Journal of Physiology (1992), 445, pp. 759-778 With 8 figures Printed in Great Britain

759

INVOLVEMENT OF SARCOPLASMIC RETICULUM 'Ca2+ RELEASE CHANNELS' IN EXCITATION-CONTRACTION COUPLING IN VERTEBRATE SKELETAL MUSCLE

BY DONALD G. BRUNDER, SANDOR GYORKE, CHRISTINE DETTBARN AND PHILIP PALADE From the Department of Physiology and Biophysics, The University of Texas Medical Branch, Galveston, TX 77550, USA (Received 7 May 1991) SUMMARY

1. Pharmacological blockers of calcium-induced calcium release from isolated skeletal sarcoplasmic reticulum (SR) vesicles have been introduced into frog skeletal muscle fibres to determine their effects on excitation-contraction coupling. 2. Among the blockers tested, Ruthenium Red, neomycin, gentamicin and 9aminoacridine inhibited the SR Ca2" release associated with excitation-contraction (E-C) coupling as much as they inhibited caffeine potentiation of that release. Protamine, certain of its derivatives, and spermine were ineffective in both in situ tests. 3. Alternative sites of polyamine action on the contractile proteins, SR Ca21 uptake or charge movements were ruled out. 4. All polyamines tested required considerably higher concentrations to inhibit excitation-contraction coupling than to block Ca2" release from isolated SR vesicles. 5. The quantitative pharmacological difference in sensitivity between isolated and intact systems serves as a reminder that results on isolated systems cannot generally be used to predict results of the same substances on more physiological systems. 6. Since caffeine is known to open the SR 'Ca2' release channels' (the ryanodine receptors that mediate Ca2+-induced Ca2" release), the equal effectiveness of these blockers at inhibiting excitation-contraction (E-C) coupling and its potentiation by caffeine suggests that the SR 'Ca2' release channels' are indeed involved in excitation-concentration coupling in skeletal muscle, although the results do not indicate how the channel is gated open during E-C coupling. INTRODUCTION

Recently several biochemistry groups (Imagawa, Smith, Coronado & Campbell, 1987; Inui, Saito & Fleischer, 1987; Lai, Erikson, Rousseau, Liu & Meissner, 1988; Takeshima, Nishimura, Matsumoto, Ishida, Kangawa, Minamino, Matsuo, Ueda, Hanaoka, Hirose & Numa, 1989; Penner, Neher, Takashima, Nishimura & Numa, 1989) have utilized [3H]ryanodine as a tool to determine the distribution of 'Ca2+ release channels' in the sarcoplasmic reticulum (SR) and then to solubilize these channels from the membrane, and isolate, purify, reconstitute, sequence and MS 9374

26-2

760

D. G. BR UNDER AND OTHERS

express them to demonstrate functional Ca2+ channel activity. While these channels appeared to mediate Ca2+-induced Ca2+ release (Lai et al. 1988), there appeared to be little new evidence suggestive that the Ca2+-induced Ca2+ release mechanism (Bianchi & Bolton, 1967; Endo, 1977) was involved in vertebrate skeletal muscle excitation-contraction (E-C) coupling. If Ca2+-induced Ca2+ release is not involved in E-C coupling, would the recently isolated SR channels that mediate such release be likely to be involved in E-C coupling? In contrast to strong evidence for a role of dihydropyridine receptors in E-C coupling of skeletal muscle (Rios & Brum, 1987; Tanabe, Beam, Powell & Numa, 1988) very little direct experimental evidence has been provided for a role of the ryanodine-sensitive 'SR Ca2+ release channels' in E-C coupling. Baylor, Hollingworth & Marshall (1989) have reported effects of Ruthenium Red on excitation-contraction coupling in fibres, but high enough concentrations were employed that possible sites of action other than the release channels warrant consideration. Most evident in favour of the ryanodine-sensitive channel as the real SR calcium release channel is considered either 'circumstantial' (Sutko & Kenyon, 1990) or based largely upon its being a Ca2+-permeable pore localized in the right place, at the triadic junction (Coronado, 1990). The present communication was intended to determine whether the SR 'Ca2+ release channels' recently isolated are indeed the physiologically relevant ones for skeletal muscle E-C coupling. To assess this, a multidisciplinary approach was initiated to identify inhibitors of SR Ca2+-induced Ca2+ release channels (Palade, 1987) and assess their effects on E-C coupling in situ. Local anaesthetics known to inhibit this channel (Palade, 1987) were avoided because of their acknowledged lack of specificity. Instead, a number of blockers of Ca2+-induced Ca2+ release, primarily polyamine (Palade, 1987), have been introduced into skeletal muscle fibres under voltage clamp conditions. Preliminary results of some of these findings have been reported (Brunder & Palade, 1989; Palade, Brunder, Dettbarn & Stein, 1990). METHODS

Cut segments of frog skeletal muscle fibres were dissected from Rana catesbeiana or pipiens semitendinosus muscles in a depolarizing relaxing solution consisting of 58 mM-K2SO4, 42 mmNa2SO4, 25 mM-KCl, 3 mM-KH2PO4, pH 72. The frogs were killed by stunning followed by decapitation. As schematized in Fig. 1, the fibre segments (shaded) were mounted in a Hille-Campbell (1976) triple Vaseline gap voltage clamp chamber with all pools (C,B,A,E, in order) filled with an 'internal' solution consisting of 120 mM-potassium-aspartate, 3 mM-MgSO4, 3 mmNa2ATP, 3 mM-Tris maleate, 5 mM-Na2phosphocreatine, and 0.1 mM-EGTA/28 /tM-CaCl2 (pCa = 7), pH 71. The A pool solution was replaced with Ringer solution (120 mM-NaCl, 2-5 mM-KCl, 1-8 mM-CaCl2, 3 mM-MOPS, pH 7 2) supplemented with 1 1uM-tetrodotoxin (TTX). All muscle fibre experiments were performed at 18-20 'C. In certain experiments the A pool solution was exchanged for TTX-Ringer solution including 0-5 mM-caffeine. In all experiments polyamine inhibitors were introduced into the E pool containing the saponin-treated fibre end. Minimum stimulus duration determinations were performed with fibre segments mounted at slack length (- 2-2 ftM sarcomere spacing). The cut end in the E pool was treated with 0-01 % saponin in 'internal' solution for 1 min and the saponin washed out. The muscle fibre was held at -90 mV, and short duration pulses to + 100 mV were applied while observing the fibre under 150 x magnification on a compound microscope stage. The minimum stimulus duration (MSD) represents the shortest duration pulse that elicits a barely detectable local contraction (Almers & Best, 1976). The MSD generally ranged from 170 to 710 Its under control conditions.

