Heat production in chemically skinned smooth muscle.
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Animals↗
Biomedical subjects
P Hellstrand
Publications and source records attributed to P Hellstrand.
Myosin composition and functional properties of smooth muscle from the uterus of pregnant and non-pregnant rats.
The myosin heavy chain stoichiometry and the force-velocity relation have been determined in the myometrium of the non-pregnant and pregnant rat. The relative proportions of the slower migrating heavy chain (MHC1) greatly exceeded that of the faster migrating heavy chain (MHC2) as shown by electrophoresis on SDS 4%-polyacrylamide gels. The ratios of MHC1/MHC2 were 2.2/1 in the non-pregnant rats, 2.6/1 in the pregnant rat, and contrasted with 0.8/1 in the rat portal vein. This stoichiometry was unchanged by extracting the myosin from the smooth muscle as native myosin in a salt extract, as dissociated myosin using sodium dodecyl sulphate (SDS) or by isolating the native myosin first by a non-dissociating (pyrophosphate) electrophoresis step and subsequently analysing the protein bands on the SDS 4%-polyacrylamide gel. Although the unequal proportions of the heavy chains suggested the possibility that the native myosin molecule may be arranged as homodimeric heavy chains, no evidence for or against the existence of native myosin isoforms could be obtained by electrophoresing native myosin extracts on pyrophosphate-polyacrylamide gels. The force-velocity relations of the intact electrically stimulated myometrium from the non-pregnant and pregnant rats gave isometric force of 45 and 135 mN/mm2 and Vmax of 0.71 and 0.52 lengths/s (37 degrees C) when measured at 95% of optimal length, whereas in chemically skinned uterine strips at 22 degrees C Vmax was 0.09 and 0.13 lengths/s, respectively. The length-force relationship was of similar shape in the non-gravid and gravid skinned tissues. The energetic tension cost (ATP-turnover/active stress) in skinned fibres was also similar. The mechanical and metabolic characteristics of the gravid and non-gravid uterus found in the present study do not suggest an obvious difference in the intrinsic properties of the myosin, although significant functional alterations in the tissue appear during pregnancy. This corresponds to the lack of a difference in the pattern of the heavy chains.
Adenosine Triphosphate↗
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 (Lövgren & Hellstrand 1985) is not in itself the major factor causing hypoxic inhibition of mechanical activity.
Adenosine Diphosphate↗
Cross-bridge behaviour in skinned smooth muscle of the guinea-pig taenia coli at altered ionic strength.
1. The effects of varied levels (25-300 mM) of ionic strength on mechanical properties and ATP hydrolysis rate of chemically skinned guinea-pig taenia coli fibres were investigated. 2. The tension development following activation by calcium (pCa 4.8), and relaxation following removal of calcium (pCa 9), were slower in 25 mM compared to 150 mM ionic strength. In fibres activated by thiophosphorylation of myosin light chains, by exposure to ATP-gamma-S, the tension development was rapid and independent of ionic strength. 3. The maximal shortening velocity (Vmax) was obtained from force-velocity relations determined by the quick-release method. The rate of ATP hydrolysis (JATP) was determined by measurement of pyruvate released from phosphoenolpyruvate (PEP). In order to obtain maximal Vmax and JATP at a Mg-ATP concentration of 1 mM, an ATP regenerating system was required. In thiophosphorylated fibres 2 mM-phosphocreatine (PCr) or 3.2 mM-PEP were adequate for maximal Vmax and JATP respectively. In calcium-activated fibres 5 mM-PCr was required for maximal Vmax. 4. The isometric force of thiophosphorylated fibres showed a biphasic dependence on ionic strength with a maximum at 90 mM. Vmax was essentially unchanged between 50 and 200 mM ionic strength. At 25 mM ionic strength, isometric force and Vmax were decreased by, respectively, about 15 and 25%. At 250 mM ionic strength, isometric force and Vmax were decreased by, respectively, 47 and 33%. 5. Vmax decreased with decreasing [Mg-ATP]. At [Mg-ATP] less than 0.1 mM there was no difference in Vmax between 35 and 150 mM ionic strength. At 250 mM ionic strength Vmax was lower than that at 150 mM at all [Mg-ATP]. 6. JATP during contraction in thiophosphorylated fibres at 35, 150 and 250 mM ionic strength was respectively, 0.62, 0.98 and 0.93 mumol g-1 min-1. The energetic tension cost (JATP/force) increased with ionic strength. 7. The force response to a quick stretch was investigated in the relaxed, contracted and rigor states at 25, 150 and 250 mM ionic strength. Stiffness in the relaxed state increased with speed of stretch and was higher the lower the ionic strength. In the contracted and rigor states, stiffness was also affected by ionic strength, but the relative effect in the contracted state was small.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenosine Triphosphate↗
Spontaneous electrical and contractile activity correlated to 86Rb+ efflux in smooth muscle of guinea-pig mesotubarium.
