Effect of Prolonged Cold Storage on the Contractile Response of Strips of Rabbit Aorta to Various Agents

Transcription

1 Effect f Prlnged Cld Strage n the Cntractile Respnse f Strips f Rabbit Arta t Varius Agents By Shji Shibata, M.D., Ph.D. With the Technical Assistance f D. D. Timmerman Dwnladed frm by n August 31, 2018 ABSTRACT After 2 days in cld strage (2.0 ± 0.5 C), the cntractile respnse f spiral strips f rabbit arta t lw cncentratins (10~!l t 10~ 10 M) f epinephrine and f nrepinephrine was ptentiated; after strage fr 4 t 7 days, an increased cntractile respnse t high cncentratins (10~ 8 t 10~ 6 M) f these catechlamines was als bserved. Reserpine-pretreated strips shwed ptentiatin f epinephrine- and nrepinephrine-induced cntractins, but nly after 2 t 7 days in cld strage. Anther result f several days f cld strage was a decrease r lss f cntractile respnse t nictine (10" 5 t 10- M), tyramine (1CH t 10~ 6 M), and ptassium (10 t 40 m\i). Ptentiatin f the cntractile respnse t epinephrine and t nrepinephrine was als bserved in ptassiumdeplarized strips at lw temperature (19 C). After incubatin f the deplarized r cld-stred strips in the Ca-free medium, n ptentiatin f epinephrine- r nrepinephrine-induced cntractins was bserved. These results indicate that cld strage, up t 7 days, des nt interfere with the activatin system related t epinephrine- r nrepinephrine-induced cntractins. Hwever, such treatment des affect the membrane system invlved in ptassium-induced cntractin. The decreased respnse t nictine and tyramine after several days f cld strage may be due t the unavailability f tissue catechlamine. N significant histpathlgic changes were bserved in any layer f the arta after cld strage up t 7 days. ADDITIONAL KEY WORDS epinephrine nrepinephrine KCL reserpine-treated strip nictine tyramine deplarized strip lw temperature histpathlgic change Even thugh the actins f catechlamine and sme ther agents n vascular smth muscle have been studied, ur knwledge f the mechanism f actin f these cmpunds is far frm cmplete. It is knwn that after smth muscle has been expsed t cld fr hurs r days, the respnsiveness which was lst during cld Frm the Department f Pharmaclgy, Schl f Medicine, University f Hawaii, Hnlulu, Hawaii This investigatin was supprted in part by research grants frm the University f Hawaii and the Hawaii Heart Assciatin. This wrk was presented in part at the meeting f the Federatin f American Scieties fr Experimental Bilgy, Atlantic City, New Jersey, April, Received fr publicatin July 1, Accepted fr publicatin December 9, strage is regained when it is rewarmed t bdy temperature (1-4). Hwever, while investigating the relatinship between temperature and mechanical activity f vascular smth muscle f rabbit artic strip, it was nticed that the cntractile respnse f the strip t epinephrine and t nrepinephrine was ptentiated after prlnged cld strage (2 C) althugh the respnses t excessive amunts f ther drugs such as ptassium, nictine, and tyramine were decreased r ablished. As yet, n reprt appears t have been made n the influence f prlnged cld strage (mre than several days) n the cntractile respnse f vascular smth muscle t chemical stimuli. Because this phenmenn might help t elucidate the mechanism and site f actins f such stim- Circulatin Research, Vlume XXIV, February

2 180 SHIBATA Dwnladed frm by n August 31, 2018 uli as well as the mechanism f the effect itself, the mdificatin f the cntractile respnse t nrepinephrine, epinephrine, ptassium, nictine, and tyramine by prlnged cld strage was studied. Methds Fr these studies, 185 rabbits f bth sexes weighing 1.5 t 2.0 g were killed by a blw n the head. The cartids were cut and the chest pened. The thracic arta was remved and placed in Ringer's slutin. Spiral artic strips were prepared after excessive fat and cnnective tissue had been remved (5). A strip f arta frm each rabbit was cut transversely and ne half used as a cntrl fr the ther half. Strips 4 t 5 mm wide and 60 mm lng were used in the experiments. The artas were munted vertically in an rgan bath in 15 ml f Ringer's slutin f the fllwing cmpsitin (HIM): NaCl, 154; KC1, 5.4; CaCL, 2.4; NaHCO :s> 