SR Ca2+ RELEASE CHANNELS IN E-C COUPLING

761

Calcium transient determinations were performed with the same voltage clamp apparatus but with fibres stretched to 3 5-40 Itm sarcomere spacing (to reduce fibre movement). Additionally the chamber was modified for optical determinations by inclusion of a fibre optic seated just underneath the A pool segment of the fibre. Antipyrylazo III (2 mM) was included in the 'internal'

Vm

Fig. 1. Diagram of the experimental cut fibre preparation. A cut fibre segment (shaded) is laid across the Vaseline threads that traverse it in the vertical direction. Additional Vaseline (or Glisseal vacuum grease) is then applied above the fibre to isolate four compartments electrically. Pools C, B and E contain internal solution, and A external solution. Only A is voltage clamped. Electrodes in C and A sense the voltage difference (VVm) between the inside and outside of the fibre relative to the grounded B compartment. Current is passed via voltage command pulses (V,) through the E electrode and recorded as Im. Polyamine substances were applied to the E pool end after permeabilization, from which they had to reach the A pool. Caffeine and 9-aminoacridine, being membrane permeant, were applied directly to the A pool in these experiments. solution applied to the saponin-treated fibre end in the E pool. Light intensity traces were recorded as described in Palade & Vergara (1982), using 710 nm for Ca2+-related absorbance changes and 790 nm to correct for any residual fibre movement. The correction involved subtraction of the 790 nm trace from the 710 nm trace after scaling to produce equal background light intensities. The results were normalized by the absorbance at 550 nm to compensate for changes in dye concentration in the region of the fibre under examination. Isolated frog sarcoplasmic reticulum terminal cisternae were prepared as described by Brunder, Dettbarn & Palade (1988) with the modification that Rana catesbeiana muscle was homogenized in an Oster blender for only 30 s at the 'grate' setting. The homogenate was then centrifuged at 6500 r.p.m. in a JA- 10 rotor (Beckman Instruments) for 10 min, the pellet rehomogenized and respun at 6500 r.p.m. for 15-30 min. The supernatant from this second homogenate was then centrifuged at 35000 r.p.m. for 1 h in a Beckman Type 35 or 45 Ti rotor to pellet microsomal membranes. These microsomes were resuspended and layered on top of a discontinuous sucrose gradient for subfractionation. Following overnight sucrose gradient centrifugation at 20000 r.p.m. in a Beckman SW 28 rotor, the final R4 fraction (terminal cisternae) at the 38%/45% sucrose interface and R2 fraction (light SR) at the 25 %/32 % interface were isolated, resuspended in 0-9 Mtrehalose and stored in liquid nitrogen until use. Calcium release studies with isolated frog terminal cisternae were performed as described in Palade (1987), using 10 mM-caffeine as the calcium-releasing agent. In these experiments the cuvette contained 88 mM-KCl, 12 mM-KMOPS, 7-5 mM-Na4P207, 1 mm-MgATP, 5 mM-Na2phosphocreatine, 20 ,ug ml-' creatine phosphokinase and 0-25 mM-Antipyrylazo III at 25 'C. Changes in [Ca2+] outside the vesicles were monitored using A710-A790. The dye responds to

D. G. BR UNVDER

762

ANVD

OTHERS

with an increase in absorbance at 710 nm. Neither the free dye nor its Ca2+ complexes absorb Ca2" at 790 nm. Thus subtraction of absorbance at 790 nm is used here to correct for possible changes

in vesicular light scattering that might take place during an experiment, just as a similar correction was applied in cut-fibre experiments to correct for fibre movement. These changes in light scattering should be relatively independent of wavelength and were extremely small under these conditions in any case. A description of a typical experiment is provided in the text in referring to Fig. 4.

Computer modelling of the time course of drug effects in muscle fibres experiments was carried out by assuming that diffusion of compounds in muscle fibres is similar to diffusion in a plane sheet. If a compound is applied to one end of a fibre of length L at time zero, the concentration of the compound, C, at any time t and distance x from that end is given by

(nnx') e-f co[(I-x'--E-sin ITn=l n eqn 4 16), where x' = x/L and

=

T = Dt/L2, C0 = the applied (derived from Crank, 1975, concentration at x 0, and D is the diffusion coefficient. To allow for strong binding and/or chemical reaction of the drug as it diffuses, the diffusion coefficient can be replaced by the apparent diffusion coefficient Dapp. The proportion of freely diffusing substance relative to the total concentration present is Dapp/D. Values of Ki in muscle fibre experiments were obtained from fits

to the relationship

percentage

block=

too

I+ (ClKi)

analysis was used to generate 95 % confidence limits. Voltage-dependent charge movements (Schneider & Chandler, 1973) were measured using the same voltage clamp system but with ionic solutions devoid of most permeant ions. In most cases, the external solution consisted of 68 mM-(TEA)2SO4, 7-5 mM-CaSO4, 5-6 mm-MOPS, 1,tMtetrodotoxin, pH 7-2, and the 'internal' solution contained 80 mM-caesium aspartate, 20 mmCs2EGTA, 3mM-MgSO4, 3 mM-Na2ATP, 5 mm-Na2phosphocreatine, 3 mM-Tris maleate, pH 7-1. With EGTA present to prevent contraction, fibres were not stretched. All pulses were delivered from a holding potential of -90 mV and were of100 ms duration. Test pulses were averaged eight times. Control pulses were of -20 mV amplitude (to -110 mV) and were averaged thirty-two times. Charge movement records were obtained by subtracting a scaled version of the control pulse current records from the test pulse current records, the scaling determined by the relative amplitude of test and control pulses. Reagents were obtained from Fisher Scientific (Pittsburgh, PA) or from Sigma Chemical Co. (St Louis, MO, USA). Perornithine-thynnine and thynnine undecapeptide were the gracious gift of F. Centro di Studi Biopolimeri del Padova, Italy. Probit