1. The spontaneous mechanical activity of guinea-pig mesotubarium consists of fused tetanic contractions lasting about 6 min, with a frequency of about four per hour. The muscle is completely relaxed between the contractions. Stretching the relaxed muscle elicits a contraction of the same appearance as the spontaneous ones. Comparison of preparations from oestrus (day 1 of the hormonal cycle), dioestrus (days 9-11) and prooestrus (days 14-15) showed no variation in the pattern of mechanical activity. 2. The resting membrane potential, measured by intracellular microelectrodes, did not differ with hormonal phase (prooestrus: -63.5 +/- 0.84 (n = 16); oestrus: -63.7 +/- 1.6 (n = 5); dioestrus: -61.6 +/- 0.77 (n = 17]. In most recordings a depolarization of a few millivolts occurred during the relaxation period (5-10 min), but in a few cells a more pronounced spontaneous depolarization of 10-15 mV was found. Ouabain (1 microM) caused depolarization by about 9 mV, both in pro- and dioestrus, leading to the initiation of maintained repetitive spiking. 3. Contraction is preceded by a depolarization lasting 10-30 s, and when a threshold is reached a train of slow waves and spikes is elicited. The frequency of slow waves and the number of spikes on each slow wave progressively decrease during the contraction, until spiking eventually ceases. 4. The spikes are resistant to tetrodotoxin (0.5 microM) and disappear in Ca2+-free medium, which also causes membrane depolarization. The duration of contractions increased with Ca2+ concentration in the range 1-5 mM. 5. The rate of 86Rb+ efflux, expressed as fractional release in 2 min intervals, showed a consistent variation during the contractile cycle in preparations with regular spontaneous activity. Relative to the value at the end of the relaxed period the efflux rate increased by about twofold during the contraction. From the beginning of the relaxed period after the contraction the efflux rate decreased by about 25% until the beginning of the next contraction. 6. It is concluded that the contractile activity in the mesotubarium, as opposed to that of the fallopian tube (Lydrup & Hellstrand, 1986a), is independent of the hormonal phase, including the period around ovulation. The mechanism for initiation of the trains of action potentials associated with spontaneous contractions may involve a gradual decrease of permeability of K+ channels or activity of the Na+-K+ pump during the relaxed period.
Action Potentials↗
Force-velocity relation and rate of ATP hydrolysis in osmotically compressed skinned smooth muscle of the guinea pig.
