. Isometric force development in response to increasing concentrations of norepinephrine (NE) was monitored before and after treatment with the type 3 phosphodiesterase inhibitor milrinone. Milrinone significantly reduced NE-induced vasoconstriction, attenuating both NE sensitivity and maximal tension generation. Inhibition of ATP-sensitive K(+) channels or voltage-gated K(+) channels did not prevent the milrinone-induced attenuation of NE responses. Blockade of inwardly rectifying K(+) channels or Ca(2+)-sensitive K(+) channels prevented the milrinone-mediated reduction in NE sensitivity, but this effect was apparently due to direct enhancement of vasoconstrictor responsiveness rather than interference with the mechanism of milrinone action. In addition, milrinone elicited substantial relaxation in vessels preconstricted with 100 mM KCl. This effect was mimicked by the adenylyl cyclase activator forskolin and was reversed by the Rp diastereomer of cAMP, which is a cAMP-dependent protein kinase (PKA) inhibitor. Our results indicate that cAMP/PKA-mediated impairment of vasoconstriction may occur without the contribution of K(+) channel regulation.

Effects of treatment with FK409, a nitric oxide donor, on collar-induced intimal thickening and vascular reactivity

Intimal thickening in arteries is considered a site of predilection for atherosclerosis. In a rabbit model of early atherosclerosis, a silastic collar was placed around the carotid artery, which resulted in the formation of intimal thickening. We investigated whether the oral application of FK409 ((±)-( E)-4-ethyl-2-[( E)-hydroxyimino]-5-nitro-3-hexenamide, 10 mg kg −1 day −1, p.o.), a nitric oxide donor, inhibited the collar-induced intimal thickening as well as accompanying reactivity changes in rabbit carotid artery. The intimal thickening was significantly inhibited by FK409. The collar treatment increased the pD 2 value of 5-hydroxytryptamine (5-HT) whereas it decreased those of phenylephrine and acetylcholine and did not significantly alter that of nitroglycerine. Maximal contractile force development in response to potassium chloride (KCl), 5-HT and phenylephrine was decreased in collared arteries. The collar did not alter the maximal relaxant effects of acetylcholine and nitroglycerine. Despite the significant reduction of intimal thickening, FK409 treatment did not affect these collar-induced modifications in vascular reactivity.

Central role of heterocellular gap junctional communication in endothelium-dependent relaxations of rabbit arteries.

1. The contribution of gap junctions to endothelium-dependent relaxation was investigated in isolated rabbit conduit artery preparations pre-constricted by 10 microM phenylephrine (PhE). 2. Acetylcholine (ACh) relaxed the thoracic aorta by approximately 60 % and the superior mesenteric artery (SMA) by approximately 90 %. A peptide possessing sequence homology with extracellular loop 2 of connexin 43 (Gap 27, 300 microM) inhibited relaxation by approximately 40 % in both artery types. Gap 27 also attenuated the endothelium-dependent component of the relaxation induced by ATP in thoracic aorta but did not modify force development in response to PhE. 3. NG-nitro-L-arginine methyl ester (L-NAME, 300 microM), an inhibitor of NO synthase, attenuated ACh-induced relaxation by approximately 90 % in the aorta but only by approximately 40 % in SMA (P < 0.05). Residual L-NAME-insensitive relaxations were almost abolished by 300 microM Gap 27 in aorta and inhibited in a concentration-dependent fashion in SMA (approximately 50 % at 100 microM and approximately 80 % at 10 mM). Gap 27 similarly attenuated the endothelium-dependent component of L-NAME-insensitive relaxations to ATP in aorta. 4. Responses to cyclopiazonic acid, which stimulates endothelium-dependent relaxation through a receptor-independent mechanism, were also attenuated by Gap 27, whereas this peptide exerted no effect on the NO-mediated relaxation induced by sodium nitroprusside in preparations denuded of endothelium. 5. ACh-induced relaxation of 'sandwich' mounts of aorta or SMA were unaffected by Gap 27 but completely abolished by L-NAME. 6. We conclude that direct heterocellular communication between the endothelium and smooth muscle contributes to endothelium-dependent relaxations evoked by both receptor-dependent and -independent mechanisms. The inhibitory effects of Gap 27 peptide do not involve homocellular communication within the vessel wall or modulation of NO release or action.