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The effect of ambient temperature on papaverine-induced relaxations in canine saphenous veins
The effect of ambient temperature on papaverine-induced relaxations in canine saphenous . vems This study was designed to measure the effect of ambient temperature (25° C) on papaverine-induced relaxations in canine saphenous veins. Segments of vein were suspended in water-jacketed tissue baths at 37° C, and isometric tension was recorded. Mter equilibration, veins were preconstricted by a median effective dose of norepinephrine 2 X 10-6moljL at either 25° C or 37° C. Consequent dose-dependent relaxations showed that papaverine (10- 7 to 1O-3moljL was three times more potent as a dilator at 37° C than at 25° C, with half-maximal relaxations occurring at 2.2 X 1O-5moljL and 6.4 X 10-5mol/L, respectively. A 10-4 mol/L dose of papaverine completely relaxed veins at 37° C, whereas veins at 25° C never fully relaxed even at ten times the standard concentration. In addition, the time for half-maximal relaxation with a 1O-4moljL dose of papaverine averaged 40 minutes at 25° C compared with 22 minutes at 37° C; this is indicative of a reduced relaxation rate at the lower temperature. These data show that papaverine is a slower and less potent dilator of canine saphenous veins at 25° C than at 37° C. This may have implications for the use of papaverine in the operating room, where it is usuaUy applied at ambient temperature to reduce vasospasm of the saphenous vein during coronary artery bypass procedures. (J 'fHORAC CARDIOVASC SURG 1992;104:1289-93)
Nancy J. Rusch, Phl)," Theresa A. Wooldridge, BA,ab Gordon N. Olinger, MD,b and Lawrence E. Boerboom, PhD,b Milwaukee, Wis.
]?paverine effectively dilates agonist-induced contractions in isolated blood vessel segments at 37° CI-6 and is therefore used as a vasodilator during coronary artery bypass procedures to reduce vasospasm of the saphenous vein. However, the effectiveness of papaverine in reducing spasm of the saphenous vein in the operating room is less than would be expected from in vitro studies." Although there are many potential explanations for this, one apparent difference is that papaverine is frequently used during surgical procedures at ambient temperature
From the Departments of Physiology" and Cardiothoracic Surgery," Medical College of Wisconsin, Milwaukee, Wis. Supported by National Institutes of Health grants HL-40474 (N.J.R.) and HL-41840 (L.E.B.).
(22° to 25° C), whereas the pharmacologic actions of papaverine have been assessed at 37° C.I-6 Papaverine causes relaxation, at least in part, by blocking the intracellular enzyme, phosphodiesterase, in vascular muscle cells. This prevents the breakdown of cyclic 3',5'adenosine monophosphate, which promotes vascular smooth muscle relaxation.f Because enzyme activity is progressively reduced at lower temperatures," blocking of phosphodiesterase by papaverine may result in less relaxation at ambient temperature than at 37° C. To investigate this possibility,we designed experiments to measure the effect of ambient temperature on papaverine-induced relaxations in vascular smooth muscle. The potency of papaverine and rate at which it induced relaxation in isolated segments of canine saphenous veins at 25° C and 37° C were studied.
Received for publication June 25, 1991. Accepted for publication Jan. 22, 1992.
Methods
Address for reprints: Nancy J. Rusch, PhD, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226.
Saphenous veins were removed from dogs anesthetized with sodium pentobarbital, 30 mg/kg given intravenously as approved by National Institutes of Health guidelines and the Institutional Animal Care and Use Committee. Veins were cut
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Control
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-. o
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765 25'C
•
Norepinephrine, 2x10- 6 M
6
5
4
3
Papaverine, -log II
4
10 min
Fig. 1. Recordings of canine saphenous veinsprecontracted by a dose of NE of 2 X 1O-6 mol/ L at 37° C or 25° C. Amplitude of NE-induced contractions was stable at both temperatures in control segments (upper traces). In experimental segments, higher concentrations of papaverine were required to relax veins at 25° C than at 37° C (lower traces). Papaverine doses are expressed as cumulative concentrations.