Chemically skinned guinea pig taenia coli fibre bundles showed a concentration-dependent decrease in width when incubated in media containing Dextran T500 (0-0.2 g ml-1). The maximal reduction in width, observed at 0.2 g ml-1 dextran, was 32%. The effect was reversible upon removal of dextran. Isometric force was slightly increased (about 10%) at the lowest dextran concentration (0.025 g ml-1) but decreased at higher concentrations (40% decrease at 0.2 g/ml-1). The energetic tension cost (ATP turnover/force) was decreased by about 40% after dextran addition. Force development and relaxation were markedly slower in 0.1 g ml-1 and absent in 0.2 g ml-1 dextran. In isotonic quick-release experiments 0.025 g ml-1 dextran did not influence maximal shortening velocity (Vmax) and relative stiffness, whereas 0.1 g ml-1 markedly increased stiffness and decreased Vmax to about 27%. Vanadate induced relaxation in the activated muscle (pCa 4.5) both in the absence and presence (0.1 g ml-1) of dextran and increased the rate of relaxation (pCa 9) at 0.1 g ml-1 dextran. The isometric rate of crossbridge turnover, as reflected by the energetic tension cost and the rate of relaxation, was decreased at all degrees of osmotic compression. Crossbridge turnover rate during shortening (Vmax) was unaffected at an osmotic compression of 12% (width) but was decreased at higher compression (32%).
Adenosine Triphosphate↗
Contractures induced by reversed Na+/Ca2+ exchange in rat portal vein: effects of calcium antagonists.
Spontaneous electrical and mechanical activity was abolished in isolated preparations of rat portal vein by exposure to K+-free Krebs solution. This procedure probably also increased the intracellular [Na+] owing to interference with the active transmembrane Na+-K+ transport. Contractures could be induced under these conditions by lowering extracellular [Na+] from the control level of 144 to 17 mM using sucrose, TrisCl, or LiCl as NaCl substitutes. Contractile force depended on the type of substitute: sucrose greater than TrisCl greater than LiCl. These contractures are thought to be caused by influx of extracellular Ca2+ through the Na+/Ca2+ exchanger, which otherwise transports Ca2+ in the opposite direction at normal transmembrane Na+ gradient. The contractile response to low Na+ was rapidly and completely abolished in nominally Ca2+-free medium; it was strongly inhibited by 0.4 mM MnCl2 but was not affected by high concentrations of the organic calcium antagonists, felodipine (10(-6) M), verapamil (10(-5) M), or diltiazem (10(-5) M). We conclude that the Na+/Ca2+-exchanger is an effective pathway for Ca2+ transport over vascular smooth muscle cell membrane; this pathway is not blocked by calcium antagonists.
Animals↗
Functional role of aerobic glycolysis in rat portal vein.
The functional role of aerobic lactate production in the rat portal vein was investigated. Changing substrate from glucose (11.5 mM) to pyruvate (11.5 mM) or beta-hydroxybutyrate (3 mM) had virtually no effect on spontaneous mechanical activity. Lactate production (FLA) was smaller with pyruvate than with glucose (0.05 +/- 0.01 vs. 0.14 +/- 0.03 mumol g-1 min-1, n = 4). Addition of 0.5 mM iodoacetate to inhibit glycolysis abolished mechanical activity in 15-20 min with glucose as substrate, whereas with pyruvate the mechanical activity was only moderately reduced over this time period. With beta-hydroxybutyrate (3 mM) as substrate no aerobic lactate production was detected during normal spontaneous activity. Inhibition of cellular respiration with increasing concentrations of cyanide in beta-hydroxybutyrate medium led to a graded decrease in mechanical activity and FO2, but only a marginal increase in lactate production. With glucose as substrate, repeated stimulation with a combination of isoproterenol (10(-5) M) and papaverine (10(-4) M) gave similar increases in lactate production at each exposure. With beta-hydroxybutyrate some lactate production was found at the first stimulation, but decreased to be abolished at the third stimulation. The mechanical inhibition caused by the stimulation was however similar at the three exposures for both substrates. Lactate production induced by cAMP-raising stimulation in beta-hydroxybutyrate could be accounted for by glycogenolysis. These results show that aerobic glycolysis leading to net lactate production is not necessary for normal spontaneous mechanical activity or the relaxing effect of hypoxia or cAMP raising stimuli in rat portal vein.
3-Hydroxybutyric Acid↗
Stiffness and the energetics of active shortening in chemically skinned smooth muscle.
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Adenosine Triphosphate↗
Influence of ATP, ADP and AMPPNP on the energetics of contraction in skinned smooth muscle.
