B~,~'himica et Bioph)wica Acta, l 133 ( I gq2) 133-141 ~) le~t/2 Elsevier Science Ppblisher.~ fiN. All righls reserved OIhT-,$gSq/t~2/Sr)5 qlt)
133
BBAMCR 131165
Vascular oxidative metabolism under different metabolic conditions C h r i s t o p h e r D. H a r d i n , R o b e r t W . W i s e m a n a n d M a r t i n 2. K u s h m e r i c k Detlurtm~,nts of Radiology, Ptt),,~ic(o&~., atul Bi.pl~vsws, Uml t,r~tly .1" |l~u~itfu~zton, S~¢ni¢, |1.~4 ( L'.S ,~ ) (Received g April 1"J911
K¢~ words: Va:~cttlar smoo~h mu,;cle: C~lntrc~l of rK:~,piration; NMP.. ~1p_: Ox'v'gctt consumplion
Control of respiration in vascular smooth muscle wa~ examined while the metabolic state of the tissue was manipulated. During KCl.indueed eontractu~s in the presence of 5 mM gln:ase, oxygen consumption increased by 10 nmul / per rain g without any decrease in phosphucreatine (PCr) or ATP as determined by 31PoNMR indicating a control of respiration which does nat involve changes in high-energy phosphates (e,g., ADP, phnsphorylatien potentialL However, when aortae with resting tone in the absence of substrate were then provided with 5 mM 2-deoxyglucose as the sole subslrate, oxygen consumption increased 7.4 n m o l / m l n per g while PCr decreased by mare than 50% (resulting in a 2-fold increase in the calculated free ADP} with no change in tension from resting tone. Outing a subsequent KCI induced contraeture in the presence of 2-deo~g]ucose, oxygen consumption increased an additional 7.2 n m o l / m i n per g while PCr continued to decline. Therefore, at least P,Yo mechanisms of l~spiratory control may exist in sheep aorta, one dependent and the other independent of changes in high-energy phn£phates.
Introduction
Our understanding of tissue respiratory control is derived primarily from studies involving skeletal and cardiac muscle. The primary regulation of cellular respiration in fast-twitch muscle was thought to be cytosolie [ADP] or [Pi] in a feedback manner [1,2], For example, Meyer demonstrated that a simple linear model [3], with the concentration of ADP as the prim a w feed-back regulator, could account for the measured steady-state energy metabolism in fast-twitch muscle. This simple view o~"mitochondrial regulation is no longer tenable as it does not account uniquely or sufficiently for observations from studies involving other muscle types. Connett and Honig [4,5] demnnstrated in highly oxidative skeletal muscle that several common models for respiratory control were redundant so that they could not be distinguished by steady-state contents of nucleotides, PL and Per. In the myocardium, metabolic conditions e~st wherein th~ rate of oxygen consumption (Jo~) can increase several-fold without any detectable change in the PCr and ATP content as
Correspondence:C. 'Aardin,F.mpl.of Radiology.SB05, Universil~of Washington,School of M~icine, $ealtle. WA 98195. U.S.A.
