Regulation of pulmonary arterial myosin phosphatase activity in neonatal circulatory transition and in hypoxic pulmonary hypertension: a role for CPI-17.

Neonatal circulatory transition is dependent upon tightly regulated pulmonary circuit relaxation. Persistent pulmonary hypertension of the newborn (PPHN) is characterized by pulmonary arterial myocyte relaxation failure. We examined the effect of short course (72 hour) in vivo normobaric hypoxia in newborn swine on smooth muscle contractile enzyme activity and regulatory phosphoprotein abundance, in tissue homogenates of 2nd to 4th generation pulmonary arteries. Myosin light chain kinase (MLCK) and phosphatase (MLCP) protein contents were unchanged in hypoxic pulmonary arteries compared to controls. MLCP activity increased in normoxic animals from birth to day 3. This was ablated by hypoxia; phosphatase activity, measured as in vitro myosin light chain dephosphorylation, was decreased significantly (P < 0.005) in the hypoxic group. Inhibitory site phosphorylations of MLCP myosin binding subunit at threonines 696 and 850 were similar in both hypoxic and normoxic subjects, suggesting that downregulation of MLCP in hypoxia does not involve this pathway. However, content of regulatory protein CPI-17 (protein kinase C-related phosphatase inhibitor) increased from birth in hypoxic subjects (P < 0.05); active (phosphorylated) CPI-17 protein abundance declined after birth in normals, but increased in hypoxic arteries (P < 0.05). This corresponded with the decrease in phosphatase activity. We speculate that CPI-17 may play a role in myosin phosphatase upregulation during neonatal circulatory transition, and in hypoxic inhibition of pulmonary phosphatase activity in PPHN.

Relaxation of tetanised smooth muscle of canine saphenous vein.

Since vascular filling depends on the cross-sectional area of the venous bed and since this is determined by the state of relaxation of the mural smooth muscle, we have studied load bearing capacity during relaxation of the smooth muscle of canine saphenous vein. The effect of load on the time course of relaxation was analysed either by comparing afterloaded contractions against various loads or by imposing abrupt alterations in load (load clamps). Unlike mammalian cardiac muscle in which relaxation was reported sensitive to loading conditions, relaxation in the smooth muscle of the saphenous vein was largely independent of loading conditions. In this it resembled frog heart muscle. This type of relaxation, which is not influenced by manipulation of loading conditions, has been termed "inactivation-dependent" relaxation. It appears to operate in muscle tissue in which the calcium sequestering apparatus is poorly developed and sequestration or some process downstream to it appears to be the rate limiting step during relaxation.

Sympathetic stimulation and hypertension in the pyridoxine-deficient adult rat.

Pyridoxal phosphate is the coenzyme of various decarboxylases involved in the formation of monoamine neurotransmitters such as gamma-aminobutyric acid, serotonin, dopamine, and norepinephrine. Adult male Sprague-Dawley rats placed on a pyridoxine-deficient diet for 8 weeks showed significant hypertension compared with pyridoxine-supplemented controls. Hypothalamic contents of pyridoxal phosphate, gamma-aminobutyric acid, and serotonin in the pyridoxine-deficient rats were significantly lower than those in pyridoxine-supplemented controls. Hypertension was associated with sympathetic stimulation. Treatment of pyridoxine-deficient rats with a single dose of pyridoxine (10 mg/kg body weight) reversed the blood pressure to normal levels within 24 hours, with concomitant restorations of hypothalamic serotonin and gamma-aminobutyric acid as well as the return of plasma norepinephrine and epinephrine to normal levels. Also, pyridoxine treatment reversed the hypothalamic hypothyroidism observed in pyridoxine-deficient rats. These results indicate an association between pyridoxine deficiency and sympathetic stimulation leading to hypertension.

Smooth muscle contractility. Effects of hypoxia.

Mechanical perturbation of smooth muscle provides information about the mechanical properties of its crossbridges. We have developed a method for identifying: (1) that normally cycling and very slowly cycling are sequentially activated, (2) the moment of this transition, and (3) the proportions of the two types of bridges recruited. Hypoxia decreases muscle shortening ability before isometric force. The former is due to depression of activity of both types of bridges.