The contraction of smooth muscle is influenced by the substrate MgATP and the product MgADP. The effects on force, shortening velocity and ATP-turnover, are consistent with an influence on the kinetics of cross-bridge cycling. Part of these effects are mediated via an influence on the regulation of contraction by myosin light chain phosphorylation. Results from preparations activated by thiophosphorylation, show that MgATP and MgADP also interact directly at the cross-bridge level, and are consistent with MgADP acting as a competitive ATP-analogue. The slow shortening velocity and decreased rate of ATP-induced relaxation from rigor in the presence of MgADP, suggest an inhibition of cross-bridge detachment. The rate of ATP-turnover was decreased in the presence of the nonhydrolyzable ATP-analogue AMPPNP. These results may contribute to the characterization of the biochemical reactions in the structurally organized smooth muscle contractile system. In addition, the influence of MgATP and MgADP on smooth muscle contraction suggest that the concentrations of substrate and products, at the level of the contractile proteins, may constitute important regulatory factors in vivo under conditions, such as hypoxia and ischemia, associated with impaired cellular energy supply.
Adenine Nucleotides↗
Isoforms of myosin in smooth muscle.
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Animals↗
Rate of oxidative and glycolytic metabolism in the guinea-pig oviduct in relation to contractility and hormonal cycle.
The rates of oxygen consumption and lactate production in the guinea-pig oviduct were measured together with registration of contractile activity during three phases of the hormonal cycle. In pro-oestrus (high oestrogen, low progesterone levels) and oestrus (time of ovulation, high oestrogen and progesterone) the rate of O2 consumption was higher than in dioestrus (low oestrogen, high progesterone). The frequency of spontaneous contractions was higher in oestrus than in the other phases. No significant differences in the proportion of the cross-sectional area occupied by smooth muscle were found between oviducts in di- and pro-oestrus. Stimulation by phenylephrine caused decreased frequency and increased amplitude of contractions in dioestrus but not in pro-oestrus, suggesting hormonal modulation of adrenergic mechanisms. The rate of relaxation of high-K+ contractures was higher in pro- than dioestrus. Lactate production and contents of ATP, ADP and phosphocreatine showed no significant variation with hormonal state. The increased rate of oxidative metabolism under oestrogenic dominance could in part reflect changes in ionic transport mechanisms, such as intracellular Ca2+ handling.
Animals↗
Role of vasoactive intestinal polypeptide (VIP) in the neurogenic vasodilatation of the portal vein in the rabbit.
A coarse network of nerve fibres displaying immunoreactivity for vasoactive intestinal polypeptide (VIP) was found in the wall of the hepatic portal vein of the rabbit. Electrical field stimulation of the rabbit portal vein in vitro, in the presence of adrenergic and cholinergic blockade, caused a marked relaxation of the vessel and a release of VIP into the perfusate. Addition of VIP to the tissue bath elicited a concentration-dependent inhibition of the mechanical activity of the portal vein. The results suggest that VIP containing neurones might participate in the non-cholinergic, non-adrenergic vasodilatation of the portal vein in the rabbit.
Animals↗
Myosin light chain phosphorylation and the cross-bridge cycle at low substrate concentration in chemically skinned guinea pig Taenia coli.