into 4 mm segments and isometric tension was recorded. Segments were suspended between stainless steel triangles in water-jacketed tissue chambers, which were filled with a standard physiologicsalt solution (PSS) consistingof the following (in millimoles per liter): NaC!, 119; KCI, 4.7; MgS04, 1.17; CaCh, 1.6; NaH 2P04, 1.18; NaHC03 24; EDTA, 0.026; and glucose 5.5. The solution was aerated with a 93% molecular oxygen-7% carbon dioxide gas mixture to maintain a pH of 7.4. The optirnallength-tension relationship was determined for each vascular segment by evaluation of the amplitude of contractions induced by potassium chloride, 60 mmol/L, at 37° C. Each segment was progressivelystretched during exposureto the depolarizing potassium chloridesolutionuntil the maximum active tension (absolute tension minus passive tension) was achieved. After a 30- to 6Q-minuteresting period, the physiologicsalt solution in some tissue chambers was allowedto equilibrate at room temperature (25° C) while solution in the other chambers was maintained at 37° C. A median effective dose (EDso, dose causing half-maximal contraction) of norepinephrine (NE) (2 X 1O-6mol/L) was used to induce a sustained contraction of venous segments at 25° or 37° c.1O In experimental segments, cumulative (l0-7mol/L to 1O-3mol/L) or single (l0-4mol/L) concentrations of papaverine were applied at the two different temperatures to determine the effect of ambient temperature on papaverine-induced relaxations. In control segments from the same vein, NE-induced contractions were maintained without the use of papaverine to determine time-dependent changes in tension. Changes in tension were displayed on an EGA monitor (Relisys Corp., Milpitas, Calif.) and simultaneously digitized and stored on an IBM-compatible 386 SX (Citus Corp., Menomonee Falls, Wis.). Data were acquired and analyzed with the use of CODAS software (Dataq Instruments, Akron, Ohio),
Fig. 2. Plot of relaxation as a function of papaverine concentration (10- 7 mol/L - 10- 3 mol/L) at 25° C and 37° C. Resulting dose-relaxation curves show that papaverine was a more potent dilator of NE-induced contractions at 37° C than at 25° C. Data are expressed as mean ± SEM (n = 6). *p < 0.05 for 37° C versus 25° C at the same papaverine concentration. and a hard copy was obtained with a Panasonic KX-P1l80 printer (Panasonic Co., Secaucaus, N.J.). Contraction measurements were expressed as mean ± standard error of the mean, and plotted as percent of responseto NE to normalizefor differences in contraction amplitude between preparations. When appropriate, one-way analysis of variance (ANOY A, p < 0.05) was used for evaluation of statistical significance. Results Cumulative relaxation curves. Segments of saphenous vein were precontracted with a median effective dose of 2 X 1O-6m ol/ L NE at 25° or 37° C. The average amplitude of contractions in response to NE was greater in segments contracted at 25° C (12.7 ± 0.6 gm) than at 37° C (8.1 ± 2.0 gm). This is consistent with many earlier reports showing enhanced a-adrenergic responsiveness of saphenous veins at lower temperatures.P'P The top tracings in Fig. I show that the amplitudes of NE-induced contractions were stable over time at 37° C and 25 ° C, with control segments showing no significant change in amplitude during 60- to 90-minute recording periods. In parallel experiments, cumulative concentrations (10-7mol/L to 10-3moljL; half-log units) of papaverine were applied to segments of the same veins precontracted with NE at the two temperatures. The lower tracings in Fig. I show that papaverine was less potent in relaxing NE-induced contractions at 25° C than at 37° C. This is evident in Fig. 2, where the average relaxation (normalized to the initial NE contraction) is plotted as a function of increasing papaverine concentrations (n = 6). The EDso (dose causing half-maximal relaxation) of papaverine was increased from 2.2 X lO-smol/L at 37° C to
Volume 104 Number S November 1992
Papaverine-induced relaxations at ambient temperature
Papaverine,
12 9 1
100
10- 4 M
37"C
-I--------------~::::-==-- I 59
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.
-Gl
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-----------------------------~---------
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Norepinephrine, 2x10- 6 M
40
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15
30
45
60
Time, minutes
Fig. 3. Recordings comparing relaxation rate for 10-4mol/L papaverine in veins precontracted by a dose of NE of 2 X 1O-6mol/ L at 37° C (uppertrace) and 25° C (lower trace). Veins exposed to papaverine at 25° C relaxedmoreslowly than those at 37° C. 6.4 X IO-smoljL at 25° C, indicating that the potency at the lower temperature was one third of the standard pharmacologic potency. At 37° C, complete relaxation was achieved at a papaverine concentration of 1O-4mol/ L. In contrast, vessels maintained at 25° C failed to relax completely despite an increase to ten times this papaverine concentration. Time-dependent relaxation. In these studies, veins were precontracted by a 2 X 1O-6moljL dose of NE at 25° C and 37° C, and a single dose of papaverine of I0-4mol/L was applied to induce relaxation. Remaining tension was measured every 5 minutes for the first 30 minutes after papaverine exposure and then at 15-minute intervals until a total of 90 minutes had elapsed. Original tracings in Fig. 3 show that the rate of relaxation with 1O-4mol/L papaverine at 25° C was about one half the rate at 37° C in segments from the same vein. This is illustrated in Fig. 4, where relaxation at both temperatures is plotted as a function of time for a 90-minute period (n = 5). The plot shows that 15 to 90 minutes after the addition of papaverine, there was significantly less relaxation at 25° C than at 37° C. For example, at 30 and 60 minutes after papaverine exposure veins at 25° C remained, respectively, 60 ± 4% and 36 ± 6% contracted, whereas at 37° Conly 21 ± 5% and 8 ± 2% of the original NE-induced contraction remained. Discussion Studies have shown papaverine to be an effective dilator of isolated animall? and human" blood vessels at 37°
o
o
10
20
..
