Mechanics of the canine diaphragm in ascites: a CT study.

Ascites causes an increase in the elastance of the abdomen and impairs the lung-expanding action of the diaphragm, but its overall effects on the pressure-generating ability of the muscle remain unclear. In the present study, radiopaque markers were attached to muscle bundles in the midcostal region of the diaphragm in five dogs, and the three-dimensional locations of the markers during relaxation and during phrenic nerve stimulation in the presence of increasing amounts of ascites were determined using a computed tomographic scanner. From these data, accurate measurements of muscle length and quantitative estimates of diaphragm curvature were obtained, and the changes in transdiaphragmatic pressure (Pdi) were analyzed as functions of muscle length and curvature. With increasing ascites, the resting length of the diaphragm increased progressively. In addition, the amount of muscle shortening during phrenic nerve stimulation decreased gradually. When ascites was 100 ml/kg body wt, therefore, the muscle during contraction was longer, leading to a 20-25% increase in Pdi. As ascites increased further to 200 ml/kg, however, muscle length during contraction continued to increase, but Pdi did not. This absence of additional increase in Pdi was well explained by the increase in the diameter of the ring of insertion of the diaphragm to the rib cage and the concomitant increase in the radius of diaphragm curvature. These observations indicate that the pressure-generating ability of the diaphragm is determined not only by muscle length as conventionally thought but also by muscle shape.     

Impact of acute ascites on the action of the canine abdominal muscles.

Although ascites causes abdominal expansion, its effects on abdominal muscle function are uncertain. In the present study, progressively increasing ascites was induced in supine anesthetized dogs, and the changes in abdominal (DeltaPab) and airway opening (DeltaPao) pressure obtained during stimulation of the internal oblique and transversus abdominis muscles were measured; the changes in internal oblique muscle length were also measured. As ascites increased from 0 to 100 ml/kg body wt, Pab and muscle length during relaxation increased. DeltaPab also showed a threefold increase (P < 0.001). However, DeltaPao decreased (P < 0.001). When ascites increased further to 200 ml/kg, resting muscle length continued to increase and muscle shortening during stimulation became very small so that active muscle length was 155% of the resting muscle length in the control condition. Concomitantly, DeltaPab returned to the control value, and DeltaPao continued to decrease. Similar results were obtained with the animals in the head-up posture, although the decrease in DeltaPao appeared only when ascites was greater than 125 ml/kg. It is concluded that 1) ascites adversely affects the expiratory action of the abdominal muscles on the lung; 2) this effect results primarily from the increase in diaphragm elastance; and 3) when ascites is severe, the abdomen cross-sectional area is also increased and the abdominal muscles are excessively lengthened so that their active pressure-generating ability itself is reduced.     

Hemodynamic effects of nicotine in canine skeletal muscle.

Studies were conducted in 36 artificially ventilated, anesthetized dogs to clarify hemodynamic effects of nicotine in resting gracilis muscle. In Series 1, effects of intravenous nicotine (36 micrograms/kg/min) were evaluated in (i) intact muscles (Condition 1), (ii) denervated muscles (Condition 2), (iii) denervated muscles following local alpha-adrenergic blockade (Condition 3), (iv) denervated muscles following combined local alpha- and beta-adrenergic blockade (Condition 4), and (v) intact muscles with aortic pressure maintained constant (Condition 5). In Series 2, nicotine was infused directly into the gracilis artery at a rate of 3.6 micrograms/kg/min. Muscle blood flow was obtained with an electromagnetic flowmeter and used to calculate vascular resistance and oxygen consumption (Fick equation). Plasma catecholamine levels were determined with a radioenzymatic method. Intravenous nicotine doubled mean aortic pressure under Conditions 1-4. In intact and denervated muscles (Conditions 1 and 2) proportional increases in vascular resistance, reflective of vasoconstriction, held blood flow constant. Muscle oxygen consumption was unchanged. alpha-Adrenergic blockade with phenoxybenzamine following denervation (Condition 3) converted muscle vasoconstriction to vasodilation during nicotine infusion. Additional beta-adrenergic blockade (Condition 4) restored muscle vasoconstriction. Nicotine-induced muscle vasoconstriction was maintained under controlled pressure (Condition 5). Intravenous nicotine significantly increased plasma catecholamine levels. Intra-arterial infusions of nicotine (Series 2) caused no hemodynamic changes in muscle. In conclusion, intravenous nicotine caused vasoconstriction in muscle, which was not due to reduced metabolic demand, pressure-flow autoregulation, or a direct [corrected] effect on vascular smooth muscle, but to stimulation of alpha-adrenergic receptors. Following denervation, this vasoconstriction was maintained by elevated plasma catecholamines. alpha-Adrenergic blockade unmasked nicotine-induced vasodilation mediated by beta-adrenergic receptors, whereas combined alpha- and beta-adrenergic blockade unmasked nicotine-induced vasoconstriction by a nonadrenergic mechanism.     

