Contractile and metabolic characteristics of creatine-depleted vascular smooth muscle of the rat portal vein

The functional consequences of phosphocreatine (PCr) depletion for mechanical properties, O2 consumption, and lactate production of the rat portal vein were investigated. After feeding rats for 8-9 weeks on a diet containing 2% beta-guanidino propionic acid (BGPA), PCr of the portal vein was reduced to 14% of control, whereas ATP was unchanged. No significant change was found in the level of spontaneous contractile activity or the force developed in a high-K+ contracture. Lactate production and the relationship between contractile force and O2 consumption were uninfluenced by BGPA treatment. The force-velocity relation of electrically stimulated portal veins showed no influence of BGPA treatment on Vmax. To investigate whether decrease in PCr influenced the response to metabolic stress, portal veins were exposed to graded concentrations (0.1-0.5 mM) of cyanide to depress cellular respiration. Veins from control and BGPA-treated rats showed the same relative decrease of contractile activity and O2 consumption, and the same increase in lactate production. Cyanide treatment resulting in a reduction of electrically stimulated force to 70-80% of the original gave a reduction of Vmax to 85-90%. The relative degree of reduction was uninfluenced by BGPA treatment. Reduction of PCr content thus does not affect the functional properties of metabolism or contractility under normoxic conditions. Furthermore, it can be inferred that the PCr reduction known to occur in smooth muscle exposed to hypoxia (L?vgren & Hellstrand 1985) is not in itself the major factor causing hypoxic inhibition of mechanical activity.     

Some properties of the smooth muscle of rabbit portal vein

1. The morphology of the smooth muscle of the rabbit portal vein and its innervation were studied with fluorescence and electron microscopy. Two layers of smooth muscle were observed in the tunica media: an inner layer of circularly arranged muscle cells and an outer layer consisting of bundles of smooth muscle cells arranged in a near longitudinal direction. The membranes of neighbouring smooth muscle cells were occasionally fused to form ;tight junctions'.2. Bundles of non-myelinated nerve fibres were observed in the adventitia, and between bundles and layers of smooth muscle cells in the media. Studies on longitudinal sections with fluorescence microscopy revealed a network of varicose noradrenergic axons.3. Electrical and mechanical activity was recorded from longitudinal strips of smooth muscle from the media of the vein with a sucrose-gap apparatus.4. The preparation was spontaneously active under minimal resting tension (less than 150 mg) and at temperatures above 28 degrees C. Slow depolarizations led to a burst of spikes (multi-spike complexes), which corresponded to rhythmic contractions. In 10% of preparations, the interval between multi-spike complexes showed a slower depolarization, suggesting the record was from a pace-maker region.5. The frequency of spontaneous activity (3-27 beats/min) was very sensitive to changes in temperature and tension.6. Noradrenaline in low doses (0.01 mug) caused an increase in frequency of the multi-spike complexes. Higher doses (0.1-0.3 mug) initiated continuous high-frequency spiking, while very high doses (0.6-2.0 mug) caused maintained depolarization.7. Responses to repetitive electrical stimulation of the vein were qualitatively similar to those in response to exogenous noradrenaline. The relation between the mechanical response and the various parameters of stimulation was consistent with the stimulation of sympathetic nerve fibres in the wall of the vein.8. The actions of isoprenaline, phentolamine and propranolol indicated the presence of alpha ;excitatory' and beta ;inhibitory' adrenotrophic receptors on the smooth muscle.     

Calcium-dependence of the calcium-activated chloride current in smooth muscle cells of rat portal vein

Ca²⁺-activated Cl^– current in freshly isolated smooth muscle cells from rat portal vein was studied using the whole-cell patch-clamp technique. Simultaneously, the free-cytosolic Ca²⁺ concentration (Ca_i) was estimated using emission from the dye Indo-1. Pretreatment of the cells with amytal and carbonyl-cyanide-m-chlorophenylhydrazone, which reduced the intracellular adenosine triphosphate concentration, was used to weaken the cellular Ca²⁺ homeostatic system. Ca_i of treated cells slowly increased during perfusion with an external Ca²⁺-containing solution. This rise in Ca_i gradually activated a Ca²⁺-dependent Cl^– current which allowed the study of the relationship between activation of this current and Ca_i levels. The threshold Ca_i for activation of Cl^– channels was around 180 nM and full activation occurred at 600 nM. The Ca_i dependence of the Cl^– channels was not changed during application of noradrenaline and did not depend on the membrane potential. The gating of Ca²⁺-dependent Cl^– channels of rat portal vein myocytes seems to be mainly controlled by intracellular Ca²⁺     