'
I
30 45607590
Time, minutes
Fig. 4. Plot of papaverine-induced relaxations at 37° C and 25° C as a function of time. Rate of relaxation for 1O-4moljL papaverine was about two times less at 25° C than at 37° C. Data are expressed as mean ± standard error of the mean (n = 5). *p < 0.05 at 37° C versus 25° C at same time interval.
C. In the operating room, however, there is controversy regarding its use and effectiveness in relieving spasms of saphenous veins stored in solutions at temperatures as low as 4° C.? The mechanism of papaverine-induced relaxation involves inhibition of enzyme activity, which is a temperature-dependent process." Although this implies that papaverine-induced relaxations might also be dependent on temperature, we could find no previous data characterizing the vasodilator properties of papaverine at lower temperatures. Therefore, this study maintained its present focus on relaxations at 25° C and 37° C incanine saphenous veins. NE was used to precontract saphenous veins in this study because it is a probable mediator of the constriction that results from excision of the saphenous vein. The saphenous vein has a rich supply of adrenergic nerve endings, and cooling the vein to 25° C is known to greatly enhance NE-induced vascular tone. 10-13 With NE as the agonist, comparison of dose-dependent relaxations with papaverine showed EDsos of 2.2 X lO- smoljL at 37° C and 6.4 X lO- smol/L at 25° C, indicating that the potency of papaverine was three times less at 25 ° ethan at 37° C under these conditions. The effectiveness of papaverine appeared stable at 25 ° C, and a concentration of 1O-3mol/L resulted in a 92% dilation of venous segments. However, this was at a concentration 10 times higher than was needed to induce full relaxation at 37° C. This reduced potency at 25 ° C may help to explain why papaverine concentrations in the range of 10-3 moljL to 1O-2mol/L are usually used to treat spasm of excised
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vessels at ambient temperature.Iv?' whereas lower concentrations of 1O-5 mol/ L to 1O-4mol/ L can be used in vitro to relax isolated blood vessels at 37° c. 1-6 At 25° C, the relaxation rate of saphenous veinsis twice as slow as the rate at 37° C. This supports previous studies showing attenuation of rates of response to other vasoactive drugs at lower temperatures.P: 23 Since cooling lowers the rate constants for many different cellular functions, the exact mechanism for this is not readily apparent.f 22, 23 However, in this study, saphenous veins maintained at 25° C never fully relaxed during a 90-minute exposure to papaverine, whereas at 37° C similar veinsrelaxed within 25 minutes. This suggests that long waiting periods may be required during surgical procedures if papaverine alone is used to dilate veins at temperatures of less than 37° C. Our findings lend credence to earlier recommendations'f- 16 that papaverine-containing solutions for distending veins be used at 37° C. Although our study does not address temperatures less than 25° C, it is likelythat papaverine-induced relaxation rates decline about twofold for every 10° C decrease in temperature, as do other enzyme-mediated responses."This calls into question the merit of adding papaverine to solutions used to store veins at low temperature, particularly in the 4° to 10° C range. 15, 16,20 In some studies, which did not directly evaluate its vasodilating action, papaverine has been used at concentrations as low as 1.6 X 1O-7mol/ L. 14 In our study, no relaxation occurred at that drug concentration, even at 37° C. However, the effectivenessof papaverine in preventing the onset of spasm may differ from its effectiveness in overcoming existing contractions. Extraction and preparation of veinsfor grafting involve consideration of spasm and hydrostatic distention, conditions implicated as causes of mechanical injury to veins. Papaverine, which has the potential for preventing spasm and thus obviating the need for distention, has itself been implicated as injurious.?:24 If papaverine is to be used as a safe and effective vasodilator, it is imperative that its pharmacodynamics be defined. In addition, it would be useful to evaluate and compare the effectivenessof calcium channel blockers, nitrates, and other vasodilators at ambient temperature. In conclusion, our findings show that the potency of papaverine as a vasodilator agent is reduced at 25° C, necessitating higher drug concentrations and longer exposure periods to achieve the levelof relaxation found at 37° C. Although this study does not address other issues surrounding the use of papaverine for venodilation, it suggests that 37° C is to be preferred over lowertemperatures