Professor

Marchiori,

CNR,

RESULTS

experiments determined the effects of different polyamines on (MSD) in voltage-clamped frog cut fibre segments. Almers and associates (Almers & Best, 1976) we reason that a critical Following amount of Ca2" must be released to reach the contractile threshold. Since the threshold is reached so quickly (< 1 ms) when pulses to very high potentials are applied, the rate of Ca2' release must greatly exceed the rate of Ca2+ reuptake at the MSD. Thus the MSD should be inversely related to the rate of Ca2+ release. Consequently, compounds which decrease the rate of Ca2' release should lengthen the MSD. The experiments shown in Fig. 2 were carried out by applying gentamicin at the concentrations indicated to one cut fibre end at t = 0. Subsequently the MSD was determined at periodic intervals to ascertain whether SR Ca2+ release in situ had Initial muscle fibre

the minimum stimulus duration

SR Ca2+ RELEASE CHANNELS IN EC COUPLING

763

been affected. The SR Ca2+ release was calculated as the inverse of the pre-drug MSD, normalized to 100%. Concentrations in the order of 50 ,ag ml-' were required to appreciably inhibit the rate of Ca2+ release. Gentamicin applied at 10 Iug ml-' or less to a cut fibre end had minimal effects on SR Ca2+ release (not shown). The lack of 100 o 20

0

0

80 8_0~~~~

Co

60\ 60° 0)~ ~

CD CD

40\=

Co

50

~ ~~~~~ 0 0

+

0 Cu

jg ml-' gentamicin

0

20 100 pg ml-' gentamicin

E

0

20

40

120 60 80 100 Time (min) Fig. 2. High concentrations of gentamicin must be applied to cut fibre ends to inhibit E-C coupling. Two voltage clamp cut fibre experiments are illustrated in which the inverse of the MSD was plotted as a function of time after application of 50 jig ml-' gentamicin (0) or 100 ,ug ml-' gentamicin (@) to the E pool fibre end. The curves through the data points represent fits to the diffusion equation outlined in the Methods, with both Dapp and C allowed to vary simultaneously. Dapp (the apparent diffusion coefficient) was calculated from the fits to be 3-26 x 10-7 cm2 s-' in both cases, and the estimated Ki was 12-4 jIg ml-' in the upper fit and 12-2 ,ig ml-' in the lower fit.

substantial effect of low concentrations of gentamicin was not due to inability of the substance to reach its site of action, since higher concentrations had significant effect in the same time frame. These data can also be modelled to determine the drug's apparent diffusion coefficient. The diffusion coefficients, so measured, were of the order of 20 % of that in free solution and will be reviewed further in discussion of results in Table 2, later. This same modelling also yields the apparent inhibitory constant (Ki) for the drug, at which concentration it inhibits SR Ca2' release in situ by 50 %. This concentration is several orders of magnitude greater than that previously reported to inhibit by 50 % caffeine-induced Ca2' release from isolated rabbit skeletal muscle triadic SR (Palade, 1987). The dose-response relation for gentamicin inhibition of SR Ca2+ release in situ is shown in Fig. 3. The scatter in the data is relatively large. The curve drawn through the data represents the best fit to a model assuming 1: 1 stoichiometry between ligand and receptor. It is possible that other steeper functions might also fit the data, but the present study is not as concerned with whether one or two

D. G. BR UNDER AND OTHERS

764

gentamicin molecules bind to the site at which E-C coupling is affected as it is with examining whether gentamicin effects on SR Ca2" release contribute to the effects on E-C coupling. Similar observations were made with a number of other polyamines, including neomycin, protamine, perornithine-thynnine (a synthetic protamine fragment) and 100 v /

O

*I

\

80

cU

X

60

.~

+0C.)

3

e-) 0 40 20~~~~~~~~~~

E

20

0

z

0

/

0

0.1

1

10

100

Gentamicin (pg ml-') Fig. 3. Inhibition of SR Ca2` release in situ as a function of estimated gentamicin concentration. Experiments were performed as described for Fig. 2. Each point was derived from a separate experiment at steady state, with the estimated gentamicin concentration assumed to be the same as that applied in the E pool.

spermine. In all cases negligible effects were seen at concentrations which inhibited Ca2+ release from isolated rabbit SR (Palade, 1987) by 50%. These data are summarized in the left-hand column of Table 1. By way of comparison, equivalent Ki values determined in isolated rabbit SR experiments (Palade, 1987) were 003 ,tg ml-' for gentamicin (> 500-fold lower), 0-06 fSM for neomycin (> 25-fold lower), 0-01 jug ml-' for protamine (> 25000-fold lower), 22 jaM for spermine (> 400fold lower) and 0 003 jaM for Ruthenium Red (> 400-fold lower). Comparable data using 9-aminoacridine, a non-polyamine blocker of the Ca2+-induced Ca21 release channel (Palade, 1987), are also provided. We have included 9-aminoacridine results because polyamines themselves might have some specific side-effects that a nonpolyamine might not exert. The 9-aminoacridine was applied in the extracellular solution in the A pool, and unlike the polyamines, exhibited similar blocking potencies in both muscle fibre (35 /AM, Table 1) and isolated SR experiments (32 jaM, Palade, 1987). Before arriving at any conclusions regarding the discrepancy in polyamine potencies we considered it necessary to test certain possible causes for it. One possible source of the discrepancy involves species differences: the comparison of fibre data obtained using frog muscle with SR data obtained originally using rabbit muscle. Accordingly, terminal cisternae were prepared from the same species of frogs that