6; dextrse, 11, all in distilled and deinized water. The slutins were maintained at 37 C and equilibrated befre and during the experiment with a gas mixture f 95% O 2 and 5% CO 2 (ph 7.3) in the tissue bath. Ligatures were placed in bth ends f the muscle strips, ne being attached t a glass rd (hlding device) and the ther t a Grass FT.03 strain gauge. During cld strage in the refrigeratr, the strips were kept in the medium withut supplying exgenus xygen, at 2.0 ± 0.5 C fr 1 t 12 days. Then strips were transferred t warmed Ringer's slutin (37 C C) and 1.5 g f tensin was applied t the strips fr 2 hurs. The laded tensin was maintained thrughut the experiment and it was peridically readjusted. Similar prcedures were carried ut n the cntrl preparatin, which did nt have the cld-strage treatment. During cld strage, there was n change in tissue medium nr was tensin applied t the strip. Ismetric cntractins were recrded by a strain-gauge transducer (Grass FT.03) and 4- channel Grass plygraph. With this equipment, it was pssible t examine fur preparatins in different tissue baths at the same time. The strips were expsed t varius dses f epinephrine, nrepinephrine, KC1, tyramine, and nictine in nrmal r mdified Ringer's slutin. Reserpine-pretreated strips were btained frm rabbits that had been injected with reserpine (Serpasil, Ciba), 4 mg/kg im, 24 hurs befre death. All rabbits treated with reserpine shwed signs f effective treatment, such as diarrhea, ptsis and a reductin in heart rate. Data is presented and analyzed in grams f cntractin. Dse-respnse relatinships were btained by adding cumulative cncentratins f the drug t the tissue bath except in the case f nictine. The vlume f each single additin was 0.5 ml. At least 1 hur was allwed between cmpletin f ne tissue stimulatin and the beginning f the next t ensure unifrm return t the previus resting tne. All drugs were prepared immediately befre use frm cncentrated stck slutins that were kept in a refrigeratr. These slutins were made with distilled and deinized water. The cncentratins f drugs tested are expressed as the final cncentratin in tissue bath. Ca-free medium refers t the nrmal Ringer's slutin frm which the calcium chlride was mitted. Results RESPONSE TO EPINEPHRINE AND TO NOREPINEPHRINE Figure 1 shws the cntractile respnse f the artic strip t epinephrine and t nrepinephrine after different duratins f cld strage at 2 C. When the strips were rewarmed at 37 C in Ringer's slutin, they regained the respnsiveness t these agents they had lst at lw temperatures. After 2 days in cld strage, nly the cntractile respnses t lw cncentratins f epinephrine r nrepinephrine (10~ t 10- ]0 M) were ptentiated (P<.01, t-test). In the fresh preparatin (nt cld stred) l(h 10 M epinephrine r nrepinephrine prduced n cntractin. The minimal effective dse f epinephrine and f nrepinephrine prducing a detectable cntractin in cntrl preparatins was apprximately 10~ 9 M. After 4 days in cld strage, ptentiated respnses were als seen with high cncentratins f these catechlamines (l(h c t HHM) (P <.02, i-test). This ptentiatin was bserved even after 7 days in cld strage, but after 10 days, ptentiatin had disappeared and the respnse was decreased. High cncentratin f these drugs (10" t 10~ S M ) culd still cause a detectable cntractin in any f seven strips frm different rabbits after 20 days in cld strage, but the amplitude f the cntractins was nly 25% t 30% f the size f thse in the fresh preparatin. After 30-minute incubatin in Ringer's slutin cntaining desipramine hydrchlride (10" t 10" 7 M) r mnamine xidase inhibitrs such Circulatin Research, Vlume XXIV, February 1969

3 > > Z > 70 n z > n H O z 00 Dwnladed frm by n August 31, 2018 (HDe 9 10 Epinephrlne (-Lg M) 7 HO Nrepinephrine (-Lg M) 3 (ISM : 1 1 m \41 ilir (71 m 1\ V: 't ' : (is) A s i\ : I' 5 ':: Mi B a i«) 7tS (96) 1 DAY AFTER COLD STORAGE FIGURE 1 Changes in the cntractile respnse f rabbit artic strip t epinephrine and t nrepinephrine after cld strage. The numbers in parentlieses are the number f strips tested. The vertical lines represent SKM. The cntractile activity n day zer is a cntrl respnse determined befre cld strage.

