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Effect of cytochalasin D on smooth muscle contraction.

Cylindrical segments of extraparenchymal pulmonary artery (essentially a preparation of smooth muscle with regard to contractile capability) were isolated from adult male rats. They were mounted in an isometric muscle bath in physiological salt solution (PSS) in an environment of 95% O2/5% CO2. After allowing 1 h for equilibration, the maximum force generated by the tissue in response to a depolarizing solution was determined. After relaxation, vessels were incubated for 1 h in one of several concentrations of cytochalasin D (CD) (0.01, 0.05, 0.5, 1, 10 micrograms/ml) and the response to stimulation retested immediately after returning to PSS, and then at 30 minute intervals up to 2 h. CD inhibited the ability of vascular smooth muscle to generate force (contract) in a concentration-dependent manner. The inhibitory effect was reversible within a short period of time. Quantitative electron microscopic examination of these vessels suggested that CD disrupts the integrity of myofilaments, especially at sites of "dense bodies." Our results indicate that a percentage of actin in smooth muscle cells is not permanently in the filamentous "F" form, but is part of the G:F actin system of the cell, labile to polymerization:depolymerization. The ability of smooth muscle cells to generate force could depend on the proper functioning of the F:G actin "treadmill."

Animals↗

Dependence of energy output on force generation during muscle contraction.

It has been proposed that the energy (heat + work) output of an isometric twitch is determined by the force that is generated under conditions of invariant activation, irrespective of muscle length. To test the effect of length and force on total energy output, muscles were stretched by increments beyond the muscle length at which twitch force is maximum (LO) and then stimulated; energy output and force then were measured. These data were compared with isovelocity twitches in which stimulated muscles, initially at different lengths, shortened (near maximum velocity) a constant distance and then redeveloped tension at lengths less than LO. If energy liberation was determined by force generation, plots of energy output versus force produced would be parallel with isovelocity twitches liberating extra energy as shortening heat. As predicted, the ratio of the slopes (n = 13) of these relations, 0.98 +/- 0.02, was not different from 1 and the shortening heat coefficient (alphaF/Pot, measured from the difference in intercepts), 0.15 +/- 0.01, was near to the expected value. Therefore, energy liberation in twitches appears to be uniquely determined by force generation and not by muscle length.

Animals↗

Mechanics and energetics of muscle contraction in normal and dystrophic chickens.

Energy liberation and isometric force generation were compared at 25 degrees C in isolated normal (n = 15) and dystrophic (n = 18) posterior latissimus dorsi muscles (PLD) from 16- to 33-day-old chickens. Twitch-energy liberation in dystrophic muscle decreased by 27 +/- 6%, and force per cross-sectional area decreased by 26 +/- 11%. Tetanic energy liberation in dystrophic PLD was depressed by 32 +/- 10% compared with a 22 +/- 9% reduction in peak force per cross-sectional area plus a 26 +/- 3% reduction in ability to maintain force. Normalizing results using the force-time integral demonstrates an unaltered force generation per unit energy liberation in dystrophic PLD (7 +/- 11% difference from normal). Kinetic properties of the mechanical responses were not significantly different in normal compared with dystrophic PLD. Dystrophic PLD demonstrated hyperexcitability to nerve transection and electrical stimulation. Although force-generating capacity is reduced in dystrophic PLD, coupling of force production to energy utilization appears unaltered.

Animals↗

Developmental changes in agonist-mediated gastric smooth muscle contraction in the rabbit.

We studied smooth muscle strips from rabbit proximal stomach to explore the age-related changes in agonist-mediated contraction. Strips from neonatal (1 d) and weanling (11 wk) rabbits were oriented to measure isometric tension in circular muscle. Bethanechol stimulated maximal tension in both age groups. Although the potencies for bethanechol were similar (ED50 approximately 5 microM), the maximal response was nearly 4-fold greater in weanling (1140 +/- 73 mN/cm2) versus neonate (305 +/- 54 mN/cm2), p less than 0.001. Maximum stress increased with age for bethanechol, high extracellular K+, and substance P, but not for serotonin, cholecystokinin octapeptide, neurotensin, or bombesin. Only bombesin stimulated larger contraction in neonates (152 +/- 37 mN/cm2) versus weanlings (86 +/- 20 mN/cm2), p less than 0.05. Potencies did not change with age, except for substance P and serotonin. Substance P and serotonin induced early phasic and prolonged tonic contractions, which were unaffected by tetrodotoxin or atropine. ED50 for the phasic and tonic components of substance P-stimulated contraction in neonates were 1.8 and 7.7 nM. Substance P was 60-70 times more potent in neonates versus weanlings (p less than 0.001). ED50 for serotonin-stimulated contraction in neonates (33 and 22 nM, respectively) were 20-30 times more potent than in weanlings (p less than 0.05). Motilin, morphine, epidermal growth factor, and gastrin did not stimulate contraction at either age. We conclude that age-dependent changes in agonist potency and efficacy may be one factor to explain in part the changes that occur in gastric motility during postnatal development.

