Effects of High-K +, Na + -Deficient Solution on Contractility of the Smooth Muscles of the Porcine Trachea

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1 FULL PAPER Pharmacology Effects of High-K +, Na + -Deficient Solution on Contractility of the Smooth Muscles of the Porcine Trachea Takeharu KANEDA 1) *, Harue KANAKURA 1), Masahiro YAMAMOTO 1), Tsuyoshi TAJIMA 1), Norimoto URAKAWA 1) and Kazumasa SHIMIZU 1) 1) Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Nippon Veterinary and Life Science University, 7 1 Kyonancho 1-chome, Musashino, Tokyo , Japan (Received 5 March 2009/Accepted 31 May 2009) ABSTRACT. A high-k +, Na + -deficient (isosmotic 154 mm K + and 0 mm Na + ; I-154 K + ) solution induced contraction followed by gradual relaxation of the smooth muscles of the porcine trachea, while hyperosmotic addition of 65 mm KCl (H-65 K + ) induced a large sustained contraction. The I-154 K + solution also induced a sustained increase in [Ca 2+ ] i level. Decreases in muscle tension and increases in cellular water content were both prevented by the addition of sucrose or NaCl in the porcine trachea. An additional application of phloridzin inhibited recoveries of I-154 K + solution by addition of NaCl in the porcine trachea. Addition of pyruvate or oxaloacetate recovered the I-154 K + solution-induced relaxation in the porcine trachea. Although application of I-154 K + solution did not affect PCr and ATP contents in the bovine trachea, the solution induced a gradual decrease of PCr content in the porcine trachea. Moreover, application of pyruvate or oxaloacetate recovered the I-154 K + solution-induced decreases of PCr content in the porcine trachea. Phloridzin inhibited H- 65 K + -induced contraction in porcine, but not in bovine trachea. In conclusion, the I-154 K + solution-induced relaxation in the porcine trachea is probably due to swelling of cells and inhibition of glucose utilization. Moreover, the inhibition of glucose utilization in I-154 K + medium in porcine trachea is different from that of bovine trachea. KEY WORDS: high-k +, Na + -deficiency, PCr, porcine trachea. J. Vet. Med. Sci. 71(9): , 2009 Application of hyperosmotically-added high-k + solution induced a sustained contraction, while an isosmotically-substituted high-k +, Na + -deficient solution induces contraction followed by gradual relaxation in various smooth muscles [5 7, 15 21]. The possible mechanism of these decreases in muscle contractility of tonic muscle is swelling of the cells, as in rabbit aorta [19]. On the other hand, the decreases in muscle contractility in phasic muscle are due to an inhibition of glucose utilization resulting from Na + deficiency in medium, as in guinea pig [15] and rat [5] urinary bladders, and the guinea pig ileum [17] and vas deferens [16]. However, the changes in some smooth muscles are due to both swelling and inhibition of glucose utilization, as in the guinea pig gall bladder [15], seminal vesicle [16], and taenia coli [18]. On tracheal smooth muscles, it has been suggested that the high-k +, Na + -deficient solution-induced decreases of muscle contraction is related to swelling of smooth muscle cells in the guinea pig trachea [15], however, relaxation was found to be related to both swelling of cells and inhibition of glucose utilization in the rabbit trachea [20]. Recently, we showed that I-154 K + solution induced-relaxation of bovine trachea is probably due to swelling of cells [7]. These data indicate that, only in the trachea, the mechanisms of high- K +, Na + -deficient solution-induced relaxation are different depending on species. Moreover, in bovine tracheal smooth muscles, it has been shown that glucose omission from *CORRESPONDENCE TO: KANEDA, T., Laboratory of Veterinary Pharmacology, Nippon Veterinary and Life Science University, 7 1 Kyonan-cho 1-chome, Musashino, Tokyo , Japan. t-kaneda@nvlu.ac.jp medium had no effect on the high-k + -induced contraction for 30 min [2]. On the other hand, there is no report to investigate the glucose utilization in high-k + -induced contraction in the