The Journal of Thoracic and Cardiovascular Surgery
for papaverine administration to induce complete and rapid relaxation of saphenous veins. REFERENCES 1. Levy JV. Papaverine antagonism of prostaglandin E2-induced contraction of rabbit aortic strips. Res Commun Chern Pathol PharmacoI1973;5:297-31O. 2. Toda N. The action of vasodilating drugs on isolated basilar,coronary and mesenteric arteries ofthedog. J Pharrnacol Exp Ther 1974;191:139-46. 3. BertiF, Fumagalli R, Folco GC, Omini C, Bernareggi V. Roleof cyclic 3' 5' -AMP oncontraction and relaxation of perfused rat caudal artery. Pharmacol Res Commun 1974;6:519-27. 4. Wells IN, Wu, YJ, Baird CE, Hardman JG. Phosphodiesterases fromporcine coronary arteries: Inhibition of separated forms by xanthines, papaverine and cyclic nucleotides. Mol PharmacoI1975;11:775-83. 5. Demesy-Waeldele F, Stocklet JC. Papaverine, cyclic AMP and the dependence of the rat aorta on extracellular calcium. Eur J PharmacoI1975;31:185-94. 6. He GW, Rosenfeldt FL, Buxton BF,Angus JA. Reactivity of humanisolated internal mammary artery to constrictor and dilatoragents. Circulation 1989;80(3 Pt 1):1140-50. 7. Olinger GN.Consequences oftraumatothesaphenous vein incurred duringpreparation for useas grafts. In: Bernhard VM, Towne JB, eds. Complications in vascular surgery. Orlando, Fla.:Grune & Stratton, 1985:143-56. 8. Weiss B, Hait WN. Selective cyclic nucleotide phosphodiesterase inhibitors as potential therapeutic agents. Annu Rev Pharmacol ToxicoI1977;17:441-77. 9. Raison JK. Temperature-induced phasechanges in membranelipids and theirinfluence onmetabolic regulation. In: Davies DD, ed. Rate control of biological processes. Cambridge: Oxford University Press, 1973:485-512. 10. Rusch NJ, Shepherd JT, Vanhoutte PM. The effect of profound cooling on adrenergic neurotransmission in canine cutaneous veins. J Physiol (Land) 1981;311 :57-65. II. Janssens WJ, Verbeuren TJ, Vanhoutte PM. Effect of moderate cooling onadrenergic neuroeffector interaction in canine cutaneous veins. Blood Vessels 1981;18:281-95. 12. Janssens WJ, Vanhoutte PM. Instantaneous changes of alpha-adrenoceptor affinity caused by moderate cooling in canine cutaneous veins. Am J Physiol 1978;234:H330-7. 13. Vanhoutte PM, Shepherd JT. Activity andthermosensitivityofcanine cutaneous veins afterinhibition ofmonoamine oxidase and catechol-O-methyl transferase. Circ Res 1969;25:607-16. 14. Haudenschild CC, Gould KE, Quist WC, LoGerfo FW. Protection of endothelium in vessel segments excised for grafting. Circulation 1981;64(2 Pt 2):101-7. 15. Baumann FG, Catinella FP, Cunningham IN, Spencer Fe. Vein contraction and smooth muscle cell extensions as causes ofendothelial damageduringgraft preparation. Ann Surg 1981; 194: 199-211.
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Papaverine-induced relaxations at ambient temperature
16. LoGerfo FW, Quist WC, Crawshaw HM, Haudenschild C. An improved technique for preservation of endothelial morphology in vein grafts. Surgery 1981;90:1015-24. 17. Catinella FP, Cunningham IN, Srungaram RK, et al. The factors influencing early patency of coronary artery bypass grafts. J THORAC CARDIOVASC SURG 1982;83:686-700. 18. LoGerfo FW, Quist WC, Cantelmo NL, Haudenschild Cc. Integrity of vein grafts as a function of initial intimal and medial preservation. Circulation 1983;68(3 Pt 2): II117-24. 19. Sottiurai VS, Sue SL, Batson RC, Frey DJ, Khaw H. Effects of papaverine on smooth muscle cell morphology and vein graft preparation. J Vase Surg 1985;2:834-42. 20. Adcock GD, Adcock OT, Wheeler JR, et al. Arterialization of reversed autogenous vein grafts: quantitative light and electron microscopy of canine jugular vein grafts harvested
21.
22.
23.
24.
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and implanted by standard or improved techniques. J Vase Surg 1987;6:283-95. Mills NL, Bringaze WL. Preparation of the internal mammary artery graft. J THORAC CARDIOVASC SURG 1989; 98:73-9. Keatinge WR. The effect of low temperatures on the responses of arteries to constrictor drugs. J Physiol (Lond) 1958;142:395-405. Keatinge WR. Mechanism of adrenergic stimulation of mammalian arteries and its failure at low temperatures. J Physiol (Lond) 1964;174:184-305. Roberts AJ, Hay DA, Mehta JL, et al. Biochemical and ultrastructural integrity of the saphenous vein conduit during coronary artery bypass grafting: preliminary results of the effect of papaverine. J THORAC CARDIOVASC SURG 1984;88:39-48.