SR Ca2+ RELEASE CHANNELS IN E-C COUPLING

765

were used for most of the muscle fibre experiments. Typical experimental results are shown in Fig. 4. The traces shown demonstrate changes in the absorbance of the Ca2+-sensitive dye Antipyrylazo III in a spectrophotometer cuvette in the presence of isolated frog terminal cisternae. The dye remains outside the vesicles and monitors

O 0

X~~~~~~~~~Control

Fig. 4. Inhibition of caffeine-induced Ca2release fromfrogterminalfcine +

Gentamicin/ 1 ,ug ml-1

a g

ml

n

Fig. 4. Inhibition of caffeine-induced Ca'+ release from frog terminal cisternae by low

concentrations of gentamicin. Experiments were carried out as described in Palade (1987) and in the Methods. Briefly, 42 ,ug frog terminal cisternae were added to 1 ml of incubation medium containing Mg, ATP, pyrophosphate and Antipyrylazo III. Upward deflections at the beginning of each trace mark 12-5 nmol CaCl2 additions to the cuvette. Following uptake of Ca2+ from fifteen such additions, 10 mM-caffeine was added (open arrow) to the cuvette, eliciting an immediate downward deflection due to an interaction between the dye and caffeine. This artifact is followed by a larger slower upward movement of the trace indicative of Ca2+ release. This absorbance change is only seen if the SR is previously loaded with Ca2+ (Palade, 1987), and this represents a true release of Ca2+. The traces underneath represent the same experiment, but carried out with 1, 5 or 20 jug ml-' gentamicin added 10-20 s before the addition of caffeine. Filled arrow-heads represent additional 12-5 nmol CaCl2 additions made to elicit Ca2+ release and to recalibrate the system in the presence of the gentamicin and caffeine.

the Ca2" levels in the solution outside the SR. In the left-hand panel each upward deflection represents a Ca2' addition to the cuvette. The record relaxes back to the baseline as the Ca21 is sequestered inside the vesicles, where the dye can no longer sense it. After ten such additions the trace is interrupted briefly (for data storage) but the experiment continues in the uppermost trace of the right-hand panel. Following five more Ca2' additions, caffeine was added to the cuvette to elicit a large upward movement in the trace indicative of release of Ca21 to the extravesicular environment where the dye can once again sense it. The three lower traces demonstrate the same experiment (in all cases with fifteen Ca2+ additions made) repeated with different concentrations of gentamicin added shortly before the caffeine additions. Gentamicin appears to completely prevent caffeine-induced Ca2' release unless further Ca2+ additions are made subsequently, triggering Ca2+-induced Ca2+ release. In these experiments gentamicin at concentrations of even 1 jtg ml-' was able to appreciably inhibit the rate of caffeine-induced Ca2+ release from frog SR. Data summarized in Table 1 and in Palade (1987) suggest that there is at most a modest

D. G. BR UNDER AND OTHERS

766

decrease in drug sensitivity when comparing results from isolated frog SR experiments with those obtained using isolated rabbit SR. As seen in Table 1, a very large discrepancy still exists when comparing frog muscle fibre results with results obtained with isolated frog SR. Another source of the discrepancy in potencies could come from impeded diffusion of the drugs into the fibres, but our computer modelling (Fig. 2) was able to fit the TABLE 1. Differential effects of selected blockers on Ca2' release from isolated SR and from in situ muscle fibre preparations

Ki Frog muscle fibres 154 (11-7-20-1) 1-68 (0-94-3-01) > 250 > 50

Isolated SR (frog terminal cisternae) 0-62 (0-21-1-30) 0-069 (0-013-0-163) 0-025 (0 007-0{059) 0-69 (0-19-1-76)

Gentamicin (,tg ml-') Neomycin (,UM) Protamine (,ug ml-') Perornithine-thynnine (,ug ml-,) 23-7 (11-6-40-1) 9700 Spermine (,UM) Ruthenium Red (,UM) 1 22 (0 66-2 26) 0-013 (0007-0{020) 9-Aminoacridine (,UM) 351 (176-541) 27-1 (220-324) Values given represent means, with 95% confidence intervals determined by probit analysis given in parentheses. Individual muscle fibre experiments averaged here are presented in Table 2, except for six experiments with 9-aminoacridine. All isolated SR experiments performed as described in Palade (1987) using isolated frog terminal cisternae, with five to nine determinations performed with each drug. All isolated SR experiments employed 10 mM-caffeine as a stimulus. Ki refers to that concentration of inhibitor which blocked Ca2' release by 50%.

data well. This suggested that the free drug concentrations finally achieved in the A pool portion of a fibre were near those applied to the fibre cut end. The apparent diffusion coefficient for those polyamines exerting appreciable effects (gentamicin, neomycin, Ruthenium Red) varied from 0-06 to 0 22 x that assumed for molecules of that molecular weight in free solution (Table 2). Among other substances tested previously, Kushmerick & Podolsky (1969) found myoplasmic diffusion of substances other than Ca21 to be 0 3 x that in free solution. The apparent diffusion coefficient for Ruthenium Red (0-09-031 x 10-6 cm2 s-1), was considerably faster than the 0 02-0)06 x 10-6 cm2 s-' obtained by Baylor et al. (1989). With protamine and its two synthetic fragments, no appreciable effects on SR Ca2' release in situ (indicative of drug entry into the portion of the fibre being voltage clamped) could be detected unambiguously. In the case of thynnine undecapeptide, there was some indication that SR Ca21 uptake might have been impaired because stimuli delivered too closely together generated MSD determinations that differed, as if the Ca2" had not been resequestered into the SR between the stimuli. These effects were, however, difficult to quantify for analysis. In the case of 9-aminoacridine, diffusion was clearly not an issue, since it was applied extracellularly, exhibited marked effects with very little lag, and exhibited no polyamine-like discrepancies in potency when comparing isolated SR results with muscle fibre results. A polyamine-induced increase in Ca21 sensitivity of the myofilaments, while not at -