porcine trachea. In the present experiment, we attempted to clarify the inhibitory mechanism of Na + deficiency on high K + -induced contraction in the porcine trachea by measuring muscle tension, wet weights of tissue, intracellular Ca 2+ ([Ca 2+ ] i ) level, and compare to our previous data in bovine trachea [7]. Moreover, we measured phosphocreatine (PCr) and ATP contents in the porcine and bovine trachea. MATERIALS AND METHODS Muscle preparations and tension measurement: Tracheas from adult porcine and bovine of either sex were obtained from a local abattoir. The smooth muscle was excised from the cartilage, and the epithelium and connective tissues were removed. The muscle strips (about 2 mm in width and 4~5 mm in length) were incubated with physiological salt solution (PSS) containing (in mm) NaCl, 5.4 KCl, 1.5 CaCl 2, 1.0 MgCl 2, 11.9 NaHCO 3, and 5.6 glucose. The PSS was aerated with 95% O 2 and 5% CO 2 at 37 C to adjust the ph to 7.2. Isosmotic 60, 77, 120.1, or mm K + solution was made by substituting an equimolar amount of K + for Na + in the PSS. In some experiments, sucrose (50 mm), NaCl (50 mm), pyruvate (5.5 mm) and oxaloacetate (5.5 mm) were added to PSS. The concentration of those substances was followed by our previous reports [5, 7, 15 18]. Muscle tension was recorded isometrically. One end of each strip was bound to a glass holder and the other end was

2 1210 T. KANEDA ET AL. connected by silk thread to a strain-gauge transducer (TB- 611T; Nihon Kohden, Tokyo) in an organ bath containing PSS with a resting tension of 2 g. The muscle strips were equilibrated for 30 min to obtain a stable contractility induced by hyperosmotic addittion of 65 mm KCl (H- 65K + ). The developed tension was expressed as a percentage by assuming the values at rest in normal PSS to be 0% and those at 15 min after addition of H-65 K + to be 100%. In this paper, we tentatively expressed the decrease of muscle contraction as the relaxation. For determining the wet weight of the tissue, muscle strips were treated with various test solutions after equilibration in PSS. After incubation, the strips were occasionally removed from the organ bath, blotted on filter paper to exclude adhering solution and weighed using a balance. The ratio of cellular water content was calculated using the following equation [15]: Ratio of cellular wet weight (treated muscle) (mg) water content = wet weight (control muscle) (mg) {1-(relative ECS+relative dry weight)}(treated muscle) {1-(relative ECS+relative dry weight)}(control muscle) Assuming that the specific gravity of the muscle was 1, the relative extracellular space (ECS) was expressed as ECS (mg)/wet weight (mg). Since the ECS of the porcine trachea was not determined in the present experiments, the value of ECS in the guinea pig trachea to be 0.56 [15] was cited in the calculation. To determine the dry weight of the strips, the samples were incubated in PSS or test solution and the was removed, blotted, weighted, dried at 95 C for 20 hr in vessels, and then weighed again. Simultaneous measurement of muscle tenison and [Ca 2+ ]i level: The [Ca 2+ ] i level was measured simultaneous to a muscle contraction as reported previously [12]. In brief, muscle strips were incubated with PSS containing 5 M fura2/am overnight at 4 C. A noncytoxic detergent, 0.02% cremophor EL, was also added to increase solubility of the fura2/am. One end of the muscle was pinned to the bottom of the organ bath which was filled with 8 ml of PSS, and the other end was attached to the transducer with silk thread. The muscle strip was kept horizontally in the organ bath. The muscle strips were alternately excited with light at 340 nm and 380 nm through a rotating filter wheel; The 500 nm emission was measured through a band-pass filter with a fluorometer (CAF-100; Japan Spectroscopic Co., Ltd., Tokyo), and their ratio (F340 / F380) was recorded as an indicator of [Ca 2+ ] i. Since Himpens and Somlyo [3] suggested that extracellular hyperosmotic pressure resulted in leakage of fura2 from the cytosol, we used isosmotic 60 mm KCl (I-60 K + ) instead of H-65 K + in this experiment. The fluorescence ratio was expressed as a percentage by assuming the values at rest in normal PSS to be 0% and those at 10 min after addition of I- 60 K + to be 100%. Assay of creatine phosphate and adenosine triphosphate: Creatine phosphate (PCr) and adenosine triphosphate (ATP) contents in the muscle strips were measured by the high-performance liquid chromatograph (HPLC) as reported previously [1]. In brief, muscles were incubated with I-154K + for 30, 60, 90 or 120 min. After the incubation, the muscles were rapidly frozen in liquid nitrogen and stored at 80 C until homogenized in 6% perchloric acid (0.9 ml). The homogenate was centrifuged at 15,000 g for 5 min and the supernatant neutralized with 0.2 ml of 2M KHCO 3. The neutralized extract were spun once more and 20 l supernatant was applied to the HPLC. The HPLC system (Shimazu Corp., Kyoto) was consisted of a pump (LC-10AT), a system controller (SCL-10A), an auto injector (SIL-10AF), a column oven (CTO-10A) and wave length-selectable detector (SPD-10Ai) set at 216 nm. Chromatography was performed by RPC C2/C18 ST (4.6 mm internal diameter and 100 mm length, Amersham Biosciences, Co., U.S.A.) using mobile phases of 50 mm KH 2 PO 4 and 5 mm tetrabutylammonium hydrogen sulphate (TBAHS) (ph 6.0, buffer A), and 50 mm KH 2 PO 4, 5 mm TBAHS and 40% methanol (ph 6.0, buffer B). Flow rate was 1.0 ml/min and the elution started with 65% buffer A. In the first 14 min, buffer B increased at a rate of 2.5%/min. This was followed by elution with 70% buffer B for 20 min and then with 100% buffer A for 10 min. These procedures were programmed with system controller. The sensitivity of the detecter was usually set at 1.0 a.u.f.s. and the oven temperature at 40 C. PCr and ATP contents are expressed as mol/ g wet weight. Chemicals: The chemicals used were phloridzin (Sigma- Aldrich, St. Louis, U.S.A.), fura2/am (Dojindo Laboratories, Kumamoto) and cremophor EL (Nacalai Tesque, Kyoto). Statistics: Values are expressed as mean SEM, and statistical analyses were performed by Student s t-test. RESULTS Changes in muscle tension and cellular water content: When H-65K + was applied to the porcine trachea, the muscle gradually increased its tension and reached a maximum after about 15 min. This muscle tension was maintained at a steady level for 120 min. Figure 1A shows the statistical results for the time course of the tension change. Isosmotic 60, 77, or 120 mm K + (I-60, -77, or -120 K + ) solution also induced contraction, and it gradually attenuated the muscle tension, at 120 min becoming , , or % of that in the H-65 K + -induced contraction at 15 min, respectively. When isosmotic 154 mm K + (I-154 K + ) solution was applied to the muscle, muscle tension increased and then decreased to % at 120 min (Fig. 1A), it was significantly smaller than that of bovine trachea ( % [7] ). Although H-65 K +, I-60 K +, and I-77 K + solution significantly decreased the ratio of cellular water content at 120 min, I-120 K + or I-154 K + solution increased it (Fig. 1B). It seems that the curve of muscle tension is drawn in the oppo-

3 HIGH-K + /LOW-Na + CONTRACTION IN THE PORCINE TRACHEA 1211 Fig. 1. Changes in tension (A) and ratio of cellular water content (B) of the porcine trachea in high-k + solution. Hyperosmotically-added 65.4 mm KCl (H-65 K + ) or isosmotically substituted 60 mm KCl (I-60 K + ), 77 mm KCl (I-77 K + ), 120 mm KCl (I-120 K + ) or 154 mm KCl (I-154 K + ) was applied at 0 min. The muscle tension induced by H-65 K + solution at 15 min was taken as 100%. Each point represents the mean of 4 6 preparations. Vertical bars indicate the SEM. **: Significant difference from control (A: H-65 K +, B: 0 min) with P<0.01. site direction to that for the ratio of cellular water content and that I-120 K + or I-154 K + solution caused swelling in the porcine trachea cells. Moreover, there was a positive correlation between the decrease of high K + -induced contraction and the increase in cellular water content (R 2 =0.86, P<0.05). These data were similar to