Bound volumes available to subscribers Boundvolumesof THEJOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY are available to subscribers (only) for the 1992 issues from the Publisher, at a cost of $71.00 for domestic, $93.97 for Canadian, and $89.00 for international subscribers for Vol. 103 (JanuaryJune) and Vol. 104 (July-December). Shipping charges are included. Each bound volume contains a subject and author index and all advertisingis removed.Copies are shipped within 60 days after publicationof the last issueof the volume.The binding is durable buckram with the JOURNAL name, volume number, and year stamped in gold on the spine. Payment must accompany all orders. Contact MosbyYear Book,Inc., SUbscription Services, 11830 Westline Industrial Drive, St. Louis, Missouri 63146-3318, USA; phone (800) 325-4177, ext. 4351, or (314) 453-4351. Subscriptioosmust be in force to qualify. Boundvolumes are not availablein place of a regular JOURNAL subscription.
Nancy J. Rusch, Phl)," Theresa A. Wooldridge, BA,ab Gordon N. Olinger, MD,b and Lawrence E. Boerboom, PhD,b Milwaukee, Wis.
]?paverine effectively dilates agonist-induced contractions in isolated blood vessel segments at 37° CI-6 and is therefore used as a vasodilator during coronary artery bypass procedures to reduce vasospasm of the saphenous vein. However, the effectiveness of papaverine in reducing spasm of the saphenous vein in the operating room is less than would be expected from in vitro studies." Although there are many potential explanations for this, one apparent difference is that papaverine is frequently used during surgical procedures at ambient temperature
From the Departments of Physiology" and Cardiothoracic Surgery," Medical College of Wisconsin, Milwaukee, Wis. Supported by National Institutes of Health grants HL-40474 (N.J.R.) and HL-41840 (L.E.B.).
(22° to 25° C), whereas the pharmacologic actions of papaverine have been assessed at 37° C.I-6 Papaverine causes relaxation, at least in part, by blocking the intracellular enzyme, phosphodiesterase, in vascular muscle cells. This prevents the breakdown of cyclic 3',5'adenosine monophosphate, which promotes vascular smooth muscle relaxation.f Because enzyme activity is progressively reduced at lower temperatures," blocking of phosphodiesterase by papaverine may result in less relaxation at ambient temperature than at 37° C. To investigate this possibility,we designed experiments to measure the effect of ambient temperature on papaverine-induced relaxations in vascular smooth muscle. The potency of papaverine and rate at which it induced relaxation in isolated segments of canine saphenous veins at 25° C and 37° C were studied.
Received for publication June 25, 1991. Accepted for publication Jan. 22, 1992.
Methods
Address for reprints: Nancy J. Rusch, PhD, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226.
Saphenous veins were removed from dogs anesthetized with sodium pentobarbital, 30 mg/kg given intravenously as approved by National Institutes of Health guidelines and the Institutional Animal Care and Use Committee. Veins were cut
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I5g
37'C
-. o
100r"=_==e::=~-~
80
.e: Ill;:
g'i
a..
60
1Ile:
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~!- 40
00
PlIP8verine. -log M 7
;/. e:
6
37'C
20
o
15g
7
765 25'C
•
Norepinephrine, 2x10- 6 M
6
5
4
3
Papaverine, -log II
4
10 min
Fig. 1. Recordings of canine saphenous veinsprecontracted by a dose of NE of 2 X 1O-6 mol/ L at 37° C or 25° C. Amplitude of NE-induced contractions was stable at both temperatures in control segments (upper traces). In experimental segments, higher concentrations of papaverine were required to relax veins at 25° C than at 37° C (lower traces). Papaverine doses are expressed as cumulative concentrations.