SR Ca2+ RELEASE CHANNELS IN E-C COUPLING

767

TABLE 2. Apparent diffusion coefficients for selected polyamines in myoplasm Estimated K, Concentration Fibre No. (,UM or ,tg ml-') Dapp x 10-7 Ruthenium Red (mol. wt 786, expected D* = 23 x 10-7 in free solution) 30 ,UM 061088E2 0-65 0 90 40,M 052688A2 3 83 2-88 060188E2 6-30 2-07 061088E1 2-68 3 05 50 /tM 052588E 1 0 95 2-47 5 20 052788A2 2-67 050188E1 080 1 15 80 UM 052488A1 003 1 10 Average 1 22 (0 66-2 26) 2-04+0 31 Gentamicin (mol. wt 463, expected D* = 35 x 10-7 in free solution) 21-1 011387E1 720 50,agml-' 012988E1 42-0 3-26 012888E3 18-9 4-10 2-4 012788A1 1 94 12-4 012688E3 3-19 032588E2 29-8 28-70 011988E3 50 1 55 012188A1 36-2 6-23 75,ug ml-' 020387E2 9-7 6-30 020387E4 35 4 20-40 100utg ml-, 12 2 011988E 1 3-26 Average 15-4 (11-7-20-1) 7-83 + 2-61 Neomycin (mol. wt 614, expected D* = 27 x 10-7 in free solution) 0-21 041087A2 0-69 50,UM 040588A1 3-77 5-80 100 ,UM 041087A1 1 01 1-84 040187E2 1-95 0-85 0-21 040187E1 0 50 033187A2 10-5 1-32 031787E2 6-02 1 00 Average 1-68 (0-94-3-01) 1-60+0-66 Spermine (mol. wt 202, expected D* = 57 x 10-7 in free solution) 10 mM 022588A2 9709 34-3 * Calculated by extrapolation or interpolation from a semilogarithmic plot relating the measured diffusion coefficients in water presented in Kushmerick & Podolsky (1969) for sulphate, sorbitol, sucrose and ATP to the molecular weights of these solutes. Two additional experiments with 30-40 ,M-Ruthenium Red, one with 75 ,ug ml-' gentamicin, and one with 3 mM-spermine, were excluded from the analysis because effects were obtained too quickly after solution changes to be attributable to drug diffusion. Average values given for Ki represent means calculated from log functions of concentrations, with values in parentheses representing the 95 % confidence limits of the means converted back from logarithmic functions to linear units. Had we merely averaged the values in linear units, the means+S.E.M. would have been 2-56+0-83,/M for Ruthenium Red, 20 5+41/,tg ml-l for gentamicin and 3 50 + 1-40 /tM for neomycin. Average values for Dapp are given in linear units + S.E.M.

all likely, could, if present, have contributed to the discrepancies between muscle fibre results and isolated SR experiments. Thus we deemed it necessary to test whether SR Ca2+ release measured directly was more sensitive to polyamine effects

D. G. BR UNDER AND OTHERS

768

than were MSD determinations. Accordingly, Ca2+ transient determinations were performed with one of the polyamines. Absorbance measurements were performed at 710 and 790 nm with Antipyrylazo III inside cut fibres before and after application of gentamicin to the E pool end of the fibre. As seen in Fig. 5, traces recorded at

+10 mV

+10 mV

0.05 25 ms

-90-

0.02L 25 ms

-90

< _ = j ~~~~~~~~~~~~~~~Bfre

60 min after 50 jig ml-' gentamicin

Fig. 5. Effects of end-pool application of gentamicin on Ca2+ release measured intracellularly with Antipyrylazo III. Ca2+ transients were recorded before and 60 min after application of 50 ,ug ml-' gentamicin to the fibre cut end. Data shown include the intensity changes at 710 (LA710) and 790 nm (A A790) (upper and lower traces, respectively, in each pair of traces to the left; upper pair = before gentamicin; lower pair = 60 min after gentamicin application), as well as the subtraction of the two and normalization by A550 (individual traces shown to the right). Control experiments without gentamicin determine that the amplitude of the Ca2+ transients remained essentially constant in these experiments when normalized by this procedure.

790 nm were essentially flat, suggesting that stretching the fibre had effectively eliminated fibre movement which could otherwise distort the absorbance measurements of intracellular Ca2+ levels. The experiment demonstrates that 50 ,ug ml-' gentamicin decreased the amplitude and rate of rise of the Ca2+ transient, but not more than 50 %. Thus gentamicin was no more effective at inhibiting SR Ca2+ release measured by Ca2+ transients than it was at lengthening the MSD, and it was much less potent at inhibiting SR Ca2+ release during E-C coupling than at inhibiting the channels that mediate Ca2+-induced Ca2+ release from isolated SR in vitro. In order to assess the degree of inhibition of Ca2+-induced Ca2+ release channels inside muscle fibres during the course of these experiments, a new protocol, illustrated in Fig. 6, was devised. MSD determinations were first performed to determine Ca2+ release rates with a muscle fibre under standard control conditions. Then the bathing solution in the A pool was replaced with one containing 0-5 mmcaffeine. This caffeine concentration is lower than that required to elicit contractures but is sufficient to potentiate twitches and Ca2+ transients (Kovacs & Szuics, 1983).

SR Ca2+ RELEASE CHANNELS IN E-C COUPLING 769 At these low concentrations, caffeine would also not be expected to affect the Ca2+ sensitivity of the myofilaments (Wendt & Stephenson, 1983). MSD determinations were again performed, revealing an increase in the rate of Ca2+ release. The effect was observed to be reversible when the caffeine was washed out. Then polyamine A 175

°-

150-

=-

125

0

0.5 mM-caffeine

50,ug

ml-' gentamicin

0)

)

75

.

50

C.)

25

0O 0

10

20

30 40 50 Time (min)

60

70

80

B 175 °

0.5 mM-caffeine

0II

150

%

125

15.