measured by "~tp-NMR, and by extension in ADP [6,7]. A number of other po~ible regulators of Jo: have been proposed (see Ref. 6 for a review) including regulation by substrate delivery (NADFI O2, TCA cycle intermediates), Ca 2÷ modulation of enzyme activities, and changes in proton motive force. The control of respiration in smooth muscle has not been systematically examined and the existing limited data lead to apparently conflicting views of the mechanisms of respiratory control. Using freeze clamped tissues, PCr decreased with contraction in ,-at portal vein [8] but not in hog carotid [9]. Using 31P-NMR, Per decreased substantially with contraction in taenia coti from guinea-pig [10~ and rabbit [ll] and a smaIler decrease in P e r was observed with contraction in rabbit bladder [I 1]. Since control of respiration involving changes in high-energy phosphates must involve changes in [PCr], these data do not provide a convineing picture of the role of changes ;n high-energy phosphates in the control of mitochondriat respiration in smooth muscle tissue. We investigated whether different metabolic states may pray a role in determining which mechanismfs) control cellular respiration. In the heart, Ugurbil etal. [7] showed that mitochondrla[ cell respiration appears to be regulated by [ADP] when pyruvatc plus glucose is
134 the substrate; but some other factor, independent of changes in high-energy phosphates, regulates respiration when glucose alone is used. In the current study, we invesligated whether more than one mechanism for the control of respiration exist in smooth muscle, one dependent and the other(s) independent of changes in high-energy phosphates, and wi',,ther the metabolic state of the tissue determines which mechanism(s) predominates. The increase in Jo2 associated with contraction in the presence of glucose occurs with no change in the levels of high energy phosphates. An adenylate mechanism of respiratory control could be unmasked in sheep aorta when we decreased the total exchangeable phosphate pool and inhibited glycolys!s when 2-deoxyglueose was the only substrate provided. Portions of this work have appeared previously as an abstract (FASEB J. 2(4), A270). Materials a n d Methods
Tissz~e handling Segments of the thoracic aorta were dissected from sheep (Suffolk) and rinsed in isotonic NaCI for 10-30 rain before placement in a physiological saline (PSS) at room temperature (pH 7.4), consisting of 4.6 mM KCi, 116 m M NaCI, 26 mM Mops, 2.5 m M CaCI 2, 1.16 mM MgCI2, 10 m g / I gentamycirt, 5 mM glucose, pre-equilibrated with a gas mixture of 95% O~ and 5% CO2. Fat, connective tissue and adventitia were removed and segments 4 mm in length were cut and placed into separate water-jacketed glass chambers filled with PSS. Another piece of approx. 5 cm in length was placed unmounted in a standard 90 mm N M R tube and s~aperfused in gas-equillbrated PSS. Tissues were all~wcd to equilibrate for at least 1 h before the experimental protocol began.
Physiological measurements: force and oxygen consumplion Oxygen consumption was measurcd on isometrically mounted aortae using Clark-type polarographie electrodes (Insteeh) with a dual channel amplifier. Oxygen tension was calibrated by flushing the chambers with PSS pre-equilibrated with 95% 0 2 / 5 % CO z. Because of daily variation in system oxygen consumption rate, it was not possible to get an accurate measure of the 'basal' oxygen consumption rate of tissue alone. ~iowever, it was always possible to have the apparatus stable to measure increases in oxygen consumption above resting rates. Zero origen tension was calibrated using a non-saturated solution of sodium dithionite. The aortic rings were held isometrically at a length 1.4 × the resting length ex vivo. Isometric force was measured with transducers from Harvard Apparatus. Force and oxygen consumption were measured on the same tissue~ in the same apparatus at 22°C. Resting
tone was considered to be tile steady-state tone maintained by aortae while superfused in PSS. Contractions were elicited by changing the bathing solution to a high KCI PSS (final concentration of 80 mM KCI in molar replacement of NaCI). Relaxation was induced by changing the bathing solution back to PSS.
31P-NMR spectroscopy 3=P-NMR Spectroscopy measurements were performed in paraIlel to the force and oxygen consumption measurements, operating at 121 MHz for alp using a General EIectrie GN300 Omega spectrometer. Data were acquired using a 00 ° pulse 02/.¢s), a predelay of 3.0 s, a sweep width of 6000 Hz, and a 2K block size. Serial spectra, consisting 0[ 200 acquisitions (approx. 10 min of data averaging), were obtained throughout the experimental protocol. Data were zero filled to 4K points and filtered using 15 Hz exponential line broadenin~; prior to Fourier transformation. Individual resonances (vithin the N M R spectra were quantified by measuring the heights of individual peaks and normalizing to total phosphate in the spectrum. Quantitation of spectra from all aortae was performed by measuring the height of each peak within the spectrum and expressing the ATP or PCr ~evels as the ratio of each to the sum of all peak heights. The normalization of individual peak heights to total phosphorous is based on the assumption that the total cellular phosphate content remains constant [5]. The use of peaks heights is valid as long as individual peak shapes do not significantly change during the course of the experiment. Peak height analysis, although simple, avoids the bias of fitting programs relating to peak shape and variations in the baseline. In some experiments, a 5 m m N M R tube filled with 2 m M methylene diphosphonate (chemical shift of I7 ppm relative to H3PO 4) was included within the 20 mm sample tube and served as an external standard. Although all data were normalized to total tissue phosphate, when peak heights were normalized to the external standard, similar results were obtained but with increased variance.
