Regulation of metabolism and contraction by cytoplasmic calcium in

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 263, No. 28,Issue of October 5, pp. 14074-14079,1988 Printed in U.S.A.

0 1988 by The American Society for Biochemistry and Molecular Biology, Inc

Regulation of Metabolism and Contraction by Cytoplasmic Calcium in the Intestinal Smooth Muscle* (Received for publication, March 25, 1988)

Hiroshi OzakiS, TakumiSatoh, Hideaki Karaki, and Yukisato Ishidas From the Department of Veterinary Pharmacology, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113 and the Department

of Muscle Physiology, Mitsubishi Life Science Institute, Machida-shi, Tokyo 194, Japan

Reduced pyridine nucleotides (PNrd) and oxidized flavoproteins (FP,,) were measured fluorometrically in the intestinal smooth muscle strip of guinea pig taenia caeci simultaneously with contractile tension. Cytoplasmic free Ca2+levels ([Ca”+],,,)were also measured by a fura-2-Ca2+fluorescence technique. PNred, FP,,, and [Ca2+],,, increased during spontaneous contraction or upon the addition of high K+ or carbachol and decreased upon the removal of these stimulants. [Ca2+Ieyt increased before the increase in muscle tension. PN,,d increased almost simultaneously with or immediately after the onset of contraction, while FP,, increased before the initiation of contraction. Both PNrd and FP, decreased a few seconds after the initiation of relaxation. In the K+-depolarized,Ca2+-depleted muscle, graded elevation of external Ca2+increased PN,a, FP,,, andmuscle tension. The sensitivity to Ca2+ was in the orderofFP,, > PNred > muscle tension. Changes in PNr.d were inhibited when glycolysis was inhibited by substitution of external glucose with oxaloacetate, pyruvate, or B-hydroxybutylate, but not when oxidative phosphorylation was inhibited by Nz bubbling or by NaCN. In contrast to this, changes in the FP,, were inhibited by NZbubbling or NaCN, but not by the inhibition of glycolysis. These results suggest that an elevation of intracellular Ca2+activates carbohydrate metabolism and contractile elements independently, resulting in the reduction of cytoplasmic pyridine nucleotides, oxidation of mitochondrial flavoproteins, and development of tension in the intestinal smooth muscle.

When smooth muscles are depolarized by an excess K+ or stimulated by receptor agonists, force is generated with an increase in [Ca2+]cy,.Therate of oxygen consumption of smooth muscle increases with the isometric force development (1-5). The rate of lactate production also correlates with the isometric force (6-9). These results suggest that the contractile response of smooth muscle is dependent on both oxidative phosphorylation and glycolytic metabolism. However,only limited information has been obtained on the temporal relationship between rapid changes in metabolic events andmuscle contraction mainly because it is quite difficult to follow

* This work was supported in partby a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture in Japan. The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore hereby be marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact. $ To whom all correspondence should be addressed Dept. of Veterinary Pharmacology, Faculty of Agriculture, The University of Tokyo, Yayoi 1-1-1,Bunkyo-ku, Tokyo 113, Japan.

precise time courses of the metabolic changes using conventional analytical methods. Reduced pyridine nucleotides (PN,,d)’ are fluorescent substances. Utilizing this characteristic, the redox state of PN has been measured fluorometrically in a variety of isolated tissues or cells (10-21) including smooth muscles (22, 23). Oxidized flavoproteins (FP,,) are also fluorescent substances which are located specifically in the inner membrane of mitochondria. Fluorometric measurements ofFP,, have also been applied to isolated tissues (19,24, 25). Recently, fluorescent dyes, such as quin2 and fura-2, have become available to measure the change in [Ca2+],, in various biological preparations (26, 27).Wehavedeveloped a method to measure muscle contraction simultaneously with fura-2-Ca2+ signals in smooth muscle tissue using dual wavelength excitation method (28, 29). Using these techniques, it may be possible to correlate the rapid changes in these metabolite contents with [Ca2+lcyt andsmooth muscle contraction, and this has been the purpose of the present study. EXPERIMENTALPROCEDURES