Cell potential and the sodium-potassium pump in vascular smooth muscle.

An electrogenic sodium-potassium pump appears to contribute materially to the steady-state potential and to certain of the transient potential responses of vascular smooth muscle. Since changes in cell potential in turn can lead to changes in contractile state, the pump is implicated in some of the constriction-dilation responses of blood vessels. The vasodilator action of potassium is explainable, for instance, through an effect on cell potential if (and only if) an electrogenic pump is assumed to be extruding sodium at a faster rate than it takes up potassium. This is supported by the observation that ouabain, an inhibitor of Na,K-ATPase activity, will eliminate or reverse the vascular effect of potassium. Furthermore, when the in vivo and in vitro effects on vascular smooth muscle of altered extracellular potassium concentration are compared to calculated cell potentials based on a model that includes an electrogenic pump, the experimental findings are shown to be logical and predictable.     

Bradykinin facilitates noradrenaline spillover during contraction in the canine gracilis muscle.

Noradrenaline spillover from skeletal muscle vascular areas increases during exercise but the underlying mechanisms are not well understood. Muscle contraction itself causes changes in many factors that could affect noradrenaline spillover. For instance, it has been reported that bradykinin is synthesized in skeletal muscle areas during contraction. Because the B2 bradykinin receptor facilitates noradrenaline spillover, it may be involved in the increase associated with contraction. In this experiment, we studied the effect of bradykinin on noradrenaline spillover in the in situ canine gracilis muscle, using the specific B2 antagonist HOE 140. The drug did not modify noradrenaline spillover at rest, but did cause a significant decrease during muscle contraction, from 558 to 181 pg min(-1). As reported previously in the literature, fractional extraction of noradrenaline decreased during muscle contraction. This effect was independent of HOE 140 treatment. In light of our results, it seems that bradykinin formation during muscle contraction may play an important part in the observed increase in noradrenaline spillover but does not affect fractional extraction.     

Analysis of excitation-contraction-coupling components in chronically stimulated canine skeletal muscle.

The chronic stimulation of predominantly fast-twitch mammalian skeletal muscle causes a transformation to physiological characteristics of slow-twitch skeletal muscle. Here, we report the effects of chronic stimulation on the protein components of the sarcoplasmic reticulum and transverse tubular membranes which are directly involved in excitation-contraction coupling. Comparison of protein composition of microsomal fractions from control and chronically stimulated muscle was performed by immunoblot analysis and also by staining with Coomassie blue or the cationic carbocyanine dye Stains-all. Consistent with previous experiments, a greatly reduced density was observed for the fast-twitch isozyme of Ca(2+)-ATPase, while the expression of the slow-twitch Ca(2+)-ATPase was found to be greatly enhanced. Components of the sarcolemma (Na+/K(+)-ATPase, dystrophin-glycoprotein complex) and the free sarcoplasmic reticulum (Ca(2+)-binding protein sarcalumenin and a 53-kDa glycoprotein) were not affected by chronic stimulation. The relative abundance of calsequestrin was slightly reduced in transformed skeletal muscle. However, the expression of the ryanodine receptor/Ca(Ca2+)-release channel from junctional sarcoplasmic reticulum and the transverse tubular dihydropyridine-sensitive Ca2+ channel, as well as two junctional sarcoplasmic reticulum proteins of 90 kDa and 94 kDa, was greatly suppressed in transformed muscle. Thus, the expression of the major protein components of the triad junction involved in excitation-contraction coupling is suppressed, while the expression of other muscle membrane proteins is not affected in chronically stimulated muscle.     