4 182 SHIBATA s CO * CD CO 00 Ol (N CO < (M -H CO tl O 00 O O5 00 O> d c4 t^ c d d cq BO c as tranylcyprmine hydrchlride (10" 4 t 10~ S M) r pheniprazine hydrchlride (10^5 t 10-"M), n ptentiatin f epinephrine r nrepinephrine was bserved n the fresh artic strips (Table 1). Hwever, desipramine (10""M) decreased the cntractile respnses t epinephrine and t nrepinephrine. These agents had n effect n the resting tensin f strips. Strips frm reserpinetreated rabbits als shwed ptentiatin f the epinephrine and nrepinephrine effects but nly after 2 t 7 days in cld strage. The time f ptentiatin after cld strage was the same in strips treated with reserpine as in thse nt s treated. Figure 2 shws the a. 5 S <D a d 2 < I RESERPINE-4 DAYS ' COLO STORAGE P<05 J RESERPiNE-BEFORE. ' COLO STORAGE Dwnladed frm by n August 31, g 5 c O a * cc cq eq cq dd O O 00 * cq cq cq O CO cq cq as cq cq in in > > in cq CO cq +1 O5 cq cq 10 in CD V >"" Q) CJ g! i si p "^?f y *^ = p g- 2 c» fly a S a III u 5 " cc 2 c-i 2 <.,i" 7cT EPINEPHRINE (-Lg M) /, / P<0.l NOREPINEPHRINE (-Lg M) FIGURE 2» RESERPINE-4 DAYS / i COLD STORAGE P«05 J RESERPINE-BEFORE - ' ' COLD STORAGE The cntractile respnse t epinephrine and t nrepinephrine in reserpine-pretreated strips befre and after cld strage. P value (t-test) indicates a significant difference between the cntractile respnses befre and thse after cld strage (4 days). Ten artic strips frm different rabbits were used in each experiment. The vertical lines represent SEM f the bservatins. Circulatin Research, Vlume XXIV, February 1969

5 COLD STORAGE AND AORTIC CONTRACTION 183 Dwnladed frm by n August 31, 2018 dse respnse curves f epinephrine and f nrepinephrine (10~ 7 t 1(H O M ) in strips frm reserpine-treated rabbits befre and after cld strage (4 days). The histpathlgic changes in artic strips after cld strage were als studied. In each experiment, fur artic strips frm different rabbits were used. The preparatins were fixed with 10& neutral frmalin, embedded in paraffin, and stained with hematxylinesin. They were then cut in lngitudinal and crss sectins (4^t) using a rtary micrtme, and histpathlgic changes were examined under light micrscpe. N significant histpathlgic changes were bserved in any layer f the artic strip after prlnged cld strage fr up t 7 days. Hwever, after 10 days in cld strage, marked vaculizatin f the intra- and intercellular space in the media (muscle layer) was bserved. These changes presumably explain the decreased cntractile respnses t epinephrine and t nrepinephrine after 10 days in cld strage. RESPONSE TO KCI Figure 3 shws the cntractile respnse t 3 z <-> I 7 DAYS COLD STORAGE WITH!5mM KCI 4 DAYS COLO STORAGE 7-10 DATS COLD (30) CONCENTRATION OF KCI (mm) FIGURE 3 - $ CO-FREE MEDIUM (7) Change in the cntractile respnse f strips f rabbit arta t excess ptassium (10 t 40 MM) after prlnged cld strage and after 1-hur incubatin in Ca-free medium. The numbers in parentheses are the number f strips tested. Cntrl respnse was tested n the preparatin befre cld strage. The vertical lines represent SEM f the bservatins. Circulatin Research, Vlume XXIV, February 1969 KCI (10 t 40 ITIM) after up t 7 days in cld strage. After 2 days in cld strage, the respnse t lw cncentratins f KCI (10 t 20 mil) was already decreased. After 4 days in cld strage, the respnse t high cncentratins f KCI (30 t 40 mm) was als diminished. In fresh preparatins, 40 mm KCI caused almst maximal cntractin. N further cntractin was bserved after additin t 80 mm KCI slutin. After 7 days in cld strage, a decrease in the cntractile respnse t the high cncentratins