Preparation and Solutions-Segments of taenia caeci, approximately 7 mm in length, were removed from male white guinea pigs weighing 300-400 g. Physiological salt solution contained(mM) NaCl 136.9, KC1 5.4, CaClz 1.5, MgCl2 1.0, NaHC03 23.8 and glucose 5.5 The solutionwas continuously bubbled with 95% 02,5% CO2 at 37°C and pH7.4. Hypoxic condition was introduced by bubbling with 95% NP, 5% COz mixture. High K+ (45.4 mM) solution was made by increasing the KC1 concentration in the above solutions. In fura-2 experiments, high K+ solution was made by replacement of 40 mM NaCl with equimolarKC1. A Ca2+-freesolution was made byomitting CaC12 from the normal solution and adding EGTA(0.1-0.5 mM). Measurement of Fluorescence-Reduced P N (PN,d, NADH, and NADPH) emits bluefluorescence(emission peak a t 470 nm) (30) when excited by 340 nm light. On the other hand, oxidized FP (FP,,) emits yellow-green fluorescence (emission peak a t 530 nm) (31, 32) when excited by 430-470 nm light. To detect PNM fluorescence, 470 nm emissionwavelength and 340 nm excitation wavelengthwere employed and to detect FP,, fluorescence, 530 nm emission wavelength and 450 nm excitation wavelength were employed (Table I). To measure the fura-2-Ca2+ fluorescence, muscle strip was alternately (48 Hz) excited a t 340 nm and 380 nm lights through the rotating filter wheel, and 500 nm emission was measured. The ratio of the fluorescence due to 340-380 nm was calculated from successive illumination periods. These experiments were performed with a fluorimeter specially designedto measure the fluorescence of living tissues (CAF-100, Japan Spectroscopic). The muscle strip was held horizontally in a temperature-controlled, 7 ml of volume organ bath. One end of the muscle strip was connected to a strain gauge transducer to monitor the mechanical activity. The time constant of the optical channels was 0.25 s. The abbreviations used are: PN,d, reduced pyridine nucleotides; PN, pyridine nucleotides; FP, flavoproteins; FP,., oxidized flavoproteins; [Ca”],,, cytoplasmic Ca2+ concentration; Fa,,, 500 nm emission at 340 nm excitation; Fam, 500 nm emission at 380 nm excitation; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraaceticacid.

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Calcium Regulation of Metabolism Fura-2 Methods-Fura-2 was loaded to the muscle by the method described previously (28, 29). Briefly, muscle strips were treated with 5 FM acetoxymethyl ester of fura-2 (fura-2/AM)(Dojindo Chemicals) for 3-5 h at room temperature. Noncytotoxic detergent, 0.5% cremophor EL (Nakarai), was added to theloading solution to solubilize fura-2/AM. In order to eliminate the possible quenching effect of heavy metal ions, N,N,N',N'-tetrakis(2-pyridilmethyl)-ethylenediamine (10 p ~ (Dojindo ) Chemicals) was added to all the experimental solutions (33). The fura-2-loaded musclewas rinsed with normal solution for 30 min at 37°C before starting the experimental period. During the rinsing, fluorescence induced by 340 nm excitation (F340) andthat induced by380 nm excitation ( F ~ I rapidly ) decreased followed by slow and steady decrease (approximately 5% of the basal fluorescence/lO min). However, the ratio ofF340 to F3W stayed almost constant indicating that the decreases in F340and F3e.0 are proportional. In accordance with the decrease in the basal level ofF340 and FSm,the changes in the fluorescence during muscle contraction also gradually decreased. The slow decrease in F340 and F3W did not apparently change either when muscle was excited continuously or when excited intermittently, suggesting that the decrease is not due to photobleaching of fura-2. Theseresults suggest that the slow decrease may be largely attributable to slow washout of uncleaved fura-B/AM and partially to leak of fura-2 from the cell. Since the decreases in these fluorescences were proportional, we used the ratio of FBMto F3m in fura-2-loaded taenia as an indicator of[Ca"],, as suggested previously (26-29, 35). Measurement of Tissue NADH Content-NADH contents of the taenia were determined by the method of Karp et al. (34) with slight modifications. After an incubation in the test solution for 30 min, muscle strips were dropped into liquid nitrogen and then powdered using amalgamator (B. Braun Melsungen A. G.). Ethanol(70%) solution containing 10 mM potassium phosphate buffer (pH 7.0) was added to thepulverized sample to extract NADH for 20 min at -20°C. This mixture was then centrifuged at 8000 X g for 1 min, and supernatant was again centrifuged for 25 min at 8000 X g at 4°C. The NADH concentration of the supernatant was measured with a purified luciferin-luciferase reagent (mixture of flavin mononucleotide, NADH:flavin mononucleotide oxidoreductase, bacterial luciferase, and myristic aldehyde) (Lumac B.V./3M). The light emission was measured in a photon counter (Biocounter M2010, Lumac B.V./3M). Materials-Drugs used were carbachol (Tokyo Kasei) and verapamil hydrochloride (Sigma). RESULTS