Fatigue of mouse diaphragm muscle in isometric and isotonic contractions.

Fatiguabilities of mouse diaphragm muscle in vitro in isometric and isotonic contractions were compared in this study. Isolated mouse diaphragm muscle was stimulated repetitively to induce fatigue during both isometric and isotonic contractions. The supramaximal electrical stimulation used was a train of 100-Hz, 0.5-ms pulses delivered to the muscle every 2 s for 0.5 s. The percentage decrease in isometric tension from beginning to end of the fatiguing process was used as the index of fatigue. The experiments were carried out at different PO2 levels in both normal and zero-glucose Ringer solutions. It was found that fatigue developed more rapidly in isotonic contractions than in isometric ones. Also, the extracellular glucose level demonstrated little effect on the muscle's short-term fatiguability, whereas reductions in the extracellular PO2 exerted a profound effect, especially in the case of isotonic fatigue.

Contractile properties of bronchial smooth muscle with and without cartilage.

The majority of in vitro studies on airway smooth muscle have used the trachealis (TSM) as a convenient substitute for muscle from airways that constitute the flow-limiting segment. The latter are technically difficult to work with. However, because the site of maximum resistance to airflow is at the third to seventh generations of the bronchial tree, the trachealis preparation is of limited value. Length-tension and force-velocity properties were therefore studied at optimal length (lo) of canine bronchial smooth muscle (BSM) from which cartilage had been carefully removed. Normalized maximum isometric tension or stress (Po x 10(4) N/m2) for BSM was 7.1 +/- 0.19 (SE), which was similar to that of BSM with cartilage (BSM+C, 6.8 +/- 0.21) but lower than for TSM (18.2 +/- 0.81). At length greater than lo, the BSM+C was stiffer than the BSM. The values of maximum shortening capacity (delta Lmax), obtained directly from isotonic shortening at a load equal to the resting tension at lo, were 0.76 lo +/- 0.03, 0.41 lo +/- 0.02, and 0.24 +/- 0.02 lo for TSM, BSM, and BSM+C, respectively. The BSM and BSM+C delta Lmaxs were different (P less than 0.05). Maximal shortening velocities (Vo) for BSM, elicited at 2, 4, and 8 s by quick release in the course of an isometric contraction were significantly higher than for the BSM+C. Vos showed gradual decreases in all three groups in the later phase of contraction, suggesting the operation of latch bridges.(ABSTRACT TRUNCATED AT 250 WORDS)

Time dependence of series elasticity in tracheal smooth muscle.

The stress-strain curve for the series elastic component (SEC) of tracheal smooth muscle was obtained by quick releasing the muscle from isometric tension to various afterloads and measuring the elastic recoils (SEC lengths) at a specific time after stimulation. A family of such curves was obtained by releasing the muscle at different points in time during contraction. Stiffnesses of the SEC (slopes of the stress-strain curves) at a specific stress level calculated from these curves (constant-stress stiffness) showed significant difference from one another. The same difference can also be characterized by the slope of the linear stiffness-stress curve, the constant A. The constant A during a 10-s isometric contraction was maximal at 2 s. It then decreased with time. This stiffness behavior is only seen when the effect of stress is held constant or eliminated. If stress is allowed to increase with time as it does during a tetanus then stiffness appears to increase monotonically. The SEC stiffness during active contraction was found to vary within the boundaries of the stiffness of muscle in rigor (upper limit) and that at resting state (lower limit).

Velocity-length-time relations in canine tracheal smooth muscle.

Zero-load velocity (V0) as a function of the length of canine tracheal smooth muscle was obtained by applying zero-load clamps to isotonically contracting muscle under various loads. The load clamps were applied at a specific time after onset of contraction. The magnitude of the isotonic load therefore determines the length of the muscle at the moment of release or at the moment the unloaded shortening velocity was measured. A family of such V0-muscle length (L) curves was obtained at 1-s intervals in the time course of contraction. The V0-L curve was fitted by a parabolic function with satisfactory goodness of fit. The maximum shortening velocity at optimum muscle length varied with time, but the minimum length at which V0 diminished to zero was time independent.