f ptassium (40 mm) was bserved. After 7 days f cld strage in Ringer's slutin cntaining excess ptassium (15 ITIM), depressin f the cntractile respnse t ptassium (40 mm) was significantly less than that after 7 t 10 days f cld strage in medium withut excess ptassium (Fig. 2). After 1-hur incubatin in Ca-free medium, ptassium-induced cntractin (20 t 40 mm) was almst cmpletely ablished, as shwn in Figure 3. When the fur strips frm different rabbits were incubated in Ringer's slutin cntaining ptassium (40 mm) fr as lng as 3 t 5 days in cld strage, the strips cmpletely lst their respnsiveness t ptassium (80 mm). This suggests that such treatment may damage the vascular tissue, since even the applicatin f BaCL (5 mm) int the tissue bath culd nt bring abut cntractin in these preparatins. In nrmal Ringer's slutin, the amplitudes f the maximal cntractins prduced by 5 mm BaCl 2 were 4.1 ± 0.5 g and 3.9 ± 0.5 g in fur fresh and fur cld-stred (4 days) strips respectively. RESPONSE TO NICOTINE AND TYRAMINE Since nictine and tyramine are believed t cause cntractin f vascular smth muscle by an indirect actin invlving liberatin f nrepinephrine frm its tissue strage site, the actin f these drugs n the preparatin befre and after different duratins f cld strage was tested. Figure 4 shws the effect f cld strage n nictine-and tyramineinduced cntractins. After 24 hurs f cld strage, the respnse t lw cncentratins f nictine and tyramine (all 10" U M) was

6 184 SHIBATA decreased. After 3 t 6 days f cld strage, the respnses t higher cncentratins f nictine (10~ 3 and 5X10~ 8 M) and tyramine (10" 4 t 10~ 3 M ) were almst cmpletely inhibited. After 10-minute incubatin in a medium cntaining alpha-receptr blcking agents (phenxybenzamine hydrchlride and phentlamine methanesulfnate, bth 5 X 10~ 7 M ), the cntractile respnse t nictine ( 10~ 5 M ) and tyramine ( KHM ) was almst cmpletely diminished in all f five fresh preparatins I - EPINEPHRINE (I9 C) NOREPINEPHRINE (I9 C),} 6 DAYS COLD STORAGE.j DEPOLARIZED MUSCLE.[CONTROL I CO-FREE MEDIUM 6 DAYS COLD STORAGE DEPOLARIZED MUSCLE.{CONTROL IC-FREE MEOIUM < I O (32) NICOTINE I CONCENTRATION OF EP 8 NE (-Lg M) FIGURE 5 The cntractile respnse f ptassium-deplarized fresh strips and strips in cld strage fr 6 days t epinephrine (EP) and nrepinephrine (NE) in nrmal Ringer's r Ca-free media at lw temperature (19 C). Cntrl respnse was tested n the undeplarized fresh preparatin. The preparatins used in Ca-free medium were the deplarized fresh strips. The vertical lines represent SEM f seven bservatins. Dwnladed frm by n August 31, 2018 r 0 L (26) TYRAMINE I 10 DAY AFTER COLD STORAGE FIGURE 4 Change in the cntractile respnse f strips f rabbit arta t nictine and tyramine after varius times in cld strage. The numbers in parentheses are the number f strips tested. The vertical lines represent SEM f the bservatins. The respnse t the drugs n day zer indicates the cntrl respnse determined befre cld strage. frm different rabbits. Als, the cntractile respnse t bth drugs in strips frm reserpine-treated rabbits was studied. The cntractile respnses f these t nictine ( 10~ 3 M ) and t tyramine (IO^M) (10 preparatins each) were diminished t abut 30% t 40% f thse in the untreated preparatin. RESPONSE TO EPINEPHRINE AND NOREPINEPHRINE OF DEPOLARIZED PREPARATION Since at 37 C, excess ptassium (40 HIM) caused almst maximal cntractin, the cntractile respnse f the ptassium-deplarized preparatin t nrepinephrine and t epinephrine culd nt be measured at that temperature. Therefre a reduced temperature (19 C) that prevented the cntractile respnse t ptassium but did nt cmpletely prevent the respnse t either epinephrine r nrepinephrine was used in this experiment. The fresh preparatin was deplarized by incubatin in a high ptassium medium (80 ITIM) at 19 C fr 2 hurs befre the start f the experiment. Figure 5 shws that the cumulative dse respnse curves fr epinephrine and nrepinephrine n the deplarized muscle were Circulatin Research, Vlume XXIV, February 1969