Changes in Cytoplasmic Ca2+Concentrations-In the furaand F380 increased by 20-302-loaded muscle strips, bothF340 muscle strips. As shown fold over those in the fura-2-unloaded in Fig. 1, addition of 45.4 mM KC1 increased F340,decreased F380, and increased the ratio, an indicatorof [Ca2+]cyl(26-29, 35), and induced muscle contraction. These results are consistent with the previous observations in the strips of rat aorta (35). Spontaneousrhythmic (28, 29) or guineapigileum change in the fluorescence ratio was also followed by the corresponding rhythmic contraction. The changes in muscle tension were preceded by the changes influorescence ratio by 0.5-1 s and when the ratio increased to a threshold level (dotted line in Fig. 1) muscle tension began to increase. Carbachol (1PM) showed similar increments in the ratio and muscle tension as KC1 whereas EGTA (4 mM) decreased the ratio and relaxed the muscle (data not shown). These results TABLE I Excitation and emission wavelengths used in the present studyto detect fluorescence of reduced pyridine nucleotides (PN), oxidized flavoproteins (FP), and fura-2-Ca2+complex Excitation wavelength wavelength

Emission

Ref.

nrn (half-wideband length)

Reduced PN Oxidized F P Fura-2-Ca2+

340 (5.5) 450 (5.5) 340 (5.5) 380 (5.5)

470 (10) 530 (10) 500 (10) 500 (10)

(30) (31) (27)

14075

FIG. 1. Change in fura-2-Ca2+fluorescence during the spontaneous contraction and 45.4 mM KCl-induced contraction in intestinal smooth muscle of guinea pig taenia caeci. FS40, the 500 nm emission at 340 nm excitation. FW, the 500 nm emission at 380 nm excitation. Ratio, F340/F380, an indicator of [Ca"]cyt. F340 and FsWare shown by relative value taking the resting fluorescence level at the startof the experiment as 100%. The dotted line indicates the level of the ratio at which muscle tension began to increase.

-

-

KC I

CCh

-- CCh

KC1

2 min

CCh

KC1

- CCh

KC1

FIG. 2. Changes in P N d fluorescence ( A )and FP,, fluorescence ( B )during muscle contraction in the guinea pig taenia during stimulation 1 CIM carbachol (CCh) and 45.4 mM K+ ( K C l ) . Fluorescence due to PN,d or FP,, are shown in the upper trace and tension changes are shown in the lower trace. Amounts of PN,d and FP,. fluorescence are shown by relative intensity of fluorescence taking the basal fluorescence as 100%.

indicate that muscle contraction in the taenia is due to the increase in [Ca2+],,. Changes in PN,,d Fluorescence and FP,, Fluorescence during Contraction-Fig. 2A shows thesimultaneous recording of PNredfluorescence (upper trace) andisometric tension development (lower trace) of the muscle strip. Carbachol (1 p M ) produceda transientcontraction followed by a sustained contraction. Fluorescence increased simultaneously or 0.5-1 s after the initiation of muscle contraction. The increase in PNredfluorescence was 42.5 f 4.6% ( n = 9) of the resting fluorescence level determined before the stimulation. When

Calcium Regulation of Metabolism

14076

carbachol was removed,the fluorescence beganto decrease 510 s after the initiation of relaxation and the fluorescence was still decreasing toward the resting level whenthe muscle was completely relaxed. The effect of 45.4 mM K+ was essentially similar to thatof carbachol (Fig. 2A). Fig. 2B shows the simultaneous recording of FP, fluorescence (upper trace) and contraction (lower trace). Carbachol (1 phi) increased FP,. fluorescence. In contrast to the PNA fluorescence,the FP,. fluorescence started toincrease slightly ( P N d fluorescence > muscle contraction. The effects of S3+ on mechanical responses and PNA and FP,. fluorescence were examined (Fig. 6). Cumulative addition of 0.1-10 mM Sr?" to the Ca2+-depleted,45.4 mM K+depolarized muscleproduced graded increase in FP, fluorescence with only a small increase in tension andP N d fluores-

loo

i

B Y

0

k

I

I

01

1

10

Ca ( m M 1

FIG. 3. Changes in P N d fluorescence ( A ) and FP,. fluorescence ( E ) ( A and E , upper traces) during spontaneous contractions ( A and B, lower traces).