Developmental differences in vascular smooth muscle mechanics in pulmonary and systemic circulations.

To evaluate the maturational changes in vascular muscle mechanics we studied and compared the isotonic half-time relaxation (t1/2P,CE) and maximal load-bearing capacity normalized to stress of pulmonary and systemic arterial muscle from perinatal and adult sheep. For the pulmonary and systemic vessels t1/2P,CE was significantly shorter in adult than in perinatal sheep (P < 0.01). In newborns t1/2P,CE of the pulmonary vessels was 185 +/- 31 (SE) s, longer than that of the systemic vessels (64 +/- 10 s; P < 0.01). In adults t1/2P,CE of the pulmonary vessels (101 +/- 14 s) was longer than that of the systemic vessels (37 +/- 5 s; P < 0.01). Maximal load-bearing capacities normalized to stress of pulmonary vessels of fetal and newborn sheep were twofold greater than those of adult sheep and of the systemic vessels of newborns and adults (P < 0.01). In conclusion, significant maturational changes in the isotonic and isometric mechanical properties of vascular pulmonary and systemic smooth muscle were observed in sheep.

Use of a new index to study relaxation in a vascular model of anaphylactic shock.

We have reported increased smooth muscle shortening ability in ragweed pollen-sensitized saphenous vein (SSV). This may account for the vascular hyperreactivity of anaphylactic shock. We have now investigated relaxation in SSV. Because isotonic relaxation is load and initial contractile element length dependent, we developed an adjusted half-relaxation time index, which was independent of these variables. Muscle activation state was monitored by measuring maximum unloaded velocity. The relaxation index showed no difference between SSV and control saphenous vein after 2.5, 10, and 15 s of electrical stimulation; however, after 1 s of stimulation it was prolonged significantly in SSV. We concluded that the cross bridges activating early in contraction demonstrated prolonged relaxation. Activation state during muscle relaxation spontaneously increased toward the end of relaxation, coincident with a slowing in isotonic re-elongation rate. This was seen only in muscles relaxing from 15 s of stimulation. Our results indicate that 1) the relaxation properties of early cycling (1 s) cross bridges are altered after sensitization; and 2) toward the end of isotonic relaxation, cross-bridge cycling rate increases spontaneously, a phenomenon not previously reported. We speculate that the rapid re-elongation in late relaxation may reactivate muscle.

A modified force-velocity equation for smooth muscle contraction.

It has been suggested that in skeletal muscle the force-velocity relationship may not be a simple hyperbolic one, as defined by Hill's equation. To determine whether smooth muscle demonstrated the same properties, quick-release force-velocity curves were obtained from canine tracheal smooth muscle. The results showed that the observed data points for tracheal smooth muscle systematically deviated from a hyperbola. Such deviation occurred at values of force (P) approaching maximum isometric force (Po) for curves elicited by quick release at 2 and 10 s in the course of isometric contractions. Shortening velocities under a given afterload were overestimated at the high-force end (P > 75% Po) by Hill's equation; this implied that a relationship more complex than a simple hyperbola was involved at high loads. We next focused on finding an equation to also fit those directly measured data points that did not conform to a hyperbola. Our rationale in developing the equation was that a plot of the linearized transform of Hill's equation should yield a straight line over the entire range of loads at which velocities were measured. The plot demonstrated that, in the low-load high-velocity portion of the curve, a peak value was reached at 70-80% Po, which decreased as load increased in the high-load low-velocity portion.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of two clusters of mechanical properties of smooth muscle along the airway tree.