Force-velocity relations, rate of ATP turnover (JATP), and phosphorylation of the 20,000 D myosin light chains (LC20) were measured in chemically skinned guinea pig Taenia coli. Relative LC20 phosphorylation at 3.2 mM MgATP was 17% in relaxed tissues at pCa 9, and increased with force at increasing [Ca2+] to a maximum of 67% at pCa 4.5. Force at pCa 4.5 was dependent on the MgATP concentration with a half-maximal response at about 0.1 mM. At 0.1 mM MgATP LC20 phosphorylation at pCa 4.5 was 38%. Both JATP and the maximal shortening velocity (Vmax) were reduced in 0.1 mM MgATP, to 32% and 43%, respectively, of their values at 3.2 mM MgATP. Low-MgATP thus inhibits both LC20 phosphorylation and the extent and rate of cross-bridge interaction. High levels of LC20 phosphorylation, independent of Ca2+ and MgATP concentrations, were obtained by treatment with ATP-gamma-S. Maximal force at 3.2 mM MgATP after LC20 thiophosphorylation was unchanged, whereas halfmaximal force occurred at 0.065 mM MgATP after thiophosphorylation, compared to 0.13 mM after activation by Ca2+. The contraction in thiophosphorylated preparations at low-MgATP (0.1 mM) was associated with submaximal Vmax (60%) and JATP (27%). The results show that LC20 phosphorylation is correlated with the degree of force development in the Ca2+ activated contraction, both when Ca2+ and MgATP concentrations are varied. The reduced force and rate of crossbridge turnover in low MgATP are however primarily mediated by an influence of MgATP on the cross-bridge cycle, which is separate from the effect on LC20 phosphorylation.
Adenosine Triphosphate↗
Graded effects of oxygen and respiratory inhibitors on cell metabolism and spontaneous contractions in smooth muscle of the rat portal vein.
The basis for the hypoxic relaxation of spontaneous activity in the rat portal vein was investigated by comparing responses to oxygen and the respiratory chain inhibitors amobarbital and cyanide. With the inhibitors, O2 consumption (FO2) is uniformly decreased throughout the cell mass, and thus O2 gradients in the tissue are avoided. Hence the effects are not to be attributed to all-or-none inhibition in anoxic regions, a possibility that might complicate the interpretation of responses to hypoxia. With stepwise reduction in PO2 (96 to o% in N2 + 4% CO2) or increasing concentration of inhibitor (o-5 mmol), FO2 decreased with a concomitant reduction in mean contractile activity (P) and increase in lactate production (FLA). The calculated ATP production (FATP) was linearly related to P for P greater than 10% of the control value in 96% O2, with the same slope for hypoxia and both inhibitors. In this range the reduced FATP with P can largely be attributed to decreased metabolic demand of contraction, as evident from a comparison with the responses to hypoxia of portal veins relaxed in nominally Ca2+-free medium. With reduced PO2 or increased amobarbital concentration the tissue content of phosphocreatine decreased, whereas ATP remained constant for P greater than 10% of control. Similar responses to hypoxia and respiratory inhibition demonstrate graded effects on metabolism and contractility in the vascular smooth muscle cells, correlating with reported vasodilatory effects of these interventions in vivo.
Adenosine Diphosphate↗
Effects of calcium and substrate on force-velocity relation and energy turnover in skinned smooth muscle of the guinea-pig.
Mechanical properties and rate of ATP breakdown (JATP) have been determined in the chemically skinned guinea-pig taenia coli at 22 degrees C. The influence of varied [Ca2+], [Mg ATP] and muscle length were investigated. The shortening response after a step decrease in force (isotonic quick release) was highly curvilinear in the first 100-200 ms. This effect was shown to be a time-dependent response to the force step and not primarily caused by the shift along the length-force relation associated with shortening. Maximal shortening velocity (Vmax) decreased gradually following the release. At pCa (= -log [Ca2+]) 4.5, Vmax at 20 and 1000 ms after release was 0.49 +/- 0.07 and 0.041 +/- 0.004 (mean +/- S.E. of mean, n = 5) lengths s-1 respectively. Unloaded shortening velocity obtained from length steps of different magnitude (slack test) also showed a gradual decrease after the release, consistent with the isotonic release results. Increasing [Ca2+] from the relaxed state at pCa 9 (1 microM-calmodulin present) gave increased isometric force to a maximum at pCa 4.5. Half-maximal response was obtained at pCa 6.1. JATP at maximal force at pCa 4.5 was about 3 times the basal rate at pCa 9. The relation between JATP and force was highly non-linear, with a marked increase in JATP with little alteration in force at the highest [Ca2+]. When force was reduced to zero at pCa 4.5 by shortening the muscle to 0.3 L0 (L0 being the length giving maximal active force), JATP decreased by about 30%. At two levels of [Ca2+] giving similar force (pCa 5.75 and 4.5) the energetic tension cost obtained by length variations was lower at the low [Ca2+]. At pCa 6.0, Vmax and force were decreased to the same extent relative to their values at pCa 4.5. At pCa 5.75, where there was no reduction in force but a 25% decrease in isometric JATP, Vmax was unchanged relative to pCa 4.5. Force, Vmax and JATP were all dependent on [Mg ATP]. Half-maximal response was obtained at 0.1 mM for force and Vmax, and at 0.5 mM for JATP. The results are discussed in relation to a possible influence of both Ca2+ and Mg ATP on kinetic properties of the cross-bridge cycle.