FIG. 5. Effects of external Ca'+ concentrations on P N d fluorescence (Closed triangle), FP.. fluorescence (closed circle) and tension development (closed square) in the IC+-depolarized taeniacaecum. Responses induced by 10 mM Ca*+was taken as 100%. Experimental protocol, see Fig. 4.

I

0-I

FIG. 4. Effect of cumulative addition of Caz+on P N d fluorescence (upper trace) and contractile force (lower trace)of high K+.After the application of 45.4 the taenia depolarized with mM K+ in normal PSS, muscle strip was treated with Ca2+-free solution (with 0.1 mM EGTA) for 15 min, and then 45.4 mMKC1 was applied. Readmission of Ca2+ produced graded increase in P N d fluorescence and contractile force.

1

011

Sr

10

(mM)

FIG. 6. Effects of external Sr*+concentrationson P N d fluorescence (closed triangle), FP, fluorescence (closed circle), and tension development (closed square) in the K+-depolarized taeniacaecum. 100% represents the response to 45.4 mM KC1 in the presence of 1.5mM Ca2+.Experimental protocol, see Fig. 4.

Regulation Calcium cence. The ability of Sr2' to activate FP,, fluorescence was approximately Y 3 of that of Ca2+. When 4 mM EGTA was applied to theprecontracted muscle with high K+ or carbachol, muscle tension rapidly decreased to theresting level. EGTA decreased PN,d and FP,, fluorescence to the level slightly higher than the resting level. The decrease in tension startedfew seconds earlier than thePN,d and FP,, fluorescence. A Ca2+channel blocker, 1 PM verapamil, showed similar effects as theCa2+-freesolution (data not shown). Effect of Metabolic Inhibition on PN,.d and FP,, Fluorescence-When the oxidative phosphorylation was inhibited by replacement of O2 with Nz, the resting P N d fluorescence gradually increased (Fig. 7). In the absence of external Ca2+, hypoxia also increased PN,d fluorescence (data not shown). Small spontaneous contractions, which occurred during the N2 bubbling, induced a large increase in P N d fluorescence. The addition of 45.4 mM K+ induced rapid phasic contraction followed by a small sustained contraction.On the otherhand, P N d fluorescence gradually increased in the presence of excess K'. When the hypoxic condition was introduced to the high K+-precontracted muscle, further increase in P N d fluorescence was observed although the muscle started to relax after some latency period (Fig. 8 A ) . Replenishment of 0 2 rapidly decreased the PN,.d fluorescence while the muscle tension began to increase after about 5 min. NaCN (1 mM) showed similar effects as hypoxia; it increased the P N d fluorescence reaching its maximum after about 1min without changing the muscle tension in resting muscle, whereas it increased PN,d fluorescence and decreased muscle tension in high K+-stimulated muscle (data not shown). Tissue NADH content was measured by an analytical method to know the absolute concentration (Table11).Under the aerobic condition, 45.4 mM K+ increased tissue NADH content from 12.0 to 36.8 nmol/g wet weight. N2 bubbling increased the NADH content to 62.8 nmol/g and high K+ further increased the content to130.0 nmol/g. In contrastto thePN,d fluorescence, N2bubbling decreased FP,, fluorescence, as shown in Fig. 9. Under this condition, 45.4 mM K+ induced rapid phasic contraction followed by a small sustained contraction which was not accompanied by the rise in FP,. fluorescence. In the high K+-precontracted muscle, N2 bubbling decreased FP,, fluorescence followed by a decrease in muscle tension and readmission of O2 increased

of Metabolism

L

14077

-J

KC1

FIG.8. Effectof

N2 bubbling on muscle tension (lower trace), P N d fluorescence ( A ) and FP,, fluorescence ( B ) (upper trace) in K+-depolarized taenia. Nz bubbling was applied during the 45.4 mM KC1-induced contraction. TABLEI1 NADH contents of the intestinal smooth muscleof taenia caeci under normoxic and hypoxic conditionsLIS determined by luciferin-luciferase method Values indicate mean f S.E., and the number of experiments is shown in parentheses. Condition

5.4 mM KC1 45.4 mM KC1

N2

0 2

nmollg wet weight 62.8 f 3.0" ( n = 4) 12.0 f 0.7 ( n = 8) 130.0 f 12.5"( n = 4) 36.8 f 5.2" ( n = 4)

"Significantly greater than the value in the presence of 5.4 mM KC1 and Oz with p < 0.01 (with Student's t test). 50

*'-[

i\$pkdf144;~~\h L i'LvLLL""L ,"

J/-""-/1 t c (

"C(

KC I

50"*

[

KCI

+

-

""

N2

15 m i n

1

KC1

t NZ

FIG.9. Effect of N2 bubbling on FP,, fluorescence (upper trace) and contractile force (lower trace).After the changes in

FP,, fluorescence reached plateau in the presence of Nz, 45.4 mM KC1 was repeatedly applied.

