• Abstract and Figures
• Public Full-text

Journal of Physiology (1993), 465, pp. 629-645 629 With 10 figures Printed in Great Britain EFFECTS OF pH AND INORGANIC PHOSPHATE ON FORCE PRODUCTION IN a-TOXIN-PERMEABILIZED ISOLATED RAT UTERINE SMOOTH MUSCLE BY CATHERINE A. CRICHTON, MICHAEL J. TAGGART*, SUSAN WRAY* AND GODFREY L. SMITH From the Institute of Physiology, University of Glasgow, Glasgow G12 8QQ and the *Physiology Laboratory, The University ofLiverpool, P0 Box 147, Liverpool L69 3BX (Received 27 July 1992) SUMMARY 1. Strips of longitudinal smooth muscle isolated from rat uterus were per- meabilized using crude a-toxin from the bacterium Staphylococcus aureus. This treatment rendered the surface membrane permeable to small molecular weight substances. Simultaneous measurements of tension and calcium concentration ([Ca2l]) (using indo-1 fluorescence) were used to investigate the effects of pH and inorganic phosphate concentration ([Pi]) on Ca2l-activated force generated by the contractile proteins. 2. Raising the [Pi] from 1 to 11 mm at a pH of 7-2 depressed both maximal and submaximal Ca2+-activated force. This effect of Pi was concentration dependent having the majority of its effect by 6 mM. 3. Further experiments at a submaximal [Ca2+] showed that Ca2+-activated force was enhanced by raising [Pi] from 6 to 11 mm suggesting that Pi increased the Ca2+ sensitivity of tension production. Based on these results, calculations indicate that the apparent affinity constant of Ca2+ for the contractile proteins increased from 4 x 106 M-1 to 6 x 106 M-1 on raising [Pi] from 1 to 11 mm. 4. Lowering pH from 7-2 to 6X7 at a [Pi] of 1 mm potentiated Ca2+-activated force with a small depression in the apparent Ca2+ sensitivity of tension production. This effect of pH on maximum (100 /M Ca2+) and submaximum (0-3 /im Ca2+) Ca2+- activated force was observed over a range of acidic pHs (7 0-6-7). 5. Increasing pH from 7-2 to 7-7 at a [Pi] of 1 mm depressed Ca2+-activated force with no effect on Ca2+ sensitivity of tension production. 6. Spontaneous contractions in intact rat myometrium are abolished under hypoxic conditions. Under these same conditions intracellular [Pi] rises and pH falls. The results of this study suggest that taken individually neither the effect of a rise in [Pi] nor a fall in pH on Ca2+-activated force generated by the contractile proteins can account for the effect of hypoxia on spontaneous contractions. INTRODUCTION Contraction of uterine smooth muscle is preceded by an increase in intracellular calcium concentration ([Ca2+]i) arising from influx across the cell membrane and/or MS 1648 21 PHY 465 630 C. A. CRICHTON AND OTHERS release from intracellular stores. Uterine hypoxia occurs during labour, as the powerful contractions occlude blood vessels (Greiss, 1965; Brinkman, 1990). Hypoxic conditions created by cyanide cause an intracellular acidification, a fall in [ATP] and a rise in inorganic phosphate concentration ([Pi]) in pregnant and non-pregnant rat myometrium. Specifically, an intracellular acidification of 0-27 pH units, a fall in [ATP] from 4 to 2 mm and a rise in [Pi] from 2 to 7 mm occurred when exposing strips of rat myometrium (non-pregnant) to cyanide (Wray, 1990). A separate study has shown that cyanide depresses spontaneous contractions in pregnant and non- pregnant rat myometrium (Wray, Duggins, Iles, Nyman & Osman, 1992). Cyanide also reduced force production in depolarized uterine preparations indicating that part of the mechanism is independent of membrane potential (Wray et al. 1992). Intracellular acidification alone has been shown to decrease or abolish spontaneous contractions in both pregnant and non-pregnant rat myometrium (Wray et al. 1992). One possible site of action for the inhibitory effects of both Pi and pH in the intact myometrium is at the contractile proteins to reduce force activated by Ca2 . However, the degree to which Pi and pH affect Ca2+-activated force in the myometrium is unknown. Evidence that [Pi] (6-10 mM) depresses Ca2+-activated force production in permeabilized vascular and visceral smooth muscle is equivocal. In Triton X-100- permeabilized taenia coli, raising [Pi] reduced maximum Ca2+-activated tension production and depressed the Ca2+ sensitivity of tension production (Gagelmann & Guth, 1987). A similar result was obtained in saponin-permeabilized rabbit mesenteric artery (Itoh, Kanmura & Kuriyama, 1986). However, other workers have demonstrated that Pi alters the rate of relaxation, but has no effect on steady-state tension (Schneider, Sparrow & Ruegg, 1981). Similarly the effect of pH on permeabilized smooth muscle is unclear. In Triton X-100-permeabilized carotid artery smooth muscle lowering pH produced a marked increase in Ca2+ sensitivity of tension production (Mrwa, Achtig & Ruegg, 1974), whereas similar studies in saponin-permeabilized taenia coli showed little effect of lowering pH (Iino, 1981; Arheden, Arner & Hellstrand, 1989). The basis of the conflicting results described above for Pi and pH is