into 4 mm segments and isometric tension was recorded. Segments were suspended between stainless steel triangles in water-jacketed tissue chambers, which were filled with a standard physiologicsalt solution (PSS) consistingof the following (in millimoles per liter): NaC!, 119; KCI, 4.7; MgS04, 1.17; CaCh, 1.6; NaH 2P04, 1.18; NaHC03 24; EDTA, 0.026; and glucose 5.5. The solution was aerated with a 93% molecular oxygen-7% carbon dioxide gas mixture to maintain a pH of 7.4. The optirnallength-tension relationship was determined for each vascular segment by evaluation of the amplitude of contractions induced by potassium chloride, 60 mmol/L, at 37° C. Each segment was progressivelystretched during exposureto the depolarizing potassium chloridesolutionuntil the maximum active tension (absolute tension minus passive tension) was achieved. After a 30- to 6Q-minuteresting period, the physiologicsalt solution in some tissue chambers was allowedto equilibrate at room temperature (25° C) while solution in the other chambers was maintained at 37° C. A median effective dose (EDso, dose causing half-maximal contraction) of norepinephrine (NE) (2 X 1O-6mol/L) was used to induce a sustained contraction of venous segments at 25° or 37° c.1O In experimental segments, cumulative (l0-7mol/L to 1O-3mol/L) or single (l0-4mol/L) concentrations of papaverine were applied at the two different temperatures to determine the effect of ambient temperature on papaverine-induced relaxations. In control segments from the same vein, NE-induced contractions were maintained without the use of papaverine to determine time-dependent changes in tension. Changes in tension were displayed on an EGA monitor (Relisys Corp., Milpitas, Calif.) and simultaneously digitized and stored on an IBM-compatible 386 SX (Citus Corp., Menomonee Falls, Wis.). Data were acquired and analyzed with the use of CODAS software (Dataq Instruments, Akron, Ohio),
Fig. 2. Plot of relaxation as a function of papaverine concentration (10- 7 mol/L - 10- 3 mol/L) at 25° C and 37° C. Resulting dose-relaxation curves show that papaverine was a more potent dilator of NE-induced contractions at 37° C than at 25° C. Data are expressed as mean ± SEM (n = 6). *p < 0.05 for 37° C versus 25° C at the same papaverine concentration. and a hard copy was obtained with a Panasonic KX-P1l80 printer (Panasonic Co., Secaucaus, N.J.). Contraction measurements were expressed as mean ± standard error of the mean, and plotted as percent of responseto NE to normalizefor differences in contraction amplitude between preparations. When appropriate, one-way analysis of variance (ANOY A, p < 0.05) was used for evaluation of statistical significance. Results Cumulative relaxation curves. Segments of saphenous vein were precontracted with a median effective dose of 2 X 1O-6m ol/ L NE at 25° or 37° C. The average amplitude of contractions in response to NE was greater in segments contracted at 25° C (12.7 ± 0.6 gm) than at 37° C (8.1 ± 2.0 gm). This is consistent with many earlier reports showing enhanced a-adrenergic responsiveness of saphenous veins at lower temperatures.P'P The top tracings in Fig. I show that the amplitudes of NE-induced contractions were stable over time at 37° C and 25 ° C, with control segments showing no significant change in amplitude during 60- to 90-minute recording periods. In parallel experiments, cumulative concentrations (10-7mol/L to 10-3moljL; half-log units) of papaverine were applied to segments of the same veins precontracted with NE at the two temperatures. The lower tracings in Fig. I show that papaverine was less potent in relaxing NE-induced contractions at 25° C than at 37° C. This is evident in Fig. 2, where the average relaxation (normalized to the initial NE contraction) is plotted as a function of increasing papaverine concentrations (n = 6). The EDso (dose causing half-maximal relaxation) of papaverine was increased from 2.2 X lO-smol/L at 37° C to
Volume 104 Number S November 1992
Papaverine-induced relaxations at ambient temperature
Papaverine,
12 9 1
100
10- 4 M
37"C
-I--------------~::::-==-- I 59
0
.
-Gl
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Gl=
00.
'" .:
60
o.Gl
Papaverine, 10- 4 M
591
-----------------------------~---------
•
Norepinephrine, 2x10- 6 M
40
"I-
20
0 0c:
2S·C
o
Glo. .. Gl
15
30
45
60
Time, minutes
Fig. 3. Recordings comparing relaxation rate for 10-4mol/L papaverine in veins precontracted by a dose of NE of 2 X 1O-6mol/ L at 37° C (uppertrace) and 25° C (lower trace). Veins exposed to papaverine at 25° C relaxedmoreslowly than those at 37° C. 6.4 X IO-smoljL at 25° C, indicating that the potency at the lower temperature was one third of the standard pharmacologic potency. At 37° C, complete relaxation was achieved at a papaverine concentration of 1O-4mol/ L. In contrast, vessels maintained at 25° C failed to relax completely despite an increase to ten times this papaverine concentration. Time-dependent relaxation. In these studies, veins were precontracted by a 2 X 1O-6moljL dose of NE at 25° C and 37° C, and a single dose of papaverine of I0-4mol/L was applied to induce relaxation. Remaining tension was measured every 5 minutes for the first 30 minutes after papaverine exposure and then at 15-minute intervals until a total of 90 minutes had elapsed. Original tracings in Fig. 3 show that the rate of relaxation with 1O-4mol/L papaverine at 25° C was about one half the rate at 37° C in segments from the same vein. This is illustrated in Fig. 4, where relaxation at both temperatures is plotted as a function of time for a 90-minute period (n = 5). The plot shows that 15 to 90 minutes after the addition of papaverine, there was significantly less relaxation at 25° C than at 37° C. For example, at 30 and 60 minutes after papaverine exposure veins at 25° C remained, respectively, 60 ± 4% and 36 ± 6% contracted, whereas at 37° Conly 21 ± 5% and 8 ± 2% of the original NE-induced contraction remained. Discussion Studies have shown papaverine to be an effective dilator of isolated animall? and human" blood vessels at 37°
o
o
10
20
..