Animals↗

Diazepam reveals different rate-limiting processes in rat skeletal muscle contraction.

The action of the tranquilizer diazepam on rat skeletal muscle showed that relaxation of isometric twitches is controlled by different processes in extensor digitorum longus (fast-twitch) and soleus (slow-twitch) muscles. Diazepam caused an increase in the amplitude of twitches in fibres from both muscles but increased the twitch duration only in soleus. The amplitude of fused tetani were reduced in both muscles and the rate of relaxation after the tetanus slowed by as much as 34% when the amplitude of the tetanus was reduced by only 11%. The slower tetanic relaxation indicated that calcium uptake by the sarcoplasmic reticulum was slower than normal in slow- and fast-twitch fibres. We conclude therefore that calcium uptake by the sarcoplasmic reticulum is rate limiting for twitch relaxation in slow-twitch but not fast-twitch fibres and suggest that calcium binding to parvalbumin controls relaxation in the fast fibres.

Animals↗

Involvement of rho and rho-associated kinase in sphincteric smooth muscle contraction by angiotensin II.

The tonic smooth muscles of lower esophageal sphincter (LES) and internal anal sphincter (IAS) are subject to modulation by the neurohumoral agents. We report that angiotensin (Ang) II-induced contraction of rat IAS and LES smooth muscle cells (SMC) was inhibited by Clostridium botulinum C3 exozyme, HA 1077 and Y 27632, suggesting a role for Rho kinase and a Rho-associated kinase (ROK). Ang II-induced contraction of the SMC was also attenuated by genistein, antibodies to the pp60(c-src), p(190) RhoGTPase-activating protein (p190 RhoGAP), carboxyl terminus of Galpha13, carboxyl terminus peptide, and ADP ribosylation factor (ARF) antibody. Ang II-induced increase in p(190) RhoGAP tyrosine phosphorylation was attenuated by genistein. Furthermore, Ang II-induced increase in smooth muscle tone and phosphorylation of myosin light chain (MLC; 20 kDa; MLC20-P) were attenuated by Y 27632 and genistein. The results suggest an important role for Galpha13 and pp60(c-src) in the intracellular events responsible for the activation of RhoA/ROK in Ang II-induced contraction of LES and IAS SMC.

ADP Ribose Transferases↗

Developmental changes in agonist-mediated colonic smooth muscle contraction in the rabbit.

We studied smooth muscle strips from rabbit distal colon to determine age-related changes in length-tension properties and agonist-mediated contraction. Strips from newborn (1-d-old) and weanling (11-wk-old) rabbits were oriented to measure isometric tension in longitudinal muscle. Active tension comprised 47 +/- 4 and 75 +/- 5% of the total tension in the newborn and weanling, respectively. Total and active tensions in the weanling were greater than in the newborn (p less than 0.001). Although the potencies for bethanechol were similar, the maximal response was nearly 9-fold greater in weanlings (6900 +/- 292 mN/cm2) versus newborns (753 +/- 112 mN/cm2), p less than 0.001. Maximal stress increased with age for bethanechol, high extracellular potassium, substance P, neurokinin A, cholecystokinin octapeptide, bombesin, and serotonin. ED50 for bethanechol, substance P, neurokinin A, and bombesin did not change with age. Serotonin was 12 times more potent in newborns versus weanlings (p less than 0.05). In contrast, cholecystokinin octapeptide was five times less potent in newborns (18.6 nM versus 3.4 nM, respectively, p less than 0.05). Substance P-induced contractions were inhibited partially by atropine. We conclude that length-tension properties of longitudinal colonic smooth muscle differ, and responses to agonists increase with age.

Animals↗