Contractile response and electrophysiological properties in enzymatically dispersed smooth muscle cells of rat vas deferens

Electrophysiological studies were performed on single smooth muscle cells isolated from the vas deferens of the rat. The tissue was preincubated in Ca-free modified Tyrode's solution for 1 h and then transferred to a high-K solution for 1 h. It was next minced and treated with the enzyme solution composed of 600–800 unit/ml collagenase and 40 unit/ml elastase. The procedure yielded about 50% spindle shaped Ca-tolerant cells (100–250 m in length and about 10 m in diameter). These cells could contract during the superfusion with the solutions containing 10^–8 to 10^–3M norepinephrine (NE) or adenosine triphosphate (ATP). The cells isolated from the epididymal portion were more sensitive to norepinephrine than were those from the prostatic part. Their basic electrical properties were studied using tight-seal suction electrode technique. The cells had resting potentials around –40 mV and their input resistance was about 0.8 G. Action potentials could be evoked by application of depolarizing current. During whole cell voltage clamp, an inward current followed by an outward current was recorded when 800 ms pulses from a holding potential of –60 mV to test potentials positive than –40 mV were applied. The transient outward current generally recorded in other smooth muscle cells was not seen in these cells. The amplitude of the inward current was Ca dependent and sensitive to a Ca antagonist, nicardipine, indicating that Ca ion is the main carrier of this component of the current. When the pipette was filled with Cs-containing solution, the outward current was abolished. In this condition, the reversal potential of Ca current was +53.4 mV, and the time course of inactivation was composed of more than one exponential component. The results suggest that these isolated cells retain many characteristics of analogous multicellular preparation and that they are a useful model of the postsynaptic properties in smooth muscle especially when studied electro-physiologically.     

Effects of calcium and sodium on contracture tension in the smooth muscle of the rat portal vein

Summary The purpose of the present study was to determine the quantitative relationship between membrane potential (or [K⁺]₀) and contracture tension in the smooth muscle of the rat portal vein, and to examine the influence of Ca⁺⁺ and Na⁺ on this relationship. However, electrical all-or-none responses were successfully abolished only in Na⁺-free sucrose-Krebs due to hyperpolarization and in K⁺-high Krebs due to depolarization. It did not seem possible to eliminate spike discharge at intermediate levels of membrane potential without the simultaneous loss of contractility. In the hyperpolarized state the muscle remained relaxed despite the very low levels of [Na⁺]₀ and despite increases in [Ca⁺⁺]₀ up to 20 mM. The depolarized portal vein developed a contracture which was intimately dependent on [Ca⁺⁺]₀, the threshold concentration being of the order of 0.1 to 0.3 mM. Spike-induced, phasic contractions showed a similar Ca⁺⁺-dependence. Variations in [Na⁺]₀ had only a slight and irregular influence on the Ca⁺⁺ dose-response curve of the depolarized muscles.Differences in the effects of Na⁺ on the rate of rise and the rate of fall of the contracture tension, respectively, suggested that Na⁺ is more important for the removal of Ca⁺⁺ from the contractile system than for the supply of Ca⁺⁺ to the system. With regard to the interaction of Ca⁺⁺ and Na⁺ in the excitation-contraction coupling the vascular smooth muscle seemed to differ from both heart muscle and skeletal muscle.The present study was supported by grants from the Swedish Medical Research Council (B 70-14x-28-06 A), from Air Force Office of Scientific Research through the European Office of Aerospace Research, OAR, United States Air Force under Contract F 1052-68-C-0044, from U.S. Public Health Service (HE-05678-08), from AB Hässle, Göteborg, and from the Deutsche Forschungsgemeinschaft (Bi 122/1).     