'
I
30 45607590
Time, minutes
Fig. 4. Plot of papaverine-induced relaxations at 37° C and 25° C as a function of time. Rate of relaxation for 1O-4moljL papaverine was about two times less at 25° C than at 37° C. Data are expressed as mean ± standard error of the mean (n = 5). *p < 0.05 at 37° C versus 25° C at same time interval.
C. In the operating room, however, there is controversy regarding its use and effectiveness in relieving spasms of saphenous veins stored in solutions at temperatures as low as 4° C.? The mechanism of papaverine-induced relaxation involves inhibition of enzyme activity, which is a temperature-dependent process." Although this implies that papaverine-induced relaxations might also be dependent on temperature, we could find no previous data characterizing the vasodilator properties of papaverine at lower temperatures. Therefore, this study maintained its present focus on relaxations at 25° C and 37° C incanine saphenous veins. NE was used to precontract saphenous veins in this study because it is a probable mediator of the constriction that results from excision of the saphenous vein. The saphenous vein has a rich supply of adrenergic nerve endings, and cooling the vein to 25° C is known to greatly enhance NE-induced vascular tone. 10-13 With NE as the agonist, comparison of dose-dependent relaxations with papaverine showed EDsos of 2.2 X lO- smoljL at 37° C and 6.4 X lO- smol/L at 25° C, indicating that the potency of papaverine was three times less at 25 ° ethan at 37° C under these conditions. The effectiveness of papaverine appeared stable at 25 ° C, and a concentration of 1O-3mol/L resulted in a 92% dilation of venous segments. However, this was at a concentration 10 times higher than was needed to induce full relaxation at 37° C. This reduced potency at 25 ° C may help to explain why papaverine concentrations in the range of 10-3 moljL to 1O-2mol/L are usually used to treat spasm of excised
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vessels at ambient temperature.Iv?' whereas lower concentrations of 1O-5 mol/ L to 1O-4mol/ L can be used in vitro to relax isolated blood vessels at 37° c. 1-6 At 25° C, the relaxation rate of saphenous veinsis twice as slow as the rate at 37° C. This supports previous studies showing attenuation of rates of response to other vasoactive drugs at lower temperatures.P: 23 Since cooling lowers the rate constants for many different cellular functions, the exact mechanism for this is not readily apparent.f 22, 23 However, in this study, saphenous veins maintained at 25° C never fully relaxed during a 90-minute exposure to papaverine, whereas at 37° C similar veinsrelaxed within 25 minutes. This suggests that long waiting periods may be required during surgical procedures if papaverine alone is used to dilate veins at temperatures of less than 37° C. Our findings lend credence to earlier recommendations'f- 16 that papaverine-containing solutions for distending veins be used at 37° C. Although our study does not address temperatures less than 25° C, it is likelythat papaverine-induced relaxation rates decline about twofold for every 10° C decrease in temperature, as do other enzyme-mediated responses."This calls into question the merit of adding papaverine to solutions used to store veins at low temperature, particularly in the 4° to 10° C range. 15, 16,20 In some studies, which did not directly evaluate its vasodilating action, papaverine has been used at concentrations as low as 1.6 X 1O-7mol/ L. 14 In our study, no relaxation occurred at that drug concentration, even at 37° C. However, the effectivenessof papaverine in preventing the onset of spasm may differ from its effectiveness in overcoming existing contractions. Extraction and preparation of veinsfor grafting involve consideration of spasm and hydrostatic distention, conditions implicated as causes of mechanical injury to veins. Papaverine, which has the potential for preventing spasm and thus obviating the need for distention, has itself been implicated as injurious.?:24 If papaverine is to be used as a safe and effective vasodilator, it is imperative that its pharmacodynamics be defined. In addition, it would be useful to evaluate and compare the effectivenessof calcium channel blockers, nitrates, and other vasodilators at ambient temperature. In conclusion, our findings show that the potency of papaverine as a vasodilator agent is reduced at 25° C, necessitating higher drug concentrations and longer exposure periods to achieve the levelof relaxation found at 37° C. Although this study does not address other issues surrounding the use of papaverine for venodilation, it suggests that 37° C is to be preferred over lowertemperatures