20 pM-9-aminoacridine 0p 30 m-9-aminoacridine

0 125[

0

100 X

*

@

50

c)

25

0-5 mM-caffeine

-IZ

75

0

0

10

20

30 40 Time (min)

50

60

70

Fig. 6. Effects of intracellularly applied gentamicin and extracellularly applied 9-aminoacridine on excitation-contraction coupling and caffeine potentiation. A, the experiment was carried out essentially as described for Fig. 2 except that 05 mM-caffeine was applied to the Ringer solution in the A pool after a baseline MSD had been established. Following determination of the effect of caffeine, it was washed out, and gentamicin subsequently applied to the E pool fibre end. After an approximate steadystate had been reached, caffeine was reapplied to the A pool to redetermine the extent of potentiation and then finally washed out. B, the experiment was performed as in A except that 9-aminoacridine was applied in the A pool solution rather than to the E pool.

D. G. BR UNDER AND OTHERS inhibitors were applied to the saponin-treated cut end in the E pool and 'basal' MSD determinations performed until an approximate steady state was reached. At this time 0 5 mM-caffeine was reintroduced into the A pool solution and the degree of potentiation of Ca2+ release remeasured (Fig. 6A). Had the Ca2+-induced Ca2+ release 770

(

100

.o

80

60 o0~~~~~~~~~ 40*,

r_

Q

/

O

o

820

00 20 40 60 80 100 Inhibition of Ca2+ release rate (% Fig. 7. Correlation between in situ effects of polyamine inhibitors on ' basal ' Ca"+ release and caffeine potentiation of that release. Experiments were performed as described for Fig. 6. Different symbols represent experiments performed with different concentrations of applied gentamicin (50 #g ml-', A) neomycin (20-50 ^ M,), Ruthenium Red (10-80,UtM, *, protamine (100-250 ug ml-', *) spermine (1-3 mm, V) and 9-aminoacridine (20-200,um, O). Two lines were drawn through the data. The dashed line represents a linear regression fit to all the data. The correlation was only 0-765. The continuous line represents the correlation obtained if the two uppermost experimental points at the left of the plot were left out. In those two exceptional fibres, caffeine elicited contractures 5-10 min after application in the presence of Ruthenium Red, and this may have affected our MSD determinations of caffeine potentiation. The correlation improved to 39.0 0

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channels been completely inhibited under these experimental conditions we should have discovered that caffeine was unable to produce any potentiation whatsoever of CaI2+ release. What we found, on the contrary, was that the potentiation of Ca2( release by caffeine was inhibited only about as much as the degree of inhibition of the 'basal' rate of Ca2+ release in the absence of caffeine. A similar experiment using a membrane-permeant blocker, 9-aminoacridine, is depicted in Fig. 6B. In this case, the 9-aminoacridine was applied to the extracellular solution in the A pool. With this non-polyamine blocker, a similar degree of inhibition was noted between effects on E-C coupling, caffeine potentiation and caffeiinduced release from isolated SR. The results of a number of other experiments using the protocols illustrated in Fig. 6 are plotted in Fig. 7. Note that with 9-aminoacridine and every polyamine studied, the degree of inhibition of caffeine potentiation was similar to the degree of inhibition of 'basal' Ca2+ release. The difference in degree of inhibition of these two processes

SR Ca2+ RELEASE CHANINELS IN E-C COUPLING

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was very small compared to that anticipated from our earlier experiments utilizing isolated SR vesicles. These results suggested strongly that the SR Ca2+ release channels that mediate Ca2+-induced Ca2' release do participate in E-C coupling, but we could not yet lay claim to having proved it until we ruled out alternative mechanisms of inhibitory action of the drugs under study. TABLE 3. Effects of polyamines and 9-aminoacridine on Ca2+ uptake by isolated frog SR Percentage of control rate of oxalate-supported Ca2" loading

Light SR Terminal cisternae Control 100 100 504uM-neomycin 91 + 11 115+ 14 100 /IM-neomycin 94+5 95+ 19 50 jtg ml-' gentamicin 90+9 107 + 12 100 ,tg ml-' gentamicin 92 + 10 95 + 5 42 + 13 50 gM-Ruthenium Red 58 + 2 100 /tM-Ruthenium Red 55+9 43+ 10 50 /tM-Ruthenium Red* 43 +6 38 + 2 100 ,um-Ruthenium Red* 51+9 30+2 50 /LM-9-aminoacridine 88 + 6 99 + 10 100 /iM-9-aminoacridine 94 + 16 83 + 11 * Ruthenium Red seemed to precipitate at the usual oxalate concentration of 6 mM; therefore additonal experiments were performed with 2 mM-oxalate. Under these conditions there may have been some release channel contribution to the net fluxes measured. Percentages given represent the mean + S.D. of three to four determinations. Control rates of Ca2" uptake for the different frog SR samples employed were 1-80-3-31 /tmol mg-' min-' for light SR and 1-93-3-15 ,umol mg-' min-' for terminal cisternae in the presence of 6 mM-oxalate (1-70 and 1-21 4umol mg-' min-1, respectively, in the presence of 2 mM-oxalate).