those obtained in bovine trachea [7]. Changes in muscle tension and [Ca 2+ ]i level in the I-154 K + - treated muscles: We showed that I-154 K + solution induced a sustained increase in the [Ca 2+ ] i level in the bovine trachea [7]. As shown in Fig. 2, application of I-60 K + solution induced a sustained contraction and an increase in the [Ca 2+ ] i level in the porcine trachea. On the other hand, application of I-154 K + solution induced a large and transient contraction but [Ca 2+ ] i increase was sustained during the stimulation (Fig. 2). The levels of tension and [Ca 2+ ] i at 40 min after application of I-154 K + solution were % and % (% of I-60K + -induced response, n=4), respectively. Effects of addition of sucrose, NaCl, pyruvate or oxaloacetate on contraction and increases in cellular water content induced by I-154 K + solution: Our previous data have shown that the 154 K + solution containing hyperosmotically-added sucrose or NaCl induced a well-maintained contraction in the bovine trachea [7]. In the present study, the 154 K + solution containing hyperosmotically-added sucrose (50 mm) or

4 1212 T. KANEDA ET AL. Fig. 2. Effects of I-154 K + solution on [Ca 2+ ] i level (F340/F380, upper trace) and muscle tension (lower trace) in porcine trachea. The increase in [Ca 2+ ] i level induced by I-60 K + solution at 10 min was taken as 100%. Trace of the typical results from 4 experiments. NaCl (50 mm) induced a well-maintained contraction in the porcine trachea. In bovine trachea, an additional application of phloridzin (1 mm), an inhibitor of the Na + -dependent d-glucose cotransporter, did not affect muscle contraction induced by 154 K + solution containing NaCl [7], but in porcine trachea, at min, phloridzin significantly inhibited muscle contraction induced by 154 K + solution containing NaCl (50 mm) (Fig. 3A). As shown in Fig. 3B, I-154 K + solution significantly increased the ratio of cellular water content in the porcine trachea. However, additional application of sucrose (50 mm) or NaCl (50 mm) decreased the I-154 K + solutioninduced increase in the ratio of cellular water content. In our previous data, application of pyruvate, a Na + -independent energy source, did not affect the I-154 K + solutioninduced contraction in bovine trachea [7], but in porcine trachea, application of pyruvate (5.5 mm) or oxaloacetate (5.5 mm) increased the I-154 K + solution-induced contraction remarkably (Fig. 4). Changes in PCr and ATP contents in the 154 K + - treated muscles: To determine the difference of glucose utilization between bovine and porcine trachea, we measured PCr and ATP contents. As shown in Fig. 5, application of I-154 K + solution did not affect PCr and ATP contents in the bovine trachea for 120 min. On the other hand, I-154 K + solution induced a gradual decreases PCr content (Fig. 6A), but not ATP (Fig. 6B) in the porcine trachea. Moreover, application of pyruvate (5.5 mm) or oxaloacetate (5.5 mm) recovered the I-154 K + solution-induced decreases of PCr content in the porcine trachea (Fig. 6A). However, application of pyruvate or oxaloacetate did not affect ATP content in the porcine trachea (Fig. 6B). Effects of addition of phloridzin on contraction induced by H-65 K + solution: We investigated that the effects of phloridzin in H-65 K + -induced contraction to compare the glucose utilization of porcine and bovine trachea in high K + medium. Phloridzin has been shown to inhibit glucose entry by blocking the Na + -dependent glucose contransporter in the intestinal brush border [8]. In our preliminary data, phloridzin at concentration lower than 1 mm had no effect on H-65 K + -induced contraction of mouse, rat and guinea pig ileal smooth muscle (data not shown). When the contractile response to H-65 K + reached a steady level (15 20 min), phloridzin (1 mm) was added and the time course of contraction was compared. Although application of phloridzin did not affect H-65 K + -induced contraction in bovine trachea (n=6), it decreased the contraction to % in porcine trachea (n=5, data not shown). DISCUSSION It has been shown that high-k + induces sustained muscle contraction in the porcine trachea [13, 14]. In the present study, however, isosmotically-substituted high-k +, Na + - deficient solution induced rapid muscle contraction followed by a gradual relaxation. Based on our observations, we determined the difference of the I-154K + solution-induced relaxation between bovine