7 COLD STORAGE AND AORTIC CONTRACTION 185 Dwnladed frm by n August 31, 2018 < tr EP "1 CO-FREE MEDIUM - NE. J 'EP-1 > NORMAL RINGER^ NE J. CO-FREE tk I MEDIUM - ME f 6 DAYS COLD STOR. /<-// ft'/ V// CONCENTRATION OF EP 8 NE (-Lg) FIGURE 6 The cntractile respnse f fresh strips and strips in cld fr 6 days t epinephrine (EP) and nrepinephrine (NE) in Ca-free medium with r withut EDTA (0.1 nui). The strip was incubated in Ca-free medium fr 1 hur. The vertical lines represent SEM f 10 bservatins. shifted t the left f the curves btained fr the cntrl muscles (undeplarized fresh strip), indicating increased sensitivity. The dse-respnse curves f these catechlamines n the deplarized fresh muscle were nt significantly different frm thse n the nndeplarized muscle after 6 days in cld strage at 19 C. RESPONSE TO EPINEPHRINE, NOREPINEPHRINE, AND POTASSIUM IN Ca-FREE MEDIUM Figure 6 shws the effect f Ca-free medium n the cntractile respnse t nrepinephrine and t epinephrine (10" t 10-*M ) befre and after cld strage. After 1-hur incubatin in this medium, n significant changes were bserved in the cntractile respnse f fresh preparatins t nrepinephrine r epinephrine as cmpared t thse in nrmal Ringer's slutin. After a similar incubatin in a Ca-free medium cntaining 0.1 HIM EDTA, the epinephrine- and nrepinephrine-induced cntractins f fresh preparatins were almst cmpletely ablished (Fig. 6). After 1-hur incubatin in the Ca-free medium, n ptentiatin f the cntractile respnse t epi- Orculatin Research, Vlume XXIV, February 1969 nephrine r nrepinephrine was bserved in the preparatins after 6 days f cld strage as shwn in Figure 6. Als, the ptentiated cntractile respnses t epinephrine and t nrepinephrine in the deplarized fresh muscle at 19 C was nt bserved after 1-hur incubatin in Ca-free medium (Fig. 5). Discussin The mechanisms f the mdificatin f respnsiveness by prlnged cld strage is nt clear; hwever, the present results indicate several pssibilities. It was fund, fr example, that reserpine-pretreated strips did nt shw supersensitivity t epinephrine r t nrepinephrine, except after prlnged cld strage. Als, the time fr develpment f supersensitivity after cld strage was identical in bth reserpine-treated and untreated strips. This might argue against supersensitivity as a depletin r releasing phenmenn f tissue catechlamine stres. Neither treatment with desipramine, which prevents catechlamine uptake frm the tissue strage site, nr treatment with mnaminxidase inhibitrs ptentiated the respnse t epinephrine and nrepinephrine in fresh artic strips. Thus, it seems unlikely that supersensitivity is caused by increased availability f catechlamine t the effectr cell as a cnsequence f the inhibitin f uptake r breakdwn f the catechlamines. I believe, hwever, that further study is necessary t rule ut either f these pssibilities cmpletely. It was als bserved that supersensitivity t epinephrine and t nrepinephrine ccurred in ptassium-deplarized fresh strips at lw temperature. Aebi (6) and Shanes (7) have indicated that cld strage will deplarize a membrane. In view f the similarity between the supersensitivity f deplarized fresh and cld-stred muscle, it seems pssible that the same mechanism is invlved in bth cases. Als, previus findings suggested that the magnitude f smth muscle cntractin is clsely related t increased Ca flux (8, 9). Therefre, if increased Ca 2+ permeability is regarded as a cmmn mechanism f supersensitivity in

