Some factors affecting the extraneuronal accumulation of adrenaline in guinea-pig trachealis smooth muscle cells

Tracheal segments from guinea-pigs pretreated with 6-hydroxydopamine were incubated in 5 or 200 mumol X 1(-1) adrenaline at 37 degrees C. Catechol-O-methyltransferase and monoamine oxidase were inhibited by U-0521 and pargyline, respectively, and the accumulation of adrenaline in trachealis smooth muscle cells was measured by fluorescence microphotometry. The effects of anoxia, anoxia plus glucose deprivation, ouabain, reduced sodium (Na+) concentration, elevated potassium (K+) concentration and calcium (Ca2+) deprivation on adrenaline accumulation were examined in tissues incubated in adrenaline for 5 min. Adrenaline accumulation in the trachealis smooth muscle cells was reduced by inhibition of oxidative metabolism (anoxia and glucose deprivation), by ouabain (100 mumol X 1(-1), by a reduction in Na+ concentration from 139 mmol X 1(-1) to 25 mmol X 1(-1), and by increases in K+ concentration, but not by omission of Ca2+. These results from guinea-pig trachealis smooth muscle cells, in combination with those obtained by other workers on various organs and tissues, indicate that the extraneuronal accumulation of catecholamines requires energy derived from oxidative metabolism and is very sensitive to inhibition by increases in K+ concentration. However, there is marked variability in the effects of ouabain and of reduced Na+ or Ca2+ concentrations on the accumulation.     

Stereoselectivity of extraneuronal uptake of catecholamines in rabbit aorta

Summary In incubated rabbit aorta, stereoselective preference of (-)isoprenaline and (-)adrenaline, but not of (-)noradrenaline, was observed with respect to extraneuronal (corticosterone-sensitive) catecholamine accumulation at relatively low amine concentrations. The ranking order (isoprenaline > adrenaline > noradrenaline) and the degree of stereoselectivity were similar to those described for the perfused rat heart. Thus, stereoselectivity of the carriermediated extrancuronal uptake process is demonstrable for different species and under conditions of incubation as well as of perfusion. On the other hand, amine distribution into the tissue that is linked neither to uptake₂ nor to uptake₁ (but which might be rather important under incubation conditions) showed no stereoselectivity in rabbit aorta.This study was supported by the Deutsche Forschungsgemeinschaft (He 813); the results were presented in a preliminary form to the German Pharmacological Society (Grohmann et al. 1983)     

Evidence from guinea-pig trachealis that Uptake2 of isoprenaline is enhanced by hyperpolarization of the smooth muscle

Summary Previous studies (Bönisch et al. 1985; Trendelenburg 1986, 1987) have provided evidence that Uptake₂ of catecholamines is inhibited by depolarization of cells. The aim of this study was to further examine the relationship between Uptake₂ and membrane potential by testing the hypothesis that Uptake₂ is, conversely, stimulated by hyperpolarization of cells. The effects of -adrenoceptor agonists (isoprenaline and salbutamol) and -adrenoceptor antagonists (propranolol and ICI 118,551) on Uptake₂ of isoprenaline were examined in guinea-pig trachealis muscle, in which stimulation of -adrenoceptors mediates hyperpolarization of the smooth muscle cells (Allen et al. 1985), and in rat heart, in which -adrenoceptor agonists do not cause hyperpolarization.In guinea-pig trachealis muscle segments, propranolol and ICI 118,551 reduced Uptake₂ (as measured by the steady-state rate of corticosterone-sensitive formation of 3-O-methylisoprenaline normalized for the isoprenaline concentration) in tissues incubated in 2.5–250 nmol/l ³H-isoprenaline (in the range over which isoprenaline causes hyperpolarization of the muscle), but not in 1 nmol/l ³H-isoprenaline (which does not hyperpolarize the muscle). The normalized rates were greater in tissues incubated in 25 nmol/l than 1 nmol/l isoprenaline, and were enhanced by 2.5 gmol/l salbutamol in tissues incubated in 1 nmol/l isoprenaline. In rat hearts perfused with 1 or 25 nmol/l ³H-isoprenaline and U-0521 to inhibit catechol-O-methyltransferase, the rate of Uptake₂ of isoprenaline, normalized for the isoprenaline concentration, was unaffected by the isoprenaline concentration or the presence of propranolol, ICI 118,551 or salbutamol.The results of the study suggest that, in guinea-pig trachealis muscle, isoprenaline and salbutamol enhance Uptake₂ due to -adrenoceptor-mediated hyperpolarization of the muscle and that propranolol and ICI 118,551 decrease Uptake₂ of isoprenaline by preventing this hyperpolarization of the smooth muscle, and not by directly inhibiting the Uptake₂ transport process. Correspondence to: L. J. Bryan-Lluka at the above addressSome of the results of this study were presented to the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (Vuocolo and Bryan-Lluka 1990) and the German Society for Pharmacology and Toxicology (Bryan-Lluka and Vuocolo 1991)     