The membrane properties of the smooth muscle of the guinea-pig portal vein in isotonic and hypertonic solutions

The membrane properties of the longitudinal smooth muscle of the guinea-pig portal vein were investigated under various experimental conditions.1. In isotonic Krebs solution, the membrane potential (-48.7 mV), the maximum rates of rise and fall of the spike (4.6 and 2.3 V/sec respectively), the space constant (0.61 mm), the conduction velocity of excitation (0.97 cm/sec) and the time constant of the foot of the propagated spike (18.4 msec) were measured.2. The various parameters of the muscle membrane in the isotonic solution were compared with those in the hypertonic solution prepared by the addition of solid sucrose (twice the normal tonicity).3. When the muscles were perfused with hypertonic solution, marked depolarization of the membrane and increased membrane resistance occurred. These were probably due to reduction of the K permeability, increased internal resistance of the muscle and shrinkage of the muscle fibre.4. The membrane potential in isotonic and hypertonic solutions was analysed into two components, i.e. the metabolic (electrogenic Na-pump) and the ionic (electrical diffusion potential) component in the various environmental conditions.(a) In isotonic and hypertonic solutions, the membrane was depolarized by lowering the temperature or by removal of K ion from the solutions. When the tissues were rewarmed or on readdition of K ion, the membrane was markedly hyperpolarized. These hyperpolarizations of the membrane were suppressed by treatment with ouabain (10(-5) g/ml.), by warming to only 20 degrees C and by K-free solution.(b) The relationships between the membrane potential and the [K](o) in isotonic Krebs, in the hypertonic (sucrose) Krebs, in the Na-free (Tris) Krebs and in the Cl-deficient (C(6)H(5)SO(3)) Krebs were observed. The maximum slopes of the membrane depolarization against tenfold changes of [K](o) were much lower than that expected if it behaved like a K electrode.(c) In Na-free (Tris) solution, the membrane was not depolarized in isotonic condition but it was depolarized in hypertonic condition.5. The low membrane potential in hypertonic solution (-37 mV) compared with isotonic solution (-49 mV) was thought to be mainly due to suppression of K permeability of the membrane and not due to suppression of the metabolic component.The electrogenic Na-pump and the membrane potential of the portal vein was discussed in relation to other excitable cell membranes.     

Effects of metabolic inhibition on cytoplasmic calcium and contraction in smooth muscle of rat portal vein

Contractions in the rat portal vein, evoked by spontaneous action potentials or depolarizing high-K⁺ solution, are rapidly and reversibly inhibited by hypoxia or respiratory blockade. Intracellular free calcium ([Ca²⁺]_i) was measured using Fura-2 to evaluate the effects of metabolic blockade on excitation–contraction coupling. Spontaneous contractions were associated with transient increases in [Ca²⁺]_i. During exposure to cyanide (0.2–0.4 mm ) or 2,4-dinitrophenol (30 μm ) the duration and amplitude of the Ca²⁺ transients were decreased, leading to a decreased mean time integral of the individual [Ca²⁺]_i transient, and corresponding decrease in the duration and amplitude of the contraction. Basal [Ca²⁺]_i was increased in the presence of the metabolic inhibitors. High-K⁺ (40 mM) contractions caused a sustained increase in [Ca²⁺]_i, which was not inhibited by exposure to cyanide, although the amplitude of the associated contraction was greatly reduced. Together with the earlier demonstration of decreased 20 kD myosin light chain phosphorylation under these conditions, this indicates that the activation of contraction is influenced by metabolism via the energy dependence of the light chain phosphorylation reaction. Thus at least three steps in the excitation–contraction sequence are influenced by inhibition of oxidative metabolism: membrane excitation, light chain phosphorylation, and the cross-bridge cycle. This provides mechanisms for a high degree of metabolic sensitivity of vascular tone, of importance for the adaptation of blood flow to tissue metabolic demands.     