8 186 SHIBATA Dwnladed frm by n August 31, 2018 bth ptassium-deplarized and cld-stred muscle, it shuld be expected that supersensitivity wuld be affected by external Ca 2+ cncentratins. The lack f supersensitivity t epinephrine and t nrepinephrine in the Ca-free medium, therefre seems t supprt the previus suggestin by Carrier and Shibata (10) that membrane permeability changes are causally related t the develpment f supersensitivity t catechlamines in artic strips. It is well knwn that ptassium-induced cntractin is prbably mediated by membrane ptential changes (11-15). In additin, since in Ca-free medium the strip lst respnsiveness t ptassium may supprt this pssibility, since external Ca 2+ in is prbably an essential factr fr the maintenance f the electrical activity f smth muscle (16-20). It may be suggested that nrmal levels f membrane permeability are required t initiate a full cntractile respnse t excess ptassium. It has als been reprted that cld strage inhibits the replarizatin prcess as well as the return f ptassium t the intracellular space, and this inhibitin becmes irreversible belw a critical level (6, 7). It was therefre speculated that the bserved decrease in the cntractile respnse t ptassium might be due t incmplete recvery f the intracellular ptassium level fllwing prlnged cld strage. This interpretatin is cnsistent with the bservatin that prlnged cld strage in Ringer's slutin cntaining excess ptassium (15 mm) decreased the inhibitin f the ptassium-induced cntractin, since such treatment might partially prevent the decrease f intracellular ptassium during prlnged cld strage. The present bservatin that reserpinepretreatment and alpha-receptr blcking agents (phentlamine and phenxybenzamine) decreased the respnse t nictine and tyramine supprts the suggestin that these agents act indirectly n vascular smth muscle by releasing available catechlamines frm tissue strage sites (21-23). It has als been reprted that prlnged cld strage allws irreversible degeneratin f islated nerve tissue, and this may lead t depletin f the tissue catechlamines (24). The ineffectiveness f nictine and tyramine n cld-stred strips may thus be due t the absence f available endgenus catechlamine in the tissue stres. The inhibitry actin f cld strage (3 t 6 days) n tyramine- and nictine-induced cntractins was greater than the effect f prir treatment with reserpine. Innes (25) has suggested that tyramine might be releasing residual amunts f tissue catechlamine after treatment with reserpine. It may be pstulated that cld strage caused a greater depletin f tissue catechlamines than prir treatment with reserpine. Thus, althugh these investigatins have shed sme light n the mechanism f the indirect actin f tyramine, the pstulated direct actin f tyramine remains unexplained (26-27). Further experiments are being carried ut t investigate this interesting phenmenn. Acknwledgments The authr wishes t express his gratitude t Dr. M. Rayner fr his advice n preparing the manuscript and t Dr. I. Sakurai fr his assistance n histpathlgic study. References 1. BAHB, L., HEADING, V. E., AND BOHB, D. F.: Ptassium and the recvery f arterial smth muscle after cld strage. J. Gen. Physil. 