Cromakalim-induced membrane current in guinea-pig tracheal smooth muscle cells

The characteristics of the cromakalim-induced membrane current were examined in single tracheal myocytes of the guinea-pig under voltage-clamp conditions. When K(+) concentrations in the pipette and bathing solutions were approximately 140 mM, cromakalim activated a membrane current (I(crom)) which was inward at -60 mV and reversed at -2 mV. I(crom) was blocked by 10 microM glibenclamide and potentiated when the ATP concentration in the pipette solution was decreased. The K(d) and Hill coefficient of glibenclamide for I(crom) block were 200 nM and 1.05, respectively. Application of the tyrosine kinase inhibitors, genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamid (ST638), reduced I(crom) in a concentration-dependent manner. Daidzein, which does not inhibit tyrosine kinase, was about 10 times less effective than genistein. Herbimycin A had no effect on I(crom). Internal application of these inhibitors from the pipette did not affect I(crom). In conclusion, cromakalim is a potent activator of the ATP-sensitive K(+) channel (K(ATP) channel) in guinea-pig tracheal myocytes. The inhibition of I(crom) by genistein and ST638 may be due to the direct block of the channel from outside.     

Mechanism of xanthine-induced relaxation of guinea-pig isolated trachealis muscle.

1. Four 3-alkylxanthines (3-methylxanthine, 3-n-propylxanthine (enprofylline), 3-n-butylxanthine and 3-iso-butylxanthine) and four 1-methyl-3-alkylxanthines (1-methyl-3-methylxanthine (theophylline), 1-methyl-3-n-propylxanthine, 1-methyl-3-n-butylxanthine and 1-methyl-3-iso-butylxanthine (IBMX], were compared in terms of cyclic AMP phosphodiesterase (PDE) inhibition and trachealis muscle relaxation. The relationship between xanthine structure and cyclic AMP PDE inhibition was also studied. 2. Xanthine induced relaxation of guinea-pig isolated trachealis muscle was measured against spontaneous tone. 3. The four 1-methyl-3-alkylxanthines were each significantly more potent than the corresponding 3-alkylxanthines in relaxing the isolated trachealis muscle. The 1-methyl-3-alkylxanthines were similarly more potent than the corresponding 3-alkyl derivatives in inhibiting low Km cyclic AMP PDE. There was a strong positive correlation between low Km cyclic AMP PDE inhibition and the tracheal smooth muscle relaxation evoked by the xanthine derivatives. 4. Since methylation of the 1-position of each 3-alkylxanthine increased the potency of the derivative in inhibiting low Km cyclic AMP PDE and in relaxing trachealis muscle and since a strong positive correlation was observed between the relaxant EC50 and the Ki value of each xanthine derivative, it is suggested that low Km cyclic AMP PDE inhibition by xanthines plays an important role in their tracheal relaxant effect.     

Temperature high sensitivity of managanese uptake in ileal longitudinal smooth muscle of guinea-pig

1. Mn²⁺ (5 mM) inhibited completely the K⁺ (60 mM)-induced ileal tonic tension to the base line, however, the tension increased progressively to the above level of the original K⁺ tonic response after 3 hr of the Mn²⁺ application at 37°C.2. At 30, 32 or 34°C, the tensions which developed after 3 hr of the addition of 5 mM Mn²⁺ in the high-K⁺ medium was 0, 21, 48% of their original K⁺ tonic levels, respectively.3. The tension development and manganese uptake in the presence of Mn²⁺ in the high-K⁺ medium was highly dependent on the temperature in very narrow range of 32–37°C in the suspending medium.     