KC1

.

4

.

.

.

N2

FIG.7. Effect of N2 bubbling on PN.4 fluorescence (upper trace) and contractile force (lower trace).After the changes in

P N d fluorescence reached plateau in the presence of Nz, 45.4 mM KC1 was repeatedly applied. Note that P N d fluorescence markedly increased following small spontaneous contractions.

FP,, fluorescence followed by an increase in muscle tension (Fig. 8B). NaCN (1 mM) induced similar changes asN2 bubbling (data not shown). When glycolysis wasinhibited by removing external glucose and adding 1mM 2-deoxyglucose,the basal PN,d fluorescence gradually decreased (Fig. 10). Increments in PN,d fluorescence and muscle tension due to high K' were also gradually inhibited. Addition of 10 mM oxaloacetate restored the high K+-induced contraction but not the PNredfluorescence. Pyr-

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FIG. 10. Effect of glucose removal on PN,d fluorescence (upper truce) Exterand contraction (lower truce). nal solution was replaced with a glucosefree solution containing 2-deoxyglucose (2-DG) and then 45.4 mM KC1 was re-

peatedlyapplied.Afterthecontractile response to high K reached a constant level, 10 mM oxaloacetate (OA) was added.

FIG. 11. Effect of glucose removal on FP,, fluorescence (upper truce) and contraction (lower truce). External solution was replaced with glucosefree solution containing 2-deoxyglucose (2-DG)and then 45.4 mM KC1 was re-

peatedly applied.

-

" " " H

CI

KC I

2-DG

free,

KC1

t

Glucose

"

H

10 m i n

U

U

n

H

Glucosefree.2-DG

uvate (10 mM) and P-hydroxybutylate (10 mM) showed similar effects as oxaloacetate. In contrast to this, the inhibition of glycolysis gradually increased the basal FP,, fluorescence. Increase in FP,. fluorescence due to high K+ was not inhibited whereas contraction was markedly suppressed (Fig. 11).

glycolysis (glycogenolysis) is stimulated either by inorganic phosphate or ADP which activates phosphofructokinase or by Ca2+which activates phosphorylase b kinase. The present findings indicate that the activation of phosphorylase might play a major rolein the regulation of glycolysis whichoperates independently of the tension developments in the smooth DISCUSSION muscle. Introduction of N2bubbling or addition of NaCN to inhibit The present results indicate that PN,d increased during contraction and decreased during relaxation in theintestinal electron flow through the respiratory chain caused a large smooth muscle. The increase in PN,.d due to stimulation has increase in P N d without changing muscle tension. Further, been reported in giant squid axon (13), rat portal vein (23), small spontaneouscontractions induced larger increase in rat hepatocyte (18), and heart muscle (21). The stimulation- PN,d under N2 bubbling than that under aerobic condition induced decrease in PN,d has also been demonstrated in eel (Fig. 3). These results may indicate that, under aerobic conditions, PN,d produced by glycolysisin the presence of stimelectric organ (10) and skeletal muscle (14, 15), andthe bubbling, biphasic effects were observed in cervical vagus nerve (16,17). ulants is oxidizedin mitochondria. In contrast to N2 When glycolysiswas inhibited by removingglucose and as expected, depletion of cellular substrates by removal of adding 2-deoxyglucose, both PN,d and muscle tension de- external glucose and addition of 2-deoxyglucose decreased creased. Oxaloacetate, pyruvate, and P-hydroxybutylate, the PN,d and increased FP,,. The present study demonstrated that FP,, increased during substrates for oxidative metabolism, were as effective as glucose in restoring tension development although these sub- contraction. Oxidation of FP does not seem to be the result strates failed to restore the decrease in PN,,d. On the other of contraction since the increase in FP,. invariably preceded increased with hand, inhibition of oxidative phosphorylation by Nz bubbling contraction. As is the case with PN,d,FP,, did not inhibit the high K+-induced increase in PN,,d. These the increase in external Ca2+ concentrations and the Ca2+ results suggest that the larger portion of P N d , measured sensitivity of FP,, was greater than either mechanical activity fluorometrically in the present study, exists in cytoplasm and or PNmd. These resultsindicate that the increase in [Ca2+lWt may directly activate the oxidation of FP in mitochondria. the amount is regulated by glycolysis. The changes in PN,d and muscle tension corresponded to Chance (36)has observed that Ca2+ increases the rate of the changes in [Ca2+],,. The increase in PN,.d preceded the respiration and electron transport of mitochondria. It was contraction for a few seconds in the presence of 0.1 mM Ca2+. also reported that oxygen consumption of heart mitochondria Further, change in PN,d was more sensitive to Ca2+ than was activated by 10 nmol Ca2+/mgmitochondrial protein (37). muscle tension. These results suggest that both mechanical Ca2+ is taken up by mitochondria with the K,,, value of activity and reduction of PN are dependent on [Ca"],, and approximately 1WM (38),and intramitochondrialkey enzymes are not sequential but independent events. It is known that for oxidative metabolism, such as cy-oxoglutaratedehydrogen-