Electrical and mechanical responses to inhibition of cell respiration in vascular smooth muscle of the rat portal vein

Metabolic regulation of contractility in vascular smooth muscle was studied in the spontaneously active rat portal vein using respiratory depression by cyanide (0.2-2.0 mM) as a model for tissue hypoxia. Intracellular recordings of electrical activity were done with concomitant registration of force development. Average membrane potential in the absence of cyanide was -61 +/- 1 mV (n = 27). Addition of cyanide to normal Krebs solution resulted in a reduction of force amplitude and the number of action potentials per burst, with a relatively more pronounced effect on the mechanical activity. At moderate levels of inhibition of force amplitude the frequency of spontaneous bursts of action potentials transiently increased concomitant with a slight depolarization, but after prolonged (15-20 min) exposure to cyanide the membrane repolarized to the level prior to cyanide addition and the burst frequency decreased to be equal to or lower than that in the absence of cyanide. Higher concentrations of cyanide totally inhibited spontaneous mechanical and electrical activity. In contrast to the results with glucose, it was found that when beta-hydroxybutyrate was used as substrate the addition of 2 mM cyanide led to a marked hyperpolarization (13 +/- 1 mV) after total inhibition of spontaneous activity. The hyperpolarization was not prevented by administration of 4-aminopyridine (2.5 mM) or tetraethylammonium (4-6 mM) prior to the addition of cyanide. To investigate the effects of increased metabolic demand on the relation between force and membrane potential in cyanide-treated muscle, high-K+ (40 mM) contractures were studied. Contractures were associated with depolarization of 34 +/- 3 mV (n = 5). 1 mM cyanide reduced the amplitude of the contractures to about 9% of control with a moderate reduction in the amount of depolarization (28 +/- 1 mV, n = 5). It is concluded that the decrease of mechanical activity during respiratory inhibition may partly reflect a reduction in the number of spikes per burst but that other mechanisms, independent of membrane activity, also contribute to the inhibition. The increase of glycolysis during respiratory inhibition seems to prevent more pronounced changes in membrane potential.     

Differential actions of exogenous and intracellular spermine on contractile activity in smooth muscle of rat portal vein

Effects of the naturally occurring polyamine spermine on electrical and contractile properties of the rat portal vein were studied. 1 mM spermine nearly abolished spike activity and spontaneous contractions and decreased the intracellular Ca²⁺ concentration ([Ca²⁺],). The phasic force responses to 0.1 and 1 μM phenylephrine were partially inhibited, but not the sustain plateau contraction caused by 5 /IM phenylephrine. The Ca²⁺-force relation in high-K⁺ (128 mM)-depolarized veins was shifted to the right, EC₅₀ for Ca²⁺ increasing from 0.50 ± 0.03 mM (control, n= 8) to 0.65 ± 0.06 and to 0.94 ± 0.03 at 1 (n – 4) and 10 (n = 3) mM spermine, respectively. However, at a Ca²⁺ concentration of 2.5 mM, giving maximal force, there was no effect of spermine (1 mM) on either force or [Ca²⁺],. Whereas extracellular spermine thus reduced contractile activity at moderate levels of stimulation, increased intracellular concentration of spermine potentiated the force response to Ca²⁺. Intracellular loading of spermine by reversible permeabilization increased its concentration by 2–3 times. The spontaneous activity and response to phenylephrine were unchanged. However, the Ca²⁺-force relation of depolarized veins was shifted to the left, EC₅₀ decreasing from 0.51 ± 0.04 mM in controls (n= 7) to 0.36 ± 0.02 mM in the loaded veins (n= 9). Spermine increased Ca²⁺-activated force in portal veins permeabilized with β-escin. The degree of potentiation was consistent with observed effects in spermine-loaded intact veins. The results suggest that spermine at physiological intracellular concentration may contribute to the determination of Ca²⁺ sensitivity in vascular smooth muscle cells.     