Heterogeneity of function of airway smooth muscle along the airways may be of great importance in regulating regional ventilation and in the pathogenesis of asthma. To investigate the distribution of mechanical properties of airway smooth muscle along the airway, muscle strips free of cartilage and epithelium from the trachea down to bronchial generation 6 were studied by employing electrical field stimulation. Results showed that smooth muscle mechanical performance decreased progressively down the airway tree. Cluster analysis further indicated that smooth muscle from these airways could be divided into two groups: 1) an extrapulmonary group, which contains muscle from the trachea and bronchial generations 1 and 2 and is characterized by higher maximum shortening capacity and zero-load velocity of shortening (V0) in early shortening, the expected decrease of V0 values (the so-called latch phase) in the later phase of shortening, and lower sensitivity to stimulation; and 2) an intrapulmonary group, which contains bronchi from generations 3-6 and has a lower maximum shortening capacity and V0 in early shortening but higher sensitivity to stimulation. The relatively lower mechanical performance of intrapulmonary bronchial smooth muscle may represent a safety device that prevents excessive smooth muscle shortening in vivo.

Studies of myofibrillar ATPase in ragweed-sensitized canine pulmonary smooth muscle.

The maximal shortening velocities of tracheal and pulmonary vascular smooth muscle from ragweed-sensitized dogs were significantly higher than those of muscles from their littermate controls. Myofibrils of tracheal and pulmonary vascular smooth muscle from ragweed-sensitized and control dogs were obtained with use of Triton X-100 homogenizing solution. The myofibrillar adenosinetriphosphatase (ATPase) activities of the sensitized tissues were significantly higher (P less than 0.05) than those of their respective controls.

Relevance of classification by size to topographical differences in bronchial smooth muscle response.

To investigate heterogeneity of airway smooth muscle response, we studied strips of large and small branches from third- to sixth-generation bronchi obtained from ragweed antigen-sensitized and control dogs. The response to electrical field stimulation and carbamylcholine chloride was greater in strips from larger branches of the same generation when expressed as "tissue stress" (force per unit cross-sectional area of the whole tissue), whereas no difference emerged with use of the more appropriate "smooth muscle stress" (force per unit cross-sectional area of the muscle tissue). The response to histamine was significantly higher in small branches than in large ones, and histamine sensitivity [mean effective concentration (EC50)] was 7.79 x 10(-6) [geometric standard error of the mean (GSEM) 1.20] and 1.49 x 10(-5) M (GSEM 1.14), respectively (P < 0.01). Strips from control and sensitized animals at each site and strips from different generations did not show any significant difference. When we clustered our preparations according to dimensions, the response to histamine was significantly higher in small bronchi than in large ones and histamine EC50 was 8.95 x 10(-6) (GSEM 1.17) and 1.57 x 10(-5) M (GSEM 1.18), respectively (P < 0.05). We conclude that evaluation of muscle response in different tissues requires appropriate normalization. Furthermore, classification into generations is inadequate to study bronchial responsiveness, inasmuch as major differences originate from airway size.

Mechanical alterations in sensitized canine saphenous vein.

Because it is likely that antigen sensitization is not restricted to airway smooth muscle but probably involves all tissues in the animal, we decided to test the hypothesis that saphenous vein from pollen extract-sensitized dogs is sensitized and is, in addition, mechanically altered. To this end, we studied responses to specific antigen challenge and length-tension and force-velocity relationships in sensitized (SSV) and control saphenous veins (CSV). The antigen challenge revealed that the venous smooth muscle was strongly sensitized and developed a Schultz-Dale response, the two main mediators of which were histamine and norepinephrine. Length-tension relationship studies showed that whereas there is no difference in maximum isometric tension development between SSV and CSV [93.95 +/- 7.34 and 87.86 +/- 4.00 (SE) mN/mm2, respectively], SSV exhibited a significantly greater maximum isotonic shortening capacity of 0.613 +/- 0.009 optional length (lo) vs. 0.578 +/- 0.012 lo for CSV. Unloaded shortening velocity (Vo), which reflects the cross-bridge cycling rate, was determined at different times after the onset of electrical stimulation. Maximum Vo was attained early (5 s) in the contraction; a 15% decline in Vo was observed at the plateau of the contraction (15 s). At 5 s, Vo of SSV (0.316 +/- 0.019 lo/s) was significantly higher than that of CSV (0.269 +/- 0.018 lo/s), although Vos were same at 15 (0.249 +/- 0.021 lo/s for SSV and 0.237 +/- 0.019 lo/s for CSV). The increase in shortening likely results from th e increase in the early cross-bridge cycling rate because our studies show that the bulk of shortening occurs in the first 5 s.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in cross-bridge properties of sensitized airway smooth muscle.