The Journal of Thoracic and Cardiovascular Surgery
for papaverine administration to induce complete and rapid relaxation of saphenous veins. REFERENCES 1. Levy JV. Papaverine antagonism of prostaglandin E2-induced contraction of rabbit aortic strips. Res Commun Chern Pathol PharmacoI1973;5:297-31O. 2. Toda N. The action of vasodilating drugs on isolated basilar,coronary and mesenteric arteries ofthedog. J Pharrnacol Exp Ther 1974;191:139-46. 3. BertiF, Fumagalli R, Folco GC, Omini C, Bernareggi V. Roleof cyclic 3' 5' -AMP oncontraction and relaxation of perfused rat caudal artery. Pharmacol Res Commun 1974;6:519-27. 4. Wells IN, Wu, YJ, Baird CE, Hardman JG. Phosphodiesterases fromporcine coronary arteries: Inhibition of separated forms by xanthines, papaverine and cyclic nucleotides. Mol PharmacoI1975;11:775-83. 5. Demesy-Waeldele F, Stocklet JC. Papaverine, cyclic AMP and the dependence of the rat aorta on extracellular calcium. Eur J PharmacoI1975;31:185-94. 6. He GW, Rosenfeldt FL, Buxton BF,Angus JA. Reactivity of humanisolated internal mammary artery to constrictor and dilatoragents. Circulation 1989;80(3 Pt 1):1140-50. 7. Olinger GN.Consequences oftraumatothesaphenous vein incurred duringpreparation for useas grafts. In: Bernhard VM, Towne JB, eds. Complications in vascular surgery. Orlando, Fla.:Grune & Stratton, 1985:143-56. 8. Weiss B, Hait WN. Selective cyclic nucleotide phosphodiesterase inhibitors as potential therapeutic agents. Annu Rev Pharmacol ToxicoI1977;17:441-77. 9. Raison JK. Temperature-induced phasechanges in membranelipids and theirinfluence onmetabolic regulation. In: Davies DD, ed. Rate control of biological processes. Cambridge: Oxford University Press, 1973:485-512. 10. Rusch NJ, Shepherd JT, Vanhoutte PM. The effect of profound cooling on adrenergic neurotransmission in canine cutaneous veins. J Physiol (Land) 1981;311 :57-65. II. Janssens WJ, Verbeuren TJ, Vanhoutte PM. Effect of moderate cooling onadrenergic neuroeffector interaction in canine cutaneous veins. Blood Vessels 1981;18:281-95. 12. Janssens WJ, Vanhoutte PM. Instantaneous changes of alpha-adrenoceptor affinity caused by moderate cooling in canine cutaneous veins. Am J Physiol 1978;234:H330-7. 13. Vanhoutte PM, Shepherd JT. Activity andthermosensitivityofcanine cutaneous veins afterinhibition ofmonoamine oxidase and catechol-O-methyl transferase. Circ Res 1969;25:607-16. 14. Haudenschild CC, Gould KE, Quist WC, LoGerfo FW. Protection of endothelium in vessel segments excised for grafting. Circulation 1981;64(2 Pt 2):101-7. 15. Baumann FG, Catinella FP, Cunningham IN, Spencer Fe. Vein contraction and smooth muscle cell extensions as causes ofendothelial damageduringgraft preparation. Ann Surg 1981; 194: 199-211.
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Papaverine-induced relaxations at ambient temperature
16. LoGerfo FW, Quist WC, Crawshaw HM, Haudenschild C. An improved technique for preservation of endothelial morphology in vein grafts. Surgery 1981;90:1015-24. 17. Catinella FP, Cunningham IN, Srungaram RK, et al. The factors influencing early patency of coronary artery bypass grafts. J THORAC CARDIOVASC SURG 1982;83:686-700. 18. LoGerfo FW, Quist WC, Cantelmo NL, Haudenschild Cc. Integrity of vein grafts as a function of initial intimal and medial preservation. Circulation 1983;68(3 Pt 2): II117-24. 19. Sottiurai VS, Sue SL, Batson RC, Frey DJ, Khaw H. Effects of papaverine on smooth muscle cell morphology and vein graft preparation. J Vase Surg 1985;2:834-42. 20. Adcock GD, Adcock OT, Wheeler JR, et al. Arterialization of reversed autogenous vein grafts: quantitative light and electron microscopy of canine jugular vein grafts harvested
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and implanted by standard or improved techniques. J Vase Surg 1987;6:283-95. Mills NL, Bringaze WL. Preparation of the internal mammary artery graft. J THORAC CARDIOVASC SURG 1989; 98:73-9. Keatinge WR. The effect of low temperatures on the responses of arteries to constrictor drugs. J Physiol (Lond) 1958;142:395-405. Keatinge WR. Mechanism of adrenergic stimulation of mammalian arteries and its failure at low temperatures. J Physiol (Lond) 1964;174:184-305. Roberts AJ, Hay DA, Mehta JL, et al. Biochemical and ultrastructural integrity of the saphenous vein conduit during coronary artery bypass grafting: preliminary results of the effect of papaverine. J THORAC CARDIOVASC SURG 1984;88:39-48.
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