46: 19, FREEMAN-NARHOD, M., AND GOODFOHD, P. J.: Sdium and ptassium cntent f the smth muscle f the guinea-pig taenia cli at different temperature and tensin. J. Physil. (Lndn) 163: 399, GOODFORD, P. J., AND HEBMANSEN, K.: Sdium and ptassium mvements in the unstriated muscle f the guinea-pig taenia cli. J. Physil. (Lndn) 158: 426, DANIEL, E. E., AND ROBINSON, K.: Secretin f sdium and uptake f ptassium by islated uterine segments made sdium rich. J. Physil. (Lndn) 154: 421, FURCHCOTT, R. F., BHADRAKOM, S.: Reactins f strips f rabbit arta t epinephrine, isprpylarterenl, sdium nitrate and ther drugs. J. Pharmacl. Exptl. Therap. 108: 129, AEBI, H.: Elektrlyt-Akkumulierung und Osmregulatin in Gewebschnitten. Helvet. Physil. Pharmacl. Acta 11: 96, Circulatin Research, Vlume XXIV, February IS)69

9 COLD STORAGE AND AORTIC CONTRACTION 187 Dwnladed frm by n August 31, SHAKES, A. H.: Effect f temperature n ptassium liberatin during nerve activity. Am. J. Physil. 177: 377, BOHR, D. F.: Vascular smth muscle: Dual effect f calcium. Science 139: 597, HARDY, F. J., SCOTT, J. B., FLORIO, M. A., JR., DOUGHERTY, R. M., AND HUIGENCA, J. N.: Lcal vascular effect f hypkalemia, alkalsis, hypercalemia and hypmagnesia. Am. J. Physil. 204: 202, CARRIER, O., JR., AND SHIBATA, S.: Pssible rle fr tissue calcium in reserpine supersensitivity. J. Pharmacl. Exptl. Therap. 155: 42, SHD3ATA, S., AND BRICCS, A. H.: Relatinship between electrical and mechanical events in rabbit artic strips. J. Pharmacl. Exptl. Therap. 153: 466, Su, C, BEVAN, J. A., AND URSILLO, R. C: Electrical quiescence f pulmnary artery smth muscle during sympathmimetic stimulatin. Circulatin Res. 15: 20, KEATING, W. R.: Mechanism f adrenergic stimulatin f mammalian arteries and its failure at lw temperature. J. Physil. (Lndn) 174: 184, AXELSSON, J., WAHLSTROM, B., JOHANSSON, B., AND Jxssx, O.: Influence f the inic mvement n spntaneus electrical and mechanical activity f the rat prtal vein. Circulatin Res. 21: 609, BARR, L.: Respnsiveness f arterial smth muscle. In Biphysics and Physilgical and Pharmaclgical Actin, edited by A. A. Shanes. Washingtn, D. C, American Assciatin fr the Advancement f Science, 1961, p BULBRINC, E.: Electrical activity in intestinal smth muscle. Physil. Rev. 42: 160, BULBRINC, E., KuRiYANtA, H.: Effects f changes in external sdium and calcium cncentratins n spntaneus electrical activity in smth muscle f guinea-pig taenia cli. J. Physil. (Lndn) 166: 29, BENNETT, M. R., BURNSTOCK, G., HOLMAN, M. E., AND WALKER, J. W.: Effect f Ca 2 + n plateau-type actin ptentials in smth muscle. J. Physil. (Lndn) 161: 47, BOZLER, E.: Cntractility f muscle in slutins f lw electrlyte cncentratin. Am. J. Physil. 199: 299, DANIEL, E. E., SEHDEV, H., AND ROBINSON, K.: Mechanisms fr activatin f smth muscle. Physil. Rev. 42: 228, MILLSON, D. R.: Nictine and the effect f antisympathmimetic agents n the arta f the rabbit. Brit. J. Pharmacl. 14: 239, KOTTEGODA, S. R.: Actin f nictine and acetylchline n the vessels f the rabbit's ear. Brit. J. Pharmacl. 8: 156, BURN, J. H., AND RAND, M. J.: Nradrenaline in artery walls and its dispsal by reserpine. Brit. Med. J. 1: 903, TRENDELENBERG, U.: Supersensitivity and subsensitivity t sympathmimetic amines. Pharmacl. Rev. 15: 225, INNES, I. R.: An actin f 5-hydrxytryptamine n adrenaline receptrs. Brit. J. Pharmacl. 19: 427, VABMA, D. R., GILLIS, R. A., AND BENETY, B. C: Reserpine as an antagnist f tyramine. J. Pharmacl. Exptl. Therap. 144: 181, FURCHGOTT, R. F., KlRPEKAR, S. S., RlCKER, M., AND SCHWAB, A.: Actins and interactins f nrepinephrine, tyramine, and ccaine n artic strip f rabbit and left atria f guinea-pig and cat. J. Pharmacl. Exptl. Therap. 142: 39, Circulatin Research, Vlume XXIV. February 1969