We have reported previously that in ovalbumin-sensitized canine tracheal smooth muscle (TSM) the maximum ability to shorten is increased. This could account for the increased airway narrowing seen in vivo in allergic bronchoconstriction. It was associated with increased velocity of shortening. We now report that, by using an electromagnetic muscle lever system, quick releases were applied to control and sensitized TSM at 0.5-s intervals throughout the course of a lightly preloaded 10-s isotonic contraction. From the records obtained it is possible to determine that, early in contraction, shortening is brought about by relatively rapidly cycling [0.35 optimal muscle length units +/- 0.033/s (SE)] cross bridges. We also report that in the sensitized TSM it is the early bridges that increase their velocity by 26.6% (P less than 0.05) compared with similar bridges in muscles from control animals. Since 70% of the maximum shortening of the muscle occurs when early bridges are operative, it is likely that these bridges are responsible for the major part of the shortening. It is thus probable that increased allergic bronchoconstriction is produced by increased activity of early, rapidly cycling bridges. The bridges that are active late in the shortening show no difference between control and sensitized airway smooth muscles.

Increased myosin light chain kinase content in sensitized canine saphenous vein.

Our previous studies revealed that smooth muscle from sensitized canine saphenous vein (SCSV) demonstrated greater active shortening capacity, maximum shortening velocity, and prolonged relaxation vis-a-vis the control muscle. These changes could be responsible for the in vivo hyperreactivity of venous smooth muscle observed in anaphylactic shock. Because smooth muscle cross-bridge cycling is regulated by myosin light chain kinase (MLCK)-dependent phosphorylation of the 20-kDa myosin light chain (MLC20), we studied MLC20 and MLCK phosphorylation in homogenates of SCSV and veins from littermate control dogs. We found that phosphorylation of MLC20 in SCSV homogenate was higher (42.26 +/- 5.10%) compared with control homogenates (26.69 +/- 3.30%; P < 0.05); MLCK content was significantly higher in SCSV homogenates [0.169 +/- 0.019 (SE) mu g/mg protein] than in control homogenates (0.075 +/- 0.004 mu g/mg protein; P < 0.05). Total MLCK activity increased from 6.16 +/- 0.60 x 10(-5) nmol Pi x mg fresh weight of tissue-1 x min-1 in control homogenates to 12.50 +/- 2.50 x 10(-5) nmol Pi x mg fresh weight of tissue-1 x min-1 in sensitized homogenates (P < 0.05). Specific MLCK activity was, however, similar in sensitized and control homogenates. The results of our study suggest that elevation of MLCK content in the homogenate could account for the increased contractility of the SCSV in anaphylactic shock.

Ca2+-dependent facilitated shortening in isotonic contraction of trachealis muscle.

We compared isotonic shortening with isometric force generation as a function of external Ca2+ in 166 tracheal smooth muscle (TSM) strips from 27 mongrel dogs in vitro. Concentration-response curves were generated with muscarinic stimulation (acetylcholine, ACh), alpha-adrenergic receptor activation (norepinephrine after beta-adrenoceptor blockade, NE), serotonin (5-HT), and KCl-substituted Krebs-Henseleit solution. The concentrations of 5-HT causing half-maximal shortening (ECS50, 1.54 +/- 0.14 X 10(-7) M) and half-maximal active isometric tension (ECT50, 1.72 +/- 0.30 X 10(-7) M) were similar (P = NS). Likewise, ECS50 (21.9 +/- 0.7 mM) and ECT50, (22.0 +/- 0.9 mM) were similar for KCl. In contrast, facilitated isotonic shortening (i.e., greater isotonic shortening for comparable degrees of force generation) was elicited with ACh and NE for all levels of force generation between 15 and 85% of maximum and for all concentrations of ACh from 3 X 10(-8) to 3 X 10(-5) M (P less than 0.05 for all points). Facilitated isotonic shortening also was elicited for all concentrations of NE from 10(-8) to 10(-6) M (P less than 0.05 for all points). Removal of Ca2+ from the perfusate substantially reduced the potency of ACh (P less than 0.001) and abolished differences between ECS50 (2.23 +/- 0.28 X 10(-5) M) and ECT50 (2.50 +/- 0.46 X 10(-5) M, P = NS). We demonstrate that for comparable degrees of force generation, muscarinic and alpha-adrenergic receptor activation cause greater isotonic shortening than KCl or 5-HT and that this facilitated shortening is associated with the concentration of external Ca2+.