Regulation Calcium ase, isocitrate dehydrogenase, and pyruvate dehydrogenase, are activated by 0.1-10 WM Ca2+(39). The contractile protein of the smooth muscle of taenia is also activated by 0.1-10p M Ca2+ (40, 41). These findings suggest that an elevation of cytoplasmic Ca2+to a physiological range triggers the activation of the mitochondrial oxidative metabolism independently of the activation of glycolysis and development of tension. Tsuda et al. (42) have reported that 0.1 mM Ca2+produces greater increase in oxygen consumption than that intension development in high K+-depolarized taenia. Balaban and Blum (18) have demonstrated in rat hepatocytes that glucagon and vasoactive intestinal polypeptide cause an transient increase in mitochondrial PN,d and sustained increase in oxygen consumption and thesechanges are dependenton external Ca2+. Sr2+ hasbeen shown to replace the role of Ca2+to support the various biological events. In smooth muscle, Sr2+ enters the cell through Ca2+entry pathways (43-45) and stimulates contractile elements(45,46).Present resultsshowed that Sr2+ substitutesCa2+ to increase FP,, more efficiently than to increase PN,d or to activate contractile elements. This result is consistent with the observation that high K+ increases oxygen consumption with little effect on muscle contraction in the taenia inthe SP-substitutedsolution (2).These results again support the conclusion that contraction and energy metabolisms are not the sequential processes. In addition, the Ca2+-regulationof FP oxidation (which is substituted by Sr2+) seems to be different from the Ca2+regulation of PN reduction or contractile elements(which are less substituted by ST2+). Allof these findings indicate that, at least in the early phase of the smooth muscle contraction, the increments in glycolysis and oxidative phosphorylation are dependent on [Ca"],, but not on the amount of ATP, ADP, or inorganic phosphate. However, thesephosphate compounds seem to play a key role in the maintained phase of the contraction because ( a ) when stimulant is removed or extracellular Ca2+ is decreased duringsustainedcontraction, muscle tension decreased faster than thedecrease in PN,d and FP,,, and ( b ) high K+ or carbachol still increased P N d and FP,, even in the absence of extracellular Ca2+.The latter effect might be attributable to the ATP consumption by Na' pump which might be activated by the above stimulants even in the absence of Ca2+. In summary, the presentresults indicate that pyridine nucleotides are reduced and flavoproteins are oxidized during smooth muscle contraction. Reduction of pyridine nucleotides is the result of an increased glycolytic flux, suggesting that large oxidation-reduction potentials are generated between cytoplasm and mitochondria during the stimulation. Further, the redox state of pyridine nucleotides and flavoproteins may be directly and independently regulated by the changes in the cytoplasmic free Ca2+ concentrations. REFERENCES 1. Saito, Y., Sakai, Y., Ikeda, M., and Urakawa, N. (1968) Jpn. J. Pharmacol. 18,321-331 2. Urakawa, N., Ikeda, M., Saito, Y., and Sakai, Y. (1968) Jpn. J. Pharmacol. 1 8 , 500-508 3. Chang, A. E., and Detar, R. (1980) Am. J. Physiol. 238, H716H728 4. Paul, R. J. (1983) Am. J . Physiol. 2 4 4 , C399-C409 5. Takai, A., and Tomita, T. (1986) J. Physiol. (Lond.) 381, 65-75 6. Peterson, J. W., and Paul, R. J. (1974) Biochim. Biophys. Acta 357, 167-176

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