Adenosine Triphosphate↗
Phosphagens and intracellular pH in intact rabbit smooth muscle studied by 31P-NMR.
Phosphorus-31 nuclear magnetic resonance (NMR) spectra (103.2 MHz) were obtained from rabbit portal vein, urinary bladder, and taenia coli smooth muscle. The muscles were mounted isometrically as strips in a horizontal probe and superfused with oxygenated medium at 23 degrees C. Under these conditions the preparations could remain in a stable metabolic state for at least 24 h. The resonances observed represented phosphocreatine (PC), adenosine triphosphate (ATP), and phosphomonoester. The PC-ATP ratio in relaxed preparations was approximately 1.9 (bladder) and 1.4 (taenia coli), approximately consistent with chemical analysis of tissue extracts by isotachophoresis. However, the levels of adenosine diphosphate (ADP) and inorganic phosphate (Pi) were considerably lower as estimated by NMR, suggesting intracellular compartmentalization. Contraction for 30 min in high-K+ medium caused a reversible 10-30% decline of PC but no change in ATP. Intracellular pH was 7.0 +/- 0.1 and was unchanged by contraction or inhibition of tissue respiration by cyanide. Treatment with 2-deoxyglucose resulted in accumulation of 2-deoxyglucose 6-phosphate, verifying the pH assignment. However, the phosphomonoester resonance of normal spectra is not glucose 6-phosphate. Treatment with Mg2+-free, high-Mg2+ (10 mM), or hyperosmotic media did not alter the Mg2+ saturation of ATP. The results obtained by the nontissue-destructive 31P-NMR measurements are consistent with the view that metabolic steady-state conditions are maintained during contraction in the smooth muscle.
Adenosine Triphosphate↗
O2 consumption, aerobic glycolysis and tissue phosphagen content during activation of the Na+/K+ pump in rat portal vein.
Oxygen consumption, lactate production and tissue contents of ATP, phosphocreatine (PCr) and lactate were measured following readdition of K+ to K+-depleted rat portal veins, in order to study the energy turnover associated with Na+/K+ pumping. During incubation in K+-free medium at 37 degrees C spontaneous contractions disappeared in 10-20 min. Readdition of K+ (5.9 mM) after 40 min K+-free incubation caused hyperpolarization of the cell membrane for the first 5-10 min and then gradual depolarization with return of spontaneous action potentials and contractions by 10-20 min. During the first 4-6 min after K+ readdition aerobic lactate production was about doubled and then gradually returned to the original level (0.17 mumol/min g) at about 20 min. The increase in glycolytic rate was prevented by 1 mM ouabain. In contrast, O2 consumption (in K+-free medium, 0.38 mumol/min g) rose by about 10% when K+ was added and this increase lasted about 5 min. By 8 min after K+ addition the increased glycolysis and oxidative phosphorylation had accounted for each about the same amount of extra ATP generation over that extrapolated from the steady rate before K+ addition. The average total increase in ATP turnover in the first 8 min was 15%. During this period there was no change in the cellular content of ATP, PCr, or extractable ADP. The results indicate that Na+/K+ pumping utilizes a relatively small share of the total energy turnover in the vascular smooth muscle but is to a large extent dependent on aerobic glycolysis and therefore a major site of carbohydrate usage.
Aerobiosis↗