Quantitative aspects of electrical and mechanical responses to anisosmolar solutions in the smooth muscle of the rat portal vein

The electrical and mechanical activity of the rat portal vein were studied quantitatively under prolonged exposure to solutions in which osmolality was varied by changes in sucrose content. Reducing osmolality by 15 or 30 mosm/kg below the control value of 290 mosm/kg caused enhanced electrical and mechanical activity whereas hyperosmolality up to 390 mosm/kg led to inhibition as demonstrated earlier. These responses faded under prolonged exposure. In hypoosmolality integrated mechanical activity returned to control after about 10–15 min while spike activity remained somewhat increased. Prolonged hyperosmolality was associated with return of spike discharge towards control frequency whereas the integrated contractile force reverted from initial inhibition to levels above control after some 10–15 min. Therefore, the integrated force per spike was increased by prolonged hyperosmolality and decreased by hypoosmolality. Variations in osmolality had similar effects on the amplitude of K⁺ contractures. The time course of the osmotic responses are discussed in relation to the dynamic effects of passive stretch and shortening in the portal vein. The relation that may exist between “inotropic” effects of osmolality and the contractures obtained in strongly hypertonic media is considered.     

Contractile effects of polycations in permeabilized smooth muscle

The polycations spermine, neomycin and polylysine potentiated Ca2+-activated force in β-escin permeabilized guinea-pig ileum strips. The effect was inhibited by the calmodulin antagonists trifluoperazine, mastoparan and W13. Potentiation was slow or absent in α-toxin permeabilized strips, indicating dependence on penetration of the polycations into cells. The effects of spermine and neomycin were maintained after extensive permeabilization by β-escin, which eliminated the contractile effect of GTPγS. Replacement of ATP by CTP, which is not a substrate for myosin light chain kinase, inhibited contractile potentiation. Potentiation of Ca2+-activated contractions was associated with increased phosphorylation of the myosin regulatory light chains (LC20). A contractile effect of polylysine and neomycin was also seen in Ca2+-free medium and after partial LC20 thiophosphorylation, indicating that phosphorylation-independent processes may contribute to the response. Although spermine does not cause contraction in Ca2+-free medium at physiological [MgATP], it did so when [MgATP] was lowered to 40μm. Similar to high-[Mg2+], the rate of contraction on addition of ATP to strips incubated with microcystin-LR to inhibit phosphatase activity was increased by the polycations, but only at [Ca2+]<0.3μm. The results suggest that polycations increase Ca2+-activated force by inhibiting myosin phosphatase activity, thereby increasing myosin LC20 phosphorylation. However, additional activation mechanisms, evident at low [Ca2+] and at low [ATP] and possibly involving direct activation of myosin, contribute to their effect. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of Ca2+ on force-velocity characteristics of normal and hypertrophic smooth muscle of the rat portal vein

Portal hypertension was induced in rats by partial ligation of the hepatic branches of the portal vein. After 5 days of hypertension the portal veins were taken out and mounted for isometric and quick-release experiments. Portal veins from sham-operated normal rats served as controls. The ligated veins had an increased cross-sectional area, indicating smooth-muscle hypertrophy. Although the absolute magnitude of active force of these veins was increased, the active force per cross-sectional area was decreased, indicating an alteration in the properties of the contractile system. No difference in the Ca2+ concentration-response relations to K+-activated intact control and hypertrophic veins was found. In chemically skinned preparations, devoid of functional plasma membranes, the hypertrophic veins had similar Ca2+ sensitivity (in the presence of I microM calmodulin) but a lower force per cross-sectional area. Force-velocity relations were determined in K+-activated intact preparations. In control veins a reduction in extracellular Ca2+ was associated with a significant reduction in both isometric force and maximal shortening velocity (Vmax). In hypertrophic veins the decreased isometric force at maximal activation was associated with a low Vmax. A comparison between hypertrophic and submaximally stimulated control vessels showed corresponding Vmax and isometric force values. We conclude that the low isometric force of hypertrophic veins is associated with a lower rate of cross-bridge turnover. This could be an effect of alterations in the activation mechanisms or in the intrinsic properties of the contractile system itself.     