5 HIGH-K + /LOW-Na + CONTRACTION IN THE PORCINE TRACHEA 1213 Fig. 3. Effects of addition of NaCl or sucrose on muscle tension (A) and ratio of cellular water content (B) in I-154 K + solution in porcine trachea. I-154K + (Control), I- 154 K mm NaCl (NaCl), I-154 K mm sucrose (Sucrose) or I-154 K mm NaCl+1 mm phlorizin (NaCl+phlorizin) were applied at 0 min. The muscle tension induced by H-65 K + solution at 15 min was taken as 100%. Each point represents the mean of 4 6 preparations. Vertical bars indicate the SEM. **, ++ : Significant difference from control or NaCl with P<0.01. and porcine trachea as follows; (1) An additional application of phloridzin significantly inhibited muscle contraction induced by 154 K + solution containing NaCl in porcine but not in bovine trachea. (2) Application of pyruvate or oxaloacetate increased the I-154 K + solution-induced contraction remarkably in porcine but not in bovine trachea. (3) I-154 K + solution induced the decreases of PCr content in porcine, but not in bovine trachea. (4) An additional application of pyruvate or oxaloacetate recovered the decreases of PCr content in porcine trachea. Suzuki et al. [19] have reported previously that high-k +, Na + -deficient solution induced rapid muscle contraction that was followed by gradual relaxation, an increase in the wet weight of tissue, and an increase in the cellular water content of the rabbit aorta. In our previous study, I-154 K + solution also induced rapid muscle contraction that was followed by gradual relaxation and an increase in the wet weight of tissue of the bovine trachea [7]. In the rabbit aorta, hyperosmotic addition of sucrose induced recovery of high-k +, Na + -deficient solution-induced relaxation and swelling of tissue [19]. In the present study, recovery of I- 154 K + solution-induced gradual relaxation and increases in

6 1214 T. KANEDA ET AL. Fig. 4. Effects of addition of pyruvate or oxaloacetate on muscle tension in I-154 K + solution in porcine trachea. I-154K + (Control), I-154 K mm pyruvate (Pyruvate) or I-154 K mm oxaloacetate (Oxaloacetate) were applied at 0 min. The muscle tension induced by H-65 K + solution at 15 min was taken as 100%. Each point represents the mean of 4 6 preparations. Vertical bars indicate the SEM. **: Significant difference from control with P<0.01. Fig. 5. Changes of phosphocreatine (PCr) and ATP contents in I-154 K + solution in bovine trachea. The data are expressed as means SEM. Each point represents the mean of 5 preparations. Vertical bars indicate the SEM. the wet weight of tissue was induced by hyperosmoticallyadded sucrose or NaCl in the porcine trachea. Similar results were obtained for the guinea pig trachea and gall bladder [15] and the rabbit [20] and bovine trachea [7], which are all classified as tonic muscles. On the other hand, it has been reported that I-154 K + solution induces gradual relaxation, but not an increase in the wet weight of tissue, in guinea pig [15] and rat [5] urinary bladders and the guinea pig ileum [17] and vas deferens [16], which are all classified as phasic muscles. It has been suggested that the relaxing mechanism in these muscles is involved in inhibition of glucose utilization. However, I-154 K + solution induced an increase of the ratio of cellular water content in some phasic muscles, such as the guinea pig taenia coli [18] and seminal vesicle [16] and the rat uterus [5]. These studies suggest the hypothesis that the relaxing mechanism of I-154 K + in tonic muscles may be related to swelling of cells. However, the relaxing mechanism in some muscles is involved with both swelling and inhibition of glucose utilization. Relaxation induced by high-k +, Na + -deficient solution