Proteolytic activation of the canine cardiac sarcoplasmic reticulum calcium pump

Mild trypsin treatment of canine cardiac microsomes consisting largely of sarcoplasmic reticulum vesicles produced a severalfold activation of oxalate-facilitated calcium uptake. The increase in calcium uptake was associated with an increase in ATP hydrolysis. Proteases other than trypsin were also effective although to a lesser degree. Trypsin produced a shift of the Ca2+ concentration dependency curve for calcium uptake toward lower Ca2+ concentrations, which was almost identical with that produced by phosphorylation of microsomes by cyclic AMP dependent protein kinase when the trypsin and the protein kinase were present at maximally activating concentrations. The Hill numbers (+/- SD) of the Ca2+ dependency after treatment of microsomes with trypsin (1.5 +/- 0.1) or protein kinase (1.7 +/- 0.1) were similar and were not significantly different from those for untreated control microsomes (1.6 +/- 0.1 and 1.8 +/- 0.1, respectively). Autoradiograms of sodium dodecyl sulfate-polyacrylamide electrophoretic gels indicate that 32P incorporation into phospholamban (Mr 27.3K) or its presumed monomeric subunit (Mr 5.5K) was markedly reduced when trypsin-treated microsomes were incubated in the presence of cyclic AMP dependent protein kinase and [gamma-32P]ATP compared to control microsomes incubated similarly but pretreated with trypsin inhibitor inactivated trypsin. The activation of calcium uptake by increasing concentrations of trypsin was paralleled by the reduction of phosphorylation of phospholamban. Trypsin treatment of microsomes previously thiophosphorylated in the presence of cyclic AMP dependent protein kinase and [gamma-35S]thio-ATP did not result in a loss of 35S label from phospholamban, which suggests that phosphorylation of phospholamban protects against trypsin attack.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasticity in canine airway smooth muscle

The large volume changes of some hollow viscera require a greater length range for the smooth muscle of their walls than can be accommodated by a fixed array of sliding filaments. A possible explanation is that smooth muscles adapt to length changes by forming variable numbers of contractile units in series. To test for such plasticity we examined the muscle length dependence of shortening velocity and compliance, both of which will vary directly with the number of thick filaments in series. Dog tracheal smooth muscle was studied because its cells are arrayed in long, straight, parallel bundles that span the length of the preparation. In experiments where muscle length was changed, both compliance and velocity showed a strong dependence on muscle length, varying by 1.7-fold and 2.2-fold, respectively, over a threefold range of length. The variation in isometric force was substantially less, ranging from a 1.2- to 1.3-fold in two series of experiments where length was varied by twofold to an insignificant 4% variation in a third series where a threefold length range was studied. Tetanic force was below its steady level after both stretches and releases, and increased to a steady level with 5-6 tetani at 5 min intervals. These results suggest strongly that the number of contractile units in series varies directly with the adapted muscle length. Temporary force depression after a length change would occur if the change transiently moved the filaments from their optimum overlap. The relative length independence of the adapted force is explained by the reforming of the filament lattice to produce optimum force development, with commensurate changes of velocity and compliance.     

Passive mechanics of muscle tendinous junction of canine diaphragm.

The diaphragmatic muscle tendon is a biaxially loaded junction in vivo. Stress-strain relations along and transverse to the fiber directions are important in understanding its mechanical properties. We hypothesized that 1) the central tendon possesses greater passive stiffness than adjacent muscle, 2) the diaphragm muscle is anisotropic, whereas the central tendon near the junction is essentially isotropic, and 3) a gradient in passive stiffness exists as one approaches the muscle-tendinous junction (MTJ). To investigate these hypotheses, we conducted uniaxial and biaxial mechanical loading on samples of the MTJ excised from the midcostal region of dog diaphragm. We measured passive length-tension relationships of the muscle, tendon, and MTJ in the direction along the muscle fibers as well as transverse to the fibers. The MTJ was slack in the unloaded state, resulting in a J-shaped passive tension-strain curve. Generally, muscle strain was greater than that of MTJ, which was greater than tendon strain. In the muscular region, stiffness in the direction transverse to the fibers is much greater than that along the fibers. The central tendon is essentially inextensible in the direction transverse to the fibers as well as along the fibers. Our data demonstrate the existence of more pronounced anisotropy in the muscle than in the tendon near the junction. Furthermore, a gradient in muscle stiffness exists as one approaches the MTJ, consistent with the hypothesis of continuous passive stiffness across the MTJ.     

