Effects of Hypothermia on Norepinephrine Release and Effector Response in Isolated Perfused Cat Spleen

Size: px

Start display at page:

Download "Effects of Hypothermia on Norepinephrine Release and Effector Response in Isolated Perfused Cat Spleen"

Giles Owens
5 years ago
Views:

Effects f Hypthermia n Nrepinephrine Release and Effectr Respnse in Islated Perfused Cat Spleen By Ake Wennmlm, Per Hedqvist, and Lennart Stjdrne ABSTRACT The effects f hypthermia n the sympathetic

1 Effects f Hypthermia n Nrepinephrine Release and Effectr Respnse in Islated Perfused Cat Spleen By Ake Wennmlm, Per Hedqvist, and Lennart Stjdrne ABSTRACT The effects f hypthermia n the sympathetic neureffectr junctin were studied in the islated perfused cat spleen. The experiments were aimed at determining the temperature dependence bth f prejunctinal events, i.e., efflux f nrepinephrine frm the sympathetic nerves in respnse t electrical stimulatin r t indirectly acting sympathmimetic amines like tyramine r phenethylamine, and f pstjunctinal events, i.e., the cntractile respnse f the smth muscle t the nrepinephrine liberated. Hypthermia was fund t depress bth the nrepinephrine efflux in respnse t stimulatin and the cntractile respnse t the endgenus nrepinephrine released, as well as t exgenus nrepinephrine. Hwever, these prcesses were fund t respnd differently t temperature change. Thus hypthermia depressed nrepinephrine release as well as the pressr respnses t tyramine and phenethylamine much mre strngly than the crrespnding respnses t electrical nerve stimulatin r the pressr respnse t exgenus nrepinephrine. The results strngly supprt the cncept that sympathmimetic amines release nrepinephrine frm sympathetic nerves by mechanisms which differ frm thse mediating nrepinephrine release induced by deplariatin. ADDITIONAL KEY WORDS sympathetic nerves nerve stimulatin tyramine temperature smth muscle The sympathetic nervus system plays an imprtant rle in thermal hmestasis during general expsure t cld, which induces increased activity in the sympathetic nerves, as reflected by a marked increase in the urinary excretin f nrepinephrine (NE) (1). Hwever, less is knwn cncerning the direct lcal effect f hypthermia n sympathetic nerve functin, such as prcesses invlved in cnductin f impulses in C fibers, neurtransmitter release, and sensitivity f the smth muscle t the transmitter released. Nerve cnductin is knwn t be temperature dependent. Thus, Fran and Igg (2) fund that in nnmyelinated nerve fibers there Frm the Department f Physilgy, Karlinska Institutet, S Stckhlm 60, Sweden. This investigatin was supprted by grants frm Stiftelsen Lars Hiertas Minne and frm the Swedish Medical Research Cuncil, Prject B 70-14X-97-06B and Prject B 70-14X Received February 11, Accepted fr publicatin May 13, was a prgressive reductin in cnductin velcity as the temperature was lwered frm 37 dwn t 8 C, at 10 C being reduced t 15 t 20$ f the 37 C value. The effect f hypthermia n neurtransmitter release has s far nt been extensively investigated. In studies f the release f NE frm the sympathetic nerves f the islated cat spleen Kirpekar et al. (3) fund that reductin f the perfusin temperature t 15 C markedly reduced the amunt f NE released in respnse t nerve stimulatin r t infusin f ptassium. Hwever, the authrs did nt present quantitative data cncerning the extent f reductin f NE release during hypthermia. The spntaneus release rate f NE frm islated bvine splenic nerve granules was fund t be reduced t abut 10 percent when the temperature f the incubatin medium was reduced frm 37 t 20 C (4, 5). Hwever, the significance f the spntaneus release f NE frm islated granules with CircnUtin Rtsurcb, Vl. XXVII, July

2 40 WENNMALM, HEDQVIST, STJARNE respect t the release f NE frm the nerves in respnse t deplariatin is nt knwn. The sensitivity f smth muscle t catechlamines has been reprted t be altered by lw temperature. Thus Smith (6) stated that vessels stimulated by epinephrine cntracted and relaxed mre slwly at 17