Bronchial smooth muscle mechanics of a canine model of allergic airway hyperresponsiveness.

Although we have reported that tracheal smooth muscle from sensitized dogs shows altered mechanical properties, we did not know, because of technical difficulties with the preparation, whether similar changes occur in the properties of sensitized central bronchial smooth muscle (BSM), the site at which the acute asthmatic response is believed to develop. We have now succeeded in developing a cartilage-free BSM preparation that retains optimal mechanical properties. Such strips were obtained from mongrel dogs that had been sensitized to ragweed pollen. Controls were littermates injected with adjuvant alone. Length-tension relationships were obtained for both control and sensitized BSM strips (CBSM and SBSM, respectively). The maximal active stresses were the same (P greater than 0.05) when normalized to muscle fraction in total tissue cross-sectional area [6.2 +/- 0.6 x 10(4) and 5.9 +/- 0.6 x 10(4) (SE) for SBSM and CBSM, respectively]. This suggests that optimal tension is an insensitive indicator of bronchial hyperresponsiveness and that isotonic studies might be more revealing. The maximal shortening velocity (Vo) for SBSM at 2 s [0.35 +/- 0.017 (SE) lo/s, where lo signifies optimal muscle length], in the course of a 10-s contraction, was significantly greater (P less than 0.05) than Vo measured for CBSM (0.27 +/- 0.015 lo/s). However, Vo did not differ at the 8-s point of contraction. The sensitized group demonstrated a statistically significantly greater maximal shortening capacity (0.67 +/- 0.04 lo) than the control group (0.51 +/- 0.04 lo). At 2 s of contraction, 80% of maximal SBSM shortening had been completed and was significantly greater than for CBSM.(ABSTRACT TRUNCATED AT 250 WORDS)

Different ontogeny of rate of force generation and shortening velocity in guinea pig trachealis.

Juveniles of many species, including humans, display greater airway responsiveness than do adults. This may involve changes in airway smooth muscle function. In the present work we studied force production and shortening velocity in trachealis from 1-wk-old (1 wk), 3-wk-old (3 wk), and 3-mo-old (adult) guinea pigs. Strips were electrically stimulated (60 Hz, 18 V) at their optimal length (l(o)) to obtain maximum active stress (P(o)) and rate of stress generation. Then, force-velocity curves were elicited at 2.5 s from the onset of the stimulus. By applying a recently developed modification of Hill's equation for airway smooth muscle, the maximum shortening velocity at zero load (V(o)) and the value alpha. gamma/beta, an index of internal resistance to shortening (Rsi), were calculated (alpha, beta, and gamma are the constants of the equation). P(o) increased little with maturation, whereas the rate of stress generation increased significantly (0.40 +/- 0.03, 0.45 +/- 0.03, 0. 51 +/- 0.03 P(o)/s for 1 wk, 3 wk, and adult animals). V(o) slightly increased early with maturation to decrease significantly later (1. 79 +/- 0.67, 2.45 +/- 0.92, and 0.55 +/- 0.09 l(o)/s for 1 wk, 3 wk, and adult animals), whereas the Rsi showed an opposite trend (14.98 +/- 5.19, 8.99 +/- 3.01, and 32.07 +/- 5.54 mN. mm(-2). l(o)(-1). s for 1 wk, 3 wk, and adult animals). This early increase of force generation in combination with late increase of Rsi may explain the changes of V(o) with age. An elevated V(o) may contribute to the incidence of airway hyperresponsiveness in healthy juveniles.