Dysfunction of calcium handling by smooth muscle in hypertension

Dysfunction of ion handling, including binding and fluxes (passive and active transport) of physiologically important ions such as potassium, sodium, calcium, and magnesium, by vascular smooth muscle cell membranes has repeatedly been reported to be associated with the pathophysiology of hypertension. The specific purpose of this review is to summarize and evaluate the evidence for alterations of calcium ion (Ca2+) handling by vascular smooth muscle in various forms of hypertension in the animal model on the basis that regulation of cytoplasmic Ca2+ concentration is a complex and yet vitally important process for a normal function of vascular smooth muscle and that derangement of such a regulation may result in excessive retention of cytoplasmic Ca2+, contribute toward increase of total peripheral resistance, and ultimately lead to elevation of blood pressure. Emphasis is placed upon the consideration of the usefulness of the subcellular membrane fractionation technique in studies of binding and transport of Ca2+ by vascular and nonvascular smooth muscle membranes from genetic as well as experimental hypertensive rats. The limitations of the interpretation of data using such an approach are also considered. Decreased active transport of Ca2+ across isolated plasma membrane vesicles from large and small arteries occurs in several but not all forms of hypertension. This membrane abnormality also occurs in nonvascular smooth muscles and other tissues or cells not confined to the cardiovascular system in genetic hypertension, but not in experimental hypertension. A hypothesis of general membrane defects in spontaneous hypertension is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple phospholipase A2 activities in canine vascular smooth muscle.

Three phospholipase A2 activities from canine vascular smooth muscle were identified and characterized including: (1) a cytosolic calcium-independent phospholipase A2 which is activated by nucleotide di- and triphosphates; (2) a cytosolic calcium-dependent phospholipase A2 which is activated by physiologic increments in calcium ion concentration; and (3) a microsomal calcium-independent phospholipase A2 which was highly selective for plasmenylcholine substrate. Vascular smooth muscle cytosolic calcium-independent phospholipase A2 was activated 338% +/- 11 (X+S.E.; n = 15) by physiologic concentrations of ATP. Similar amounts of activation were also present utilizing other nucleotide di- and triphosphates (e.g., ADP, CTP, GDP and GTP) as well as non-hydrolyzable nucleotide triphosphate analogs (e.g., ATP-gamma-S, AMP-PNP and GTP-gamma-S). Vascular smooth muscle cytosolic calcium-dependent phospholipase A2 was purified 455-fold by sequential DEAE-Sephacel, Phenyl-Sepharose, Mono Q, hydroxyapatite and Superose 12 chromatographies. The partially purified calcium-dependent phospholipase A2 was activated by physiologic increments in calcium ion concentration (e.g., 1 microM) and possessed an apparent native molecular weight of 95 kDa, an acidic isoelectric point (pI = 4.8) and a neutral pH optimum (pH 7.0). Vascular smooth muscle microsomal phospholipase A2 activity was predominantly calcium-independent and was over six-fold selective for hydrolysis of plasmenylcholine substrate. Taken together, these results demonstrate the existence of three separate and distinct phospholipase A2 activities in vascular smooth muscle and identify ATP and calcium ion as independent modulators of discrete phospholipase A2 activities in vascular smooth muscle cells.     

Immediate exercise hyperemia: contributions of the muscle pump vs. rapid vasodilation.

A striking characteristic of the blood flow adaptation at exercise onset is the immediate and substantial increase in the first few (0-5 s) seconds of exercise. The purpose of this mini-review is to put into context the present evidence regarding mechanisms responsible for this phase of exercise hyperemia. One potential mechanism that has received much attention is the mechanical effect of muscle contraction (the muscle pump). The rapid vasodilatory mechanism(s) is another possible mechanism that has recently been shown to exist. This review will provide the reader with 1) an understanding of the basic physics of blood flow and the theories of muscle pump function, 2) a critical examination of evidence both for and against the contribution of the muscle pump or rapid vasodilatory mechanisms, and 3) an awareness of the limitations and impact of experimental models and exercise modes on the contribution of each of these mechanisms to the immediate exercise hyperemia. The inability to measure microvenular pressure continues to limit investigators to indirect assessments of the muscle pump vs. vasodilatory mechanism contributions to immediate exercise hyperemia in vivo. Future research directions should include examination of muscle-contraction-induced resistance vessel distortion as a trigger for rapid smooth muscle relaxation and further investigation into the exercise mode dependency of muscle pump vs. rapid vasodilatory contributions to immediate exercise hyperemia.