Changes of the potassium currents in rat aortic smooth muscle cells during postnatal development

This study was designed to investigate the role and regulation of arterial K+ channels during postnatal development. Rat thoracic aortic segments were suspended for isometric tension and resting membrane potential (RMP) recording. Contraction in response to 4-aminopyridine (4-AP) was similar in 4-, 8- and 12-week-old rats but was higher in 1-day-old rats. Contraction in response to tetraethylammonium (TEA) increased after 4 weeks. TEA increased the contractions evoked by noradrenaline in the aorta from 8- and 12-week-old rats but not from 1-day- and 4-week-old rats. RMP did not change during development. Patch-clamp studies of freshly isolated smooth muscle cells from the same aortas bathed in Ca2+-free medium showed a voltage-dependent K+ current (IK) sensitive to 4-AP. This current remained stable at all ages whereas the density of the total IK, recorded in the presence of Ca2+, showed a twofold increase between 4 and 8 weeks. This current was highly sensitive to TEA and charybdotoxin. The binding site density of 125I-labelled charybdotoxin was threefold higher in the membranes of aortas from 12-week-old compared to 4-week-old rats. These results indicate that changes in K+ channel distribution occur in the rat aorta during postnatal development. These are related to an increase in the expression of charybdotoxin-sensitive Ca2+-activated K+ channels.     

Comparison of VIP and beta 2-adrenoceptor-induced relaxations in the circular and longitudinal smooth muscle layers of the rat portal vein

The relative importance of VIP in reduction of vascular tone was studied in circular and longitudinal preparations of the VIP-innervated rat portal vein. Exogenous VIP inhibited the methoxamine-evoked contractures in the atropine-blocked preparations with a lower potency in the inner, circular (pD2 = 6.4 +/- 0.5, n = 6) than in the outer, longitudinal layer (pD2 = 7.7 +/- 0.1, n = 6). VIP was also a less efficient relaxant (intrinsic activity (alpha) = 0.60 +/- 0.16, n = 6) of the inner than of the outer layer (alpha = 1.00). The selective (salbutamol) and the non-selective (isoproterenol) beta 2-agonists completely relaxed the methoxamine contractures in both layers and the potency (isoproterenol) was higher in the inner (pD2 = 6.39 +/- 0.32, n = 6) than in the outer layer (pD2 = 5.67 +/- 0.34, n = 6). Plasma from the portal-mesenteric vein of anaesthetized, fasting rats contained 0.036 nM VIP (median, n = 17), that is, several orders of magnitude lower than the range of VIP concentrations relaxing the methoxamine contracted vein preparations via VIP receptors of the apamin-blockable category. The results support the hypothesis that alpha 1-adrenoceptor-induced contractions in the circular layer are predominately relaxed via beta 2-adrenoceptors while relaxation of the outer layer may occur via VIP receptors, probably activated by local release of the neuropeptide.     

Contractile properties of the human triceps surae muscle during simulated weightlessness

The effect of a 120-day period of bed rest on the mechanical properties of human triceps surae muscle was studied in a group of male volunteers (n = 6, mean age 38 years). The results shows that the contractile properties of skeletal muscle in response to disuse change considerably. Time to isometric peak tension of the triceps surae muscle increased from 120 (SEM 3.0) ms to 136 (SEM 2.9) ms (P < 0.01), half relaxation time from 92 (SEM 2.1) ms to 100 (SEM 1.6) ms (P < 0.05) and total contraction time from 440 (SEM 9.9) ms to 540 (SEM 18.7) ms (P < 0.001). Isometric twitch force (F _t) decreased by a mean of 36.7% (P < 0.05), maximal voluntary contraction (MVC) and maximal force (F _max) by a mean of 45.5% and 33.7%, respectively (P < 0.05-0.01). The valueF _max:F _t ratio increased by 3.6% (nonsignificant). The difference betweenF _max and MVC, expressed as a percentage ofF _max and referred to as force deficiency, has also been calculated. Force deficit increased by a mean of 60% (P < 0.001) after bed rest. Force-velocity properties of the triceps surae muscle calculated according to an absolute scale of voluntary and electrically evoked contraction development decreased considerably. The calculations of the same properties on a relative scale did not differ substantially from the initial physiological state. The results would suggest that muscle disuse is associated with both atrophy and a reduction in contractility in the development ofF _max and decreased central (motor) drive. The change in the triceps surae muscle contractile velocity properties may indicate changes in the kinetically active state in the muscles.