unknown, but may arise from the different permeabilizing techniques or the different smooth muscle types. a-Toxin from Staphylococcus aureus has been previously shown to permeabilize smooth muscle: rabbit mesenteric artery (Nishimura, Klober & van Breemen, 1988); guinea-pig ileum (Kitazawa, Kobayashi, Horiuti, Somlyo & Somlyo, 1989) and guinea-pig portal vein and rat anococcygeus (Crichton & Smith, 1991). The advantage of a-toxin over other permeabilization techniques is that the small pore size prevents loss of the cytosolic proteins involved in contraction. The aim of this study was to investigate the effect of altering pH and [Pi] on Ca2+_ activated force in a-toxin-permeabilized non-pregnant rat myometrium smooth muscle while simultaneously measuring [Ca2+] using indo-1 fluorescence. Acidi- fication at constant [Ca2+] was found to increase Ca2+-activated force and raising [Pi] reduced force. These effects occurred with only small changes in the Ca2+ sensitivity of force production. A preliminary account of some of this work has been presented to the Physiological Society (Crichton, Taggart, Wray & Smith, 1993). pH AND Pi ON PERMEABILIZED MYOMETRIUM 631 METHODS Tissue Adult virgin Wistar rats weighing 200-250 g were killed by a blow to the head followed by cervical dislocation. After taking a vaginal smear to determine the oestrus cycle stage, the uterus was rapidly removed. Small strips of longitudinal myometrium (approximately 400 ,/m wide, 100 ,sm thick and 3 mm long) were dissected, and attached to a force transducer (Akers, AE17625; SensoNor a.s., Norway) and a fixed point with snares, in a small tissue bath. A small amount of resting tension (0-1 mN) was applied. All diagrams show active isometric tension generated by the preparation. a-Toxin permeabilization procedure The preparation was transferred from Tyrode solution (solution C, Table 1) to a mock intracellular solution (solution A + 70 /M CaCl2, Table 1) with a [Ca2M] of 100 EM, containing crude a-toxin (2 mg/ml) from the bacterium Staphylococcus aureus (Crichton & Smith, 1991). Tension rose slowly over a 10-15 min period as the Ca2+ gained access to the myofilaments. When tension had plateaued the a-toxin was removed and the [Ca2+] lowered to 1 nm (solution B, Table 1). Lowering the [Ca2+] caused the muscle to relax. The maximum Ca2+-activated tension generated by the permeabilized myometrial strips (in 100 /FM Ca2+) was 97 + 3 % of the peak tension observed on exposure of the intact muscle to high-K+ solution (solution A). In experiments where significant deterioration in force production was observed, linear extrapolation was used to predict the level of maximum Ca2+-activated force. Frequently Ca2+-activated force showed both a phasic and tonic component. Under these circumstances experimental interventions and measurements ([Ca2+] and tension) were made at the end of the tonic phase. Experiments were carried out at room temperature (20-22 0). Measurement of [Ca2+] within permeabilized preparations using indo-1 [Ca2+] was measured using the fluorescent dye indo-1. The tissue bath was placed on the stage of a Nikon Diaphot inverted microscope. The middle 1 mm length of the preparation was brought into focus using a 20 x fluor objective (Nikon CF Fluor, numerical aperture 0 75). A perspex column (6 mm diameter) was lowered onto the muscle to minimize the volume ofthe solution above the preparation. The perfusing solutions containing indo-1 (4/M) were pumped through the central bore of the column at a constant rate of 1 ml/min onto the permeabilized muscle strip. The volume surrounding the preparation was approximately 4 #1. The preparation was illuminated with light (360 nm) and the average [Ca2+], within the visual field containing the preparation, was calculated from the ratio of the emitted light intensities at 405 and 495 nm. Light emitted from areas of the visual field not occupied by the image of the muscle was reduced using a variable rectangular window on the side camera port of the microscope before the TV camera and photomultipler tubes. The focus was adjusted to provide an image at the level of maximum cross-section of the preparation. The depth of focus of the 20 x objective is approximately 0-3 /m. However, fluorescence from the volume above and below the plane of focus is also collected by the objective. This is minimized by lowering a perspex column to within 5-10 #sm of the top surface of the muscle as described above. Preliminary experiments with this technique have established that indo-1 binds to structures within the preparation and yet remains able to signal [Ca2+] with a Ca2+ sensitivity similar to that of free solution (G. L. Smith, D. S. Steele & C. A. Crichton, unpublished observation). Thus a larger proportion of the signal comes from the volume containing the preparation than from the surrounding perfusing solution. Estimates based on total fluorescent measurements suggest at least 66 % of the total fluorescent signal is from the volume occupied by the preparation.

Load more

  17

Copy Link