If a substance happened to deplete the SR of Ca21 it could in principle generate a similar correlation to that shown in Fig. 7. While low concentrations of these polyamines had no discernible effect on Ca2+ pumping in isolated rabbit SR vesicles (Palade, 1987). Two additional tests were performed here. The first was to assess the effects of high concentrations of polyamines and 9-aminoacridine on oxalatesupported Ca2+ uptake by purified frog light SR. Since only Ruthenium Red, neomycin and gentamicin exhibited large effects on Ca2+ release in situ, these were the only polyamines tested. The results are shown in Table 3. With Ruthenium Red there were severe effects on the rate of SR Ca21 uptake, but the effects with neomycin, gentamicin and 9-aminoacridine were much more moderate at the concentrations employed in situ. Furthermore, none of these compounds appeared to reduce the final extent of Ca2+ uptake or to leave elevated [Ca21] levels extravesicularly. The effect of these three agents on Ca2+ uptake could not possibly by itself have produced the effects seen on E-C coupling. Partial inhibition of Ca2+ uptake by Ruthenium Red would probably have been reflected in impairment of Ca2+ release only with stimuli spaced more closely together than in our experiments. Under our experimental conditions at least 30 s were allotted for fibres to resequester released Ca2+ back into the SR, much more than required for this reuptake to be completed. A second test involved examination for possible in situ effects on Ca2+

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COUPLING 773 sequestration. Inspection of the Ca2+ transient in Fig. 5 reveals no obvious gentamicin-induced impairment in Caa2+ sequestration after the stimulatory pulse was turned off. Thus in situ effects on uptake are no more marked than in vitro effects. Consequently, polyamine effects on SR Ca2+ uptake are unlikely to contribute (via SR Ca2+ depletion) to the polyamine effects on Ca2+ release. If the same concentration of polyamine substances happened to affect the voltagedependent charge movement in the transverse tubule membrane that controls SR Ca2+ release, inhibition of SR Ca2+ release we have observed could be due to charge movement inhibition rather than release channel blockade. In this case, the correlation between the degree of block of Ca2+-induced Ca2+ release channels and the degree of block of in situ electrically stimulated Ca2+ release might have been fortuitous. Accordingly, experiments with these blockers were performed to assess their effects on the charge movement that might be responsible for gating the SR Ca2+ release channels. As seen in Fig. 8, there was very little discernible effect of the applied gentamicin over a period of 50 min. These experiments were performed in the presence of elevated EGTA in the end-pool solutions to prevent fibre movement. We did not notice any evidence for a hump in any of our charge movement traces, but neither did we systematically look for one. Thus it remains possible at a Q, component of charge movement may have been absent in our records. A compendium of results from numerous other charge movements experiments with neomycin, gentamicin, Ruthenium Red and 9-aminoacridine is shown in Fig. 8, demonstrating that none of these compounds had appreciable effects on charge movements at concentrations where SR Ca2+ release was seriously impaired. In all, fourteen separate experiments were performed in which fibres yielding acceptable charge movement records were followed for suitable periods of time. In only one of the neomycin experiments (that labelled with open triangles) was there evidence for a large reduction. Even in this case, though, the inhibition was transient despite the continued presence of the drug. Most probably this represented an artifact of some sort. Otherwise, inspection of the data would suggest conservatively that at most 20% of the charge movement is affected by any of these compounds. Therefore by far the most likely site of polyamine and 9-aminoacridine action on SR Ca2+ release is at the level of the SR Ca2+ release channel. DISCUSSION

The discovery of polyamines as inhibitors of SR Ca2+-induced Ca2+ release channels (Palade, 1987) prompted the current investigation into the role of these channels in physiological excitation-contraction coupling. The approach taken here has been a pharmacological one intended to determine whether blockade of these SR 'Ca2+ release channels' inhibits E-C coupling. If the channels were not involved in E-C coupling, little inhibition would have been observed. Conversely, if the channels were involved in E-C coupling a high degree of correlation should have been (and was) observed between channel blockade (assessed by effects on caffeine potentiation) and inhibition of E-C coupling. A similar study to ours arriving at similar conclusions to those reported here has been presented by Baylor et al. (1989) working with the polycationic dye Ruthenium

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Red. Their study was by no means definitive in determining the site of action of their Ruthenium Red effects, however. Ruthenium Red has been criticized as 'by no means a specific tool' (Volpe, Salviati & Chu, 1986) in view of its staining of SR and calsequestrin, its effects on the force-pCa relationship and its known inhibition of both mitochondrial and SR Ca2" uptake. The results of Baylor et al. (1989) could also have been due to SR depletion of Ca2" due to SR Ca2+ pump inhibition (or even to irreversible local fibre damage at the injection site) at the high Ruthenium Red concentrations injected. The much lower apparent diffusion coefficient calculated by Baylor et al. relative to that determined here may indicate that Ruthenium Red applied at high concentrations becomes immobilized by binding to numerous sites of moderate affinity for the dye, producing additional untoward functional effects. The identification of other inhibitors of SR Ca2+-induced Ca2+ release channels has enabled the issue to be examined more critically here. Specifically, we have utilized several inhibitors, demonstrated their in situ effectiveness against caffeine effects, and documented effects on Ca2+ transients as well as lack of effects on charge movement.

Specificity of drug action As with any such study, the specificity of action of the substances utilized is critical. Aside from the SR Ca2+ release channels believed responsible for the effects observed here, several additional possible sites of action of the drugs utilized have been addressed in the Results, including the contractile proteins that generate the contractions being monitored, the dipoles in the transverse tubule membranes believed responsible for physiological gating of the SR Ca2+ release channels, and SR Ca2+ uptake. Sites of polyamine action at the level of the contractile proteins did not appreciably affect our results, since Ca2+ transients were equally sensitive to gentamicin as were MSD determinations. With respect to neomycin, Vergara, Tsien & Delay (1985) demonstrated direct effects on Ca2+ transients at concentrations (150-300 uM) similar to those utilized here to increase the MSD. The results of Volpe et al. (1986) suggest that effects of 5 /IM-Ruthenium Red at the level of the contractile proteins are minor. Sites of action at the level of the charge movements reportedly affecting SR Ca2+ release have been excluded by direct examination. A similar study by Baylor et al. (1989) also provided evidence that Ruthenium Red did not affect subthreshold charge movements. None of the compounds effectively inhibiting E-C coupling appeared to stimulate SR Ca2+ uptake, and only Ruthenium Red inhibited SR Ca2+ uptake markedly. The rapidity of 9-aminoacridine action further suggested that SR Ca2+ depletion due to diminished uptake was not contributing to its effects on E-C coupling. We restricted our analysis with Ruthenium Red to concentrations of 50 tM and below since our experience with higher concentrations suggested additional deleterious effects probably unrelated to Ca2+ release channel blockade. Additional possible sites of drug action could include other SR proteins. Several other forms of relatively slow SR Ca2+ release have been reported from isolated light SR subfractions derived from longitudinal elements of the SR which lack caffeine sensitivity (Volpe et al. 1983; Brunder et al. 1988; Dettbarn & Palade, 1991), but in