7 HIGH-K + /LOW-Na + CONTRACTION IN THE PORCINE TRACHEA 1215 Fig. 6. Changes of PCr (A) and ATP (B) contents in I-154 K+ solution in porcine trachea. I-154K + (Control), I-154 K mm pyruvate (Pyruvate) or I-154 K mm oxaloacetate (Oxaloacetate) were applied at 0 min. Each point represents the mean of 4 5 preparations. Vertical bars indicate the SEM. **: Significant difference from control with P<0.01. was significantly reversed by application of pyruvate (5.5 mm) or oxalacetate (5.5 mm) as an energy source in the guinea pig taenia coli [18], urinary bladder [15], vas deferens [16] and seminal vesicle [16]. In the present experiment, application of pyruvate or oxalacetate recovered the relaxation induced by I-154 K + solution in porcine trachea. On the other hand, application of pyruvate did not affect the I-154 K + solution-induced relaxation in the bovine trachea [7]. Moreover, phloridzin, an inhibitor of the Na + -dependent d-glucose cotransporter, inhibited the recovery of contraction induced by application of NaCl or H-65 K + -induced contraction in porcine trachea, but it did not affect these contractions in bovine trachea. It has been reported that glycogen contents gradually decreased in Na + -free medium for 60 min in vascular smooth muscle [10]. More study is required to clarify the high- K + -induced contraction depends on an endogenous energy source in the bovine trachea. These results suggested that the energy utilization in the I-154 K + solution is different between bovine and porcine trachea. In the guinea pig ileum, we have previously reported relaxation induced by I-154 K + solution with decreases in PNred fluorescence represented glycolysis activity, but not FPox fluorescence represented mitochondrial respiration activity, and the application of Na + induced recovery of the I-154 K + solution-induced contraction and PNred fluorescence [17]. These results suggest that Na + -dependent d-glucose cotransporter is present in the plasma membrane and that inhibition of glucose utilization is the relaxing mechanism of the guinea pig ileum. In bovine trachea, I-154 K + solution induced relaxation without changes in the increases of FPox fluorescence or PNred fluorescence [7]. Moreover, it has been thought that PCr/creatine kinase system plays a role in the transport of high energy phosphates from the mitochondrial compartment to the sites of energy utilization, correlating with oxidative metabolism in mammalian smooth muscle [4, 9]. In the present study, application of I- 154 K + solution did not affect PCr and ATP contents in bovine trachea. On the other hand, I-154 K + solution decreased PCr content and application of pyruvate or oxaloacetate recovered the I-154 K + solution-induced decreases of PCr content in porcine trachea. These results suggest that glucose uptake may be related to Na + -dependent d-glucose cotransporter in porcine but not in bovine trachea. More study is required to clarify the expression of Na + -dependent d-glucose cotransporter in porcine trachea and other smooth muscles. These results suggest that I-154 K + solution induced-relaxation in the porcine trachea may be due to inhibition of glucose utilization resulting from Na + deficiency in medium. In the guinea pig taenia coli, hypoxia decreased PCr content in high K + solution, but did not affect high K + -induced sustained increase of [Ca 2+ ] i level [11]. In our experiments, I-154 K + solution induced a sustained increase of [Ca 2+ ] i level in the porcine trachea. These data suggest that the inhibition of glucose utilization does not involve in the changes of high K + -induced increase of [Ca 2+ ] i level. In our present results, it shows that the sodium ions mediate tracheal smooth muscle contraction for long time, as previous reports [7, 15, 20]. These data may supply new concept that the sodium ions mediate the chronic airway constriction diseases such as asthma and chronic obstructive lung disease. In conclusion, the I-154 K + solution induced-relaxation in the porcine trachea may be due to swelling of cells and inhibition of glucose utilization. These data imply that the inhibition of glucose utilization in I-154 K + medium in porcine trachea is different from that of bovine trachea. REFERENCES 1. Dickenson, K Separation of tissue metabolites with a PepRPC HR 5/5 column and FPLC system. Science Tools 36: Hai, C.M., Watson, C., Wallach, S.J., Reyes, V., Kim, E. and Xu, J Effects of substrate and inhibition of oxidative