Uptake kinetics and ion requirements for extraneuronal uptake of noradrenaline by arterial smooth muscle and collagen

1. The ionic requirements for noradrenaline uptake into vascular smooth muscle cells were studied by perfusing rabbit isolated ear arteries with noradrenaline (10(-3)M) either in Krebs solution or in Krebs solution modified by altering the concentration of one or more ion. Noradrenaline uptake was measured by quantitative microphotometry.2. Some uptake into smooth muscle continued in isotonic sucrose in the absence of all ions. Omission of Na(+) from the Krebs solution partially inhibited uptake as did high (100 mM) K(+). Omission of K(+), Ca(++) or Mg(++) had no effect on uptake. Lithium was able completely to substitute for Na(+).3. Alteration in ion concentration did not affect the binding of noradrenaline to collagen.4. The kinetics of uptake of noradrenaline into smooth muscle were analysed and found to be saturable with a Km of 4.9 x 10(-4)M.5. It is concluded that the ionic requirements of the transport mechanism for the uptake of noradrenaline by vascular smooth muscle show a relatively low specificity.     

The force driving the extraneuronal transport mechanism for catecholamines (uptake2)

Summary Recently, uptake₂ was shown to exist in the clonal Caki-1 cell line. The aim of this study was two-fold: a) to determine, in Caki-1 cells, the intracellular fate of ³H-noradrenaline after its translocation by uptake₂ and b) to analyse the force driving uptake₂.Caki-1 cells have the characteristics of a metabolizing system in which the activity of catechol-O-methyl transferase (COMT) greatly exceeds that of monoamine oxidase (MAO). In all subsequent experiments these enzymes were inhibited. The determination of initial rates of uptake₂ into Caki-I cells at an extracellular pH between 6.9 and 7.9 indicated that the protonated species of ³H-noradrenaline is transported. Depolarization of Caki-1 cells (by three different procedures) inhibited the inward transport. Determination of the time course of the specific accumulation of ³H-noradrenaline in Caki-I cells and of ³H-isoprenaline in the perfused rat heart (both mediated by uptake₂) revealed that depolarization (by high K⁺) reduced the rate constant for inward transport (k_IN) and increased that for outward movement (k_OUT). Consequently, depolarization reduced the steady-state factor of accumulation.It is proposed that, as the protonated species of the substrates of uptake₂ is transported, the membrane potential is likely to provide the driving force for uptake₂. The fact that depolarization decreased k_IN and increased k_OUT agrees with this proposal, as do the magnitudes of the steady-state accumulation factors determined in Caki-I cells and perfused rat heart.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - MAO monoamine oxidase - NMN normetanephrine - OMDA O-methylated and deaminated metabolites Supported by the Deutsche Forschungsgemeinschaft (SFB 176) and the Dr. Robert Pfleger-Stiftung Send offprint requests to E. Schömig at the above address     

Effect of cromakalim on smooth muscle cells of guinea-pig taenia caeci

Cromakalim caused hyperpolarization and reduction of the electrotonic potential in a concentration-dependent manner in smooth muscle cells of guinea-pig taenia caeci. There was a relatively constant change in the electrotonic potential under calcium-free, low-sodium and low-chloride conditions in the presence of cromakalim as compared to control conditions with Krebs solutiuon. The effect of cromakalin (10(-5) M) was inhibited by glibenclamide (5 X 10(-5) M). These results indicate that cromakalim specifically promotes potassium efflux in smooth muscle cells of the guinea-pig taenia caeci via glibenclamide-sensitive potassium channels, to cause hyperpolarization, suppression of spike activity and relaxation.     

Relaxant effects of mercury and mercury uptake in the smooth muscle of guinea-pig taenia coli

Hg2+ (0.005-0.5 mM) induced a concentration-dependent reduction on high-K+-induced contraction of taenia coli. The K+-induced increase in 45Ca uptake was significantly reduced in the presence of Hg2+ (0.05-0.5 mM). The contractions of the glycerinated taenia coli were inhibited by an increase in Hg2+ concentration. Mercury uptake increased along the duration of Hg2+ incubation. When muscles were rinsed with a medium containing 5 mM EDTA after 0.5 mM Hg2+ treatment for 90 min about 30% of the original level of tissue mercury was retained. Possible mechanism for the tension inhibitory action of Hg2+ include: (1) that Hg2+ bound to cell membrane initially interferes with the permeability of Ca2+ and that (2) the cellular mercury fraction that is not eliminated by EDTA subsequently correlates with the tension inhibitory action.     

Effects of vasopressin on smooth muscle cells of guinea-pig mesenteric vessels

1 The effects of vasopressin on the membrane and contractile properties of smooth muscle cells of guinea-pig mesenteric arteries, and mesenteric and portal veins were investigated in various ionic environments by means of a micro-electrode technique and an isometric tension recording method. The results were compared with those obtained with oxytocin and noradrenaline (NA).