SR Ca+2 RELEASE CHANNELS IN E-C COUPLING

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frog SR these releases are less sensitive to neomycin and gentamicin (C. Dettbarn & P. Palade, unpublished results). Similarly, phosphatidyl-inositol 4,5 bisphosphate (PIP2)-induced Ca2' release from isolated frog SR (Ogawa & Harafuji, 1989), while properly sensitive to neomycin and Ruthenium Red and insensitive to spermine and protamine, was not greatly affected by 9-aminoacridine (100 /M) (C. Dettbarn & P. Palade, unpublished results). Thus involvement of these forms of Ca2+ release in our muscle fibre results is unlikely. A possible role of inositol 1,4,5-trisphosphate (InsP3) in skeletal muscle E-C coupling has been suggested, at least in part on the basis of polyamine effects on E-C coupling (Vergara et al. 1985). However, polyamine inhibition of caffeine potentiation of release can only be explained by such a theory if InsP3 and caffeine act on the same SR Ca2+ release channels, which appears not to be the case (Palade, 1987). Thus we continue to consider effects of polyamine on E-C coupling more likely to be related to their inhibition of caffeine-sensitive Ca2+ release channels than to interference with any InsP3-associated phenomena (Palade, 1987).

Correlations between in vitro and in situ studies The conclusion that the polyamine effects on muscle fibres described here are due to direct action of Ca2+-induced Ca2+ release channels could not have been drawn from a superficial comparison of the data shown in Table 1, where drug effects on isolated SR were apparent at much lower concentrations than in muscle fibres. Had we concluded on that basis that the Ca2+-induced Ca2+ release channels were not involved in E-C coupling, we would have been incorrect, as our later experiments including assessment of drug effects on caffeine potentiation demonstrated. Thus our results also have some implications for experimenters utilizing isolated SR. Sensitivity to channel modulators like those polyamines appears to be quite dependent on the experimental conditions. This may explain why muscle fibres still contract despite the presence of millimolar concentrations of spermine, another polyamine, inside. Since it may never be possible to exactly duplicate myoplasmic conditions in vitro, inferences made from isolated SR experiments concerning muscle SR physiological function should always be subjected to the additional scrutiny of certain physiological experiments when possible. The sensitivity of Ca2+ release channels to polyamines can evidently be modulated by various factors (Cifuentes, Ronjat & Ikemoto, 1989; Calviello & Chiesi, 1989; Palade, Dettbarn, Brunder, Stein & Hals, 1989) which may account for the decreased sensitivity to Ruthenium Red reported in fibre studies (Volpe et al. 1986; Baylor et al. 1989; present communication). The differential sensitivity of fibres to various polyamines affords an opportunity to match experimental conditions appropriate for isolated SR experiments with conditions found in situ, even if not ionically correct. Thus isolated SR should under 'physiological' conditions be only moderately responsive to Ruthenium Red, neomycin and gentamicin; and protamine and spermine should neither induce Ca2+ release (Cifuentes et al. 1989) nor inhibit caffeine-induced Ca2+ release (Palade, 1987). A set of conditions which approximates this behaviour is outlined in Palade et al. (1989).

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SR Ca2+ release channels and the release mechanism Endo (1977) utilized experiments with Mg2+ and procaine to argue against the involvement of the Ca2+-induced Ca2+ release mechanism in E-C coupling. His results would also tend to cast doubt on involvement of those SR Ca2+ release channels mediating such Ca2+-induced release. However, the actions of Mg2+ in inhibiting Ca2+_ or caffeine-induced Ca2+ release may not be as complete at physiological Mg2+ concentrations as Endo (1977) had suggested. We have found that elevated Mg2+ (3-10 mM) applied to both end-pools does inhibit Ca2+ release (not shown). In intact fibres, 10 mM-procaine blocked caffeine contractures but not K+ contractures (Endo, 1977), perhaps because the K+ contractures involved a less procaine-sensitive entry of extracellular Ca2+ through surface membrane/T-tubule Ca2+ channels (Palade & Almers, 1985). The small procaine effects noted by Heistracher & Hunt (1969) in voltage-clamped fibres after short-duration exposures may have been due to limited permeability at neutral pH (Gonzalez, Brum & Pizarro, 1991), since stronger procaine effects on Ca2+ transients have been reported (Klein, Simon & Schneider, 1992). Our results are interpreted as indicative of involvement of Ca2+-induced Ca2+ release channels in E-C coupling in skeletal muscle, but they do not permit a conclusion to be drawn about whether Ca2+ is indeed the physiological trigger. Since polyamines seem to inhibit all or most releases involving this channel, they are most likely to interfere with ion movement through the pore part of the channel rather than solely by competing with any one agonist such as Ca2+ for a binding site (Palade, 1987). Other effectors have recently been claimed to be capable of opening these same channels: inositol 1,4,5-trisphosphate (Suarez-Isla et al. 1988), alkalinization (Ma, Fill, Knudson, Campbell & Coronado, 1988), sulphydryl oxidation (Salama & Abramson, 1984; Brunder et al. 1988), prior SR Ca2+ release (Rios & Pizarro, 1988) as well as the presumed mechanical linkage affected by charge movements (Schneider & Chandler, 1973). If these claims are true, then any of these effectors could represent the physiological activator of SR Ca2+ release, so long as it was produced in situ in sufficient quantities locally to overcome any endogenous inhibitors (e.g. Brunder et al. 1988). The authors gratefully acknowledge Lynette Durant for typing of the manuscript. This work was supported by NIH AR34377 (to P. P.) and a grant-in-aid from the American Heart Association, Texas Affiliate (D. G. B.). REFERENCES

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