Experimen tel protocol After equilibration, tissues were placed in PSS with 5 mM glucose for 1 h (relaxation), contracted with PSS with glucose and 80 mM KCI (in molar replacement for NaCI) for 1 h, and relaxed in PSS with glucose for 1 h. Then, the same relaxation/contraction/relaxation sequence was performed in the absence of substrate (glucose-free) and then in the presence of 5 mM 2-deoxyglueo~e. At the end of the protocol of some experiments, the tissues from the oxygen chambers were rinsed with PSS and rapidly frozen. These tissues were stored at - 8 9 ° C until further chemical analysis was performed.
135
Extracr.¢ and HPLC analysis Neutralized perehlorie acid extracts of tissues frozen by submersion in liquid nitrogen were analyzed for PCr and ATP by HPLC on a strong anion exchange column (Vydae model 303 NT305) using a phosphate gradient from 25 (pH 4.5) to 0.5 M phosphate buffer (pH 2.7). Absorbance was measured at 210 nm and quantification was accomplished by comparison with standards of ATP and PCr previously calibrated speetrophotometricaily using standard enzyme-linked metabo[itc assays
[~2]. Results Aortae were contracted and relaxed once in the presence of glucose, then in the absence of glucose, and then in the presence of 2-deoxyglucose. The KCtinduced eontra,'ture of the mounted aortic rings required approx, l h to reach peak isometric force (Fig. t) and approx. 1 h for full relaxation. This slow development of contraction and relaxation allowed 12 acquisition blocks (10 min each) to be obtained during the contraction/relaxation cycle.
2.Deoxyglucose as ~ubstrate under resting conditions After the I h with the aorta at resting tension in substrate-free PSS, we included 5 m M 2-deoxyglucose as the only exogenous substrate in the PSS. Ehosphorylation of 2-deoxyglucose by hexokinase re~iults in appreciable accumulation of 2-deoxyglueose 6-phosphate. Smooth muscle has an appreciable rate of glucose transport [13-16] and since cellular phosphate pools remain constant in smooth muscle [5] 2-dco~q,glueose acts as a phosphate sink in this tissue. Provision of 2-deox~,glueose for 1 h resulte,.t in a de-
creased level of PCr in the aortae compared to level~ during superfusiou with substrate-free PSS (Fig. 2A). However, during the hour of superfusion with 2-deoxy;21ueose in PSS, no decrease in the levels of ATP was oL~served (Fig. 2B), consistent with PCr acting to buffer changes in the [ATP] via the creatine kinase reaction. Sho~n in Fig. 2 are the normalized peak heights of PCr and 3,ATP during the transition from PSS wi~h(~ut any sabstrate to PSS with 5 m M 2-deoxyglucose a~ substr,'2_te. In thd presence of 2-deo:wglucose, isometric force remained at the unstimulate(] level. Stiil, Jo, increased by 7.4 5: I.i. n m o l / m i n per g blot wt. ( ± S.I~., n = 5) above that in PSS withoul substrate ITable I). Thus, the increase in Jo, asse-:ialed with the addition of 2-deoxvglucos¢ to the bath occurs when PCr levels in the aorta decrease, and hence t~ee [ADP] increases, without force developmeTlt,
Calculation of the free A;.,'P concentration In the subset of tissues frozen at the end of the experimental protocol, ATP, PCr and total creatine were measured by HPLC_ The measured concentrations of PCr and A T P of the tissues at the end of the experiment were assigned to the average peak heights of the PCr and -yATP peaks at the end of the experiment for two experiments. Concentration~ of ATP and PCr during the experiment were extrapolated by scaling to the changes in peak heights of ATP and PCr during the experiment. Using this subset of tissues, we estimated the range of free [ADP} during the experiment. The free [ADP] was calculated for those cxtrapolazed concentrations of PCr and ATP, and measured total ereatine (PCr + Cr) by using the ereatine kinase equilibrium constant measured in smooth muscle [17]
co RTRACI'I NO SOLUTION
VO
REL~Xl NG SCLU'rlO~
_/
I blOUR
~
....
--
---~-.~
(IlmQi~m Irl gml (1~)
(14)
FiB. L Tracing o[ a force record of a sheep aorta during a KCI-induced conll-~.clure and sd~equcnl i{:li~(aticsn, A~¢ra~.~ ~,~-gl~n ~onsumplion v a l u ~ for Ih~ pr¢-contzactcd and coelracting aorta arc give in parentheses. ]?or delails see t¢~.
13fi 0 25
25
0;:O"
020'
pCrlP t
ATP/P t
0 i =~..
®
OIC"
0 05 OC0 ---
~,.,cose
IL.
g oQ
J
2-Deoxygr~'cose
- glu;~;e
T|i~e ( 1 0 r~lr~J;C,5 I~CF D01n r-)
JL, 2-D~.C~xygltecose J
Time (10 mtt~utes per p~lnt)
Fi~. 2. Mean (±S.E., , = 5) peak hei&hT~ or" PCr (A) and 7ATP (B) qorma]izcd Io total t]~su~ ph0~phatc in ~ort~e ut re~ling trine in the ab.'ience of exogennus ~ub~tral¢ (open) or in the pre~coce of 5 m[~ 2-deoxy~;lucose as the nnly stibstr.:]le (slanted ]inc,~).
PME
PCr
133
BBAMCR 131165
Vascular oxidative metabolism under different metabolic conditions C h r i s t o p h e r D. H a r d i n , R o b e r t W . W i s e m a n a n d M a r t i n 2. K u s h m e r i c k Detlurtm~,nts of Radiology, Ptt),,~ic(o&~., atul Bi.pl~vsws, Uml t,r~tly .1" |l~u~itfu~zton, S~¢ni¢, |1.~4 ( L'.S ,~ ) (Received g April 1"J911
K¢~ words: Va:~cttlar smoo~h mu,;cle: C~lntrc~l of rK:~,piration; NMP.. ~1p_: Ox'v'gctt consumplion
Control of respiration in vascular smooth muscle wa~ examined while the metabolic state of the tissue was manipulated. During KCl.indueed eontractu~s in the presence of 5 mM gln:ase, oxygen consumption increased by 10 nmul / per rain g without any decrease in phosphucreatine (PCr) or ATP as determined by 31PoNMR indicating a control of respiration which does nat involve changes in high-energy phosphates (e,g., ADP, phnsphorylatien potentialL However, when aortae with resting tone in the absence of substrate were then provided with 5 mM 2-deoxyglucose as the sole subslrate, oxygen consumption increased 7.4 n m o l / m l n per g while PCr decreased by mare than 50% (resulting in a 2-fold increase in the calculated free ADP} with no change in tension from resting tone. Outing a subsequent KCI induced contraeture in the presence of 2-deo~g]ucose, oxygen consumption increased an additional 7.2 n m o l / m i n per g while PCr continued to decline. Therefore, at least P,Yo mechanisms of l~spiratory control may exist in sheep aorta, one dependent and the other independent of changes in high-energy phn£phates.
Introduction
Our understanding of tissue respiratory control is derived primarily from studies involving skeletal and cardiac muscle. The primary regulation of cellular respiration in fast-twitch muscle was thought to be cytosolie [ADP] or [Pi] in a feedback manner [1,2], For example, Meyer demonstrated that a simple linear model [3], with the concentration of ADP as the prim a w feed-back regulator, could account for the measured steady-state energy metabolism in fast-twitch muscle. This simple view o~"mitochondrial regulation is no longer tenable as it does not account uniquely or sufficiently for observations from studies involving other muscle types. Connett and Honig [4,5] demnnstrated in highly oxidative skeletal muscle that several common models for respiratory control were redundant so that they could not be distinguished by steady-state contents of nucleotides, PL and Per. In the myocardium, metabolic conditions e~st wherein th~ rate of oxygen consumption (Jo~) can increase several-fold without any detectable change in the PCr and ATP content as
Correspondence:C. 'Aardin,F.mpl.of Radiology.SB05, Universil~of Washington,School of M~icine, $ealtle. WA 98195. U.S.A.
measured by "~tp-NMR, and by extension in ADP [6,7]. A number of other po~ible regulators of Jo: have been proposed (see Ref. 6 for a review) including regulation by substrate delivery (NADFI O2, TCA cycle intermediates), Ca 2÷ modulation of enzyme activities, and changes in proton motive force. The control of respiration in smooth muscle has not been systematically examined and the existing limited data lead to apparently conflicting views of the mechanisms of respiratory control. Using freeze clamped tissues, PCr decreased with contraction in ,-at portal vein [8] but not in hog carotid [9]. Using 31P-NMR, Per decreased substantially with contraction in taenia coti from guinea-pig [10~ and rabbit [ll] and a smaIler decrease in P e r was observed with contraction in rabbit bladder [I 1]. Since control of respiration involving changes in high-energy phosphates must involve changes in [PCr], these data do not provide a convineing picture of the role of changes ;n high-energy phosphates in the control of mitochondriat respiration in smooth muscle tissue. We investigated whether different metabolic states may pray a role in determining which mechanismfs) control cellular respiration. In the heart, Ugurbil etal. [7] showed that mitochondrla[ cell respiration appears to be regulated by [ADP] when pyruvatc plus glucose is
134 the substrate; but some other factor, independent of changes in high-energy phosphates, regulates respiration when glucose alone is used. In the current study, we invesligated whether more than one mechanism for the control of respiration exist in smooth muscle, one dependent and the other(s) independent of changes in high-energy phosphates, and wi',,ther the metabolic state of the tissue determines which mechanism(s) predominates. The increase in Jo2 associated with contraction in the presence of glucose occurs with no change in the levels of high energy phosphates. An adenylate mechanism of respiratory control could be unmasked in sheep aorta when we decreased the total exchangeable phosphate pool and inhibited glycolys!s when 2-deoxyglueose was the only substrate provided. Portions of this work have appeared previously as an abstract (FASEB J. 2(4), A270). Materials a n d Methods
Tissz~e handling Segments of the thoracic aorta were dissected from sheep (Suffolk) and rinsed in isotonic NaCI for 10-30 rain before placement in a physiological saline (PSS) at room temperature (pH 7.4), consisting of 4.6 mM KCi, 116 m M NaCI, 26 mM Mops, 2.5 m M CaCI 2, 1.16 mM MgCI2, 10 m g / I gentamycirt, 5 mM glucose, pre-equilibrated with a gas mixture of 95% O~ and 5% CO2. Fat, connective tissue and adventitia were removed and segments 4 mm in length were cut and placed into separate water-jacketed glass chambers filled with PSS. Another piece of approx. 5 cm in length was placed unmounted in a standard 90 mm N M R tube and s~aperfused in gas-equillbrated PSS. Tissues were all~wcd to equilibrate for at least 1 h before the experimental protocol began.
Physiological measurements: force and oxygen consumplion Oxygen consumption was measurcd on isometrically mounted aortae using Clark-type polarographie electrodes (Insteeh) with a dual channel amplifier. Oxygen tension was calibrated by flushing the chambers with PSS pre-equilibrated with 95% 0 2 / 5 % CO z. Because of daily variation in system oxygen consumption rate, it was not possible to get an accurate measure of the 'basal' oxygen consumption rate of tissue alone. ~iowever, it was always possible to have the apparatus stable to measure increases in oxygen consumption above resting rates. Zero origen tension was calibrated using a non-saturated solution of sodium dithionite. The aortic rings were held isometrically at a length 1.4 × the resting length ex vivo. Isometric force was measured with transducers from Harvard Apparatus. Force and oxygen consumption were measured on the same tissue~ in the same apparatus at 22°C. Resting
tone was considered to be tile steady-state tone maintained by aortae while superfused in PSS. Contractions were elicited by changing the bathing solution to a high KCI PSS (final concentration of 80 mM KCI in molar replacement of NaCI). Relaxation was induced by changing the bathing solution back to PSS.
31P-NMR spectroscopy 3=P-NMR Spectroscopy measurements were performed in paraIlel to the force and oxygen consumption measurements, operating at 121 MHz for alp using a General EIectrie GN300 Omega spectrometer. Data were acquired using a 00 ° pulse 02/.¢s), a predelay of 3.0 s, a sweep width of 6000 Hz, and a 2K block size. Serial spectra, consisting 0[ 200 acquisitions (approx. 10 min of data averaging), were obtained throughout the experimental protocol. Data were zero filled to 4K points and filtered using 15 Hz exponential line broadenin~; prior to Fourier transformation. Individual resonances (vithin the N M R spectra were quantified by measuring the heights of individual peaks and normalizing to total phosphate in the spectrum. Quantitation of spectra from all aortae was performed by measuring the height of each peak within the spectrum and expressing the ATP or PCr ~evels as the ratio of each to the sum of all peak heights. The normalization of individual peak heights to total phosphorous is based on the assumption that the total cellular phosphate content remains constant [5]. The use of peaks heights is valid as long as individual peak shapes do not significantly change during the course of the experiment. Peak height analysis, although simple, avoids the bias of fitting programs relating to peak shape and variations in the baseline. In some experiments, a 5 m m N M R tube filled with 2 m M methylene diphosphonate (chemical shift of I7 ppm relative to H3PO 4) was included within the 20 mm sample tube and served as an external standard. Although all data were normalized to total tissue phosphate, when peak heights were normalized to the external standard, similar results were obtained but with increased variance.
Experimen tel protocol After equilibration, tissues were placed in PSS with 5 mM glucose for 1 h (relaxation), contracted with PSS with glucose and 80 mM KCI (in molar replacement for NaCI) for 1 h, and relaxed in PSS with glucose for 1 h. Then, the same relaxation/contraction/relaxation sequence was performed in the absence of substrate (glucose-free) and then in the presence of 5 mM 2-deoxyglueo~e. At the end of the protocol of some experiments, the tissues from the oxygen chambers were rinsed with PSS and rapidly frozen. These tissues were stored at - 8 9 ° C until further chemical analysis was performed.
135
Extracr.¢ and HPLC analysis Neutralized perehlorie acid extracts of tissues frozen by submersion in liquid nitrogen were analyzed for PCr and ATP by HPLC on a strong anion exchange column (Vydae model 303 NT305) using a phosphate gradient from 25 (pH 4.5) to 0.5 M phosphate buffer (pH 2.7). Absorbance was measured at 210 nm and quantification was accomplished by comparison with standards of ATP and PCr previously calibrated speetrophotometricaily using standard enzyme-linked metabo[itc assays
[~2]. Results Aortae were contracted and relaxed once in the presence of glucose, then in the absence of glucose, and then in the presence of 2-deoxyglucose. The KCtinduced eontra,'ture of the mounted aortic rings required approx, l h to reach peak isometric force (Fig. t) and approx. 1 h for full relaxation. This slow development of contraction and relaxation allowed 12 acquisition blocks (10 min each) to be obtained during the contraction/relaxation cycle.
2.Deoxyglucose as ~ubstrate under resting conditions After the I h with the aorta at resting tension in substrate-free PSS, we included 5 m M 2-deoxyglucose as the only exogenous substrate in the PSS. Ehosphorylation of 2-deoxyglucose by hexokinase re~iults in appreciable accumulation of 2-deoxyglueose 6-phosphate. Smooth muscle has an appreciable rate of glucose transport [13-16] and since cellular phosphate pools remain constant in smooth muscle [5] 2-dco~q,glueose acts as a phosphate sink in this tissue. Provision of 2-deox~,glueose for 1 h resulte,.t in a de-
creased level of PCr in the aortae compared to level~ during superfusiou with substrate-free PSS (Fig. 2A). However, during the hour of superfusion with 2-deoxy;21ueose in PSS, no decrease in the levels of ATP was oL~served (Fig. 2B), consistent with PCr acting to buffer changes in the [ATP] via the creatine kinase reaction. Sho~n in Fig. 2 are the normalized peak heights of PCr and 3,ATP during the transition from PSS wi~h(~ut any sabstrate to PSS with 5 m M 2-deoxyglucose a~ substr,'2_te. In thd presence of 2-deo:wglucose, isometric force remained at the unstimulate(] level. Stiil, Jo, increased by 7.4 5: I.i. n m o l / m i n per g blot wt. ( ± S.I~., n = 5) above that in PSS withoul substrate ITable I). Thus, the increase in Jo, asse-:ialed with the addition of 2-deoxvglucos¢ to the bath occurs when PCr levels in the aorta decrease, and hence t~ee [ADP] increases, without force developmeTlt,
Calculation of the free A;.,'P concentration In the subset of tissues frozen at the end of the experimental protocol, ATP, PCr and total creatine were measured by HPLC_ The measured concentrations of PCr and A T P of the tissues at the end of the experiment were assigned to the average peak heights of the PCr and -yATP peaks at the end of the experiment for two experiments. Concentration~ of ATP and PCr during the experiment were extrapolated by scaling to the changes in peak heights of ATP and PCr during the experiment. Using this subset of tissues, we estimated the range of free [ADP} during the experiment. The free [ADP] was calculated for those cxtrapolazed concentrations of PCr and ATP, and measured total ereatine (PCr + Cr) by using the ereatine kinase equilibrium constant measured in smooth muscle [17]
co RTRACI'I NO SOLUTION
VO
REL~Xl NG SCLU'rlO~
_/
I blOUR
~
....
--
---~-.~
(IlmQi~m Irl gml (1~)
(14)
FiB. L Tracing o[ a force record of a sheep aorta during a KCI-induced conll-~.clure and sd~equcnl i{:li~(aticsn, A~¢ra~.~ ~,~-gl~n ~onsumplion v a l u ~ for Ih~ pr¢-contzactcd and coelracting aorta arc give in parentheses. ]?or delails see t¢~.
13fi 0 25
25
0;:O"
020'
pCrlP t
ATP/P t
0 i =~..
®
OIC"
0 05 OC0 ---
~,.,cose
IL.
g oQ
J
2-Deoxygr~'cose
- glu;~;e
T|i~e ( 1 0 r~lr~J;C,5 I~CF D01n r-)
JL, 2-D~.C~xygltecose J
Time (10 mtt~utes per p~lnt)
Fi~. 2. Mean (±S.E., , = 5) peak hei&hT~ or" PCr (A) and 7ATP (B) qorma]izcd Io total t]~su~ ph0~phatc in ~ort~e ut re~ling trine in the ab.'ience of exogennus ~ub~tral¢ (open) or in the pre~coce of 5 m[~ 2-deoxy~;lucose as the nnly stibstr.:]le (slanted ]inc,~).
PME
PCr