8 1216 T. KANEDA ET AL. metabolism on contraction and myosin phosphorylation in ASM. Am. J. Physiol. 264: L Himpens, B. and Somlyo, A.P Free-calcium and force transients during depolarization and pharmacomechanical coupling in guinea-pig smooth muscle. J. Physiol. 395: Ishida, Y. and Paul, R.J Effects of hypoxia on highenergy phosphagen content, energy metabolism and isometric force in guinea-pig taenia caeci. J. Physiol. 424: Kamizaki, J., Shimizu, K., Nakajyo, S. and Urakawa, N A comparative study on the contraction induced by high-k + / Na + -deficient solution in rat uterus or urinary bladder. Jpn. J. Pharmacol. 47: Kamizaki, J., Shimizu, K., Nakajyo, S. and Urakawa, N A change in inhibitory mechanism of Na + deficiency on high K + -induced contraction in rat uterus with the progress of pregnancy. J. Smooth Muscle Res. 26: Kaneda, T., Sakaguchi, R., Shimizu, K., Urakawa, N. and Nakajyo, S Effects of high-k +, Na + -deficient solution on contractility of the smooth muscles of the bovine trachea. J. Vet. Med. Sci. 68: Lostao, M.P., Hirayama, B.A., Loo, D.D. and Wright, E.M Phenylglucosides and the Na + /glucose cotransporter (SGLT1): analysis of interactions. J. Membr. Biol. 142: Lövgren, B. and Hellstrand, P Graded effects of oxygen and respiratory inhibitors on cell metabolism and spontaneous contractions in smooth muscle of the rat portal vein. Acta Physiol. Scand. 123: Lynch, R.M., Kuettner, C.P. and Paul, R.J Glycogen metabolism during tension generation and maintenance in vascular smooth muscle. Am. J. Physiol. 257: C Obara, K., Bowman, P.S., Ishida, Y. and Paul, R.J Effects of hypoxia on [Ca 2+ ] i, phi and myosin light chain phosphorylation in guinea-pig taenia caeci. J. Physiol. 503: Ozaki, H., Sato, K., Sato, T. and Karaki, H Simultaneous recording of calcium signals and mechanical activity using fluorescent dye fura2 in isolated strips of vascular smooth muscle. Jpn. J. Pharmacol. 45: Ozaki, H., Hori, M., Takeo, J., Hata, J., Jinno, S., Okita, T., Yamashita, S. and Karaki, H Mechanisms responsible for the in vitro relaxation of a novel dibenzothiepine derivative (NSU-242) on tracheal and vascular smooth muscles. Eur. J. Pharmacol. 488: Sanagi, M., Ozaki, H., Mitsui, M. and Karaki, H Mechanism of relaxing action of the antiasthmatic drug, azelastine, in isolated porcine tracheal smooth muscle. Eur. J. Pharmacol. 222: Shimizu, K., Yaguchi, S., Kometani, A., Nakajyo, S., Ozaki, H. and Urakawa, N A comparative study on the contraction induced by high-k + /Na + -deficient solution in the trachea, gall bladder and urinary bladder in the guinea pig. Jpn. J. Pharmacol. 34: Shimizu, K., Tahara, K., Nakajyo, S. and Urakawa, N Effects of the high-k + /Na + -deficient solution on mechanical response and wet weight of tissue in vas deferens and seminal vesicle in guinea-pig. J. Smooth Muscle Res. 24: Shimizu, K., Kaburagi, T., Nakajyo, S. and Urakawa, N Decrease in muscle tension and reduced pyridine nucleotides of the guinea pig ileal longitudinal smooth muscle in high K +, Na -deficient solution. Jpn. J. Pharmacol. 56: Suzuki, T., Karaki, H. and Urakawa, N Mechanism of inhibition of contraction in high K, Na deficient solution in smooth muscle of guinea-pig taenia coli. Arch. Int. Pharmacodyn. 248: Suzuki, T., Karaki, H. and Urakawa, N Inhibition of contraction by swelling of vascular smooth muscle in high KCl, low Na solution. Arch. Int. Pharmacodyn. 250: Ueda, F., Kishimoto, T., Karaki, H. and Urakawa, N HighK-induced contractions in rabbit and monkey tracheal smooth muscle. J. Smooth Muscle Res. 19: Urakawa, N., Karaki, H. and Ikeda, M Ca uptake and tissue Ca of guinea pig taenia coli in isotonic high-k/na-deficient medium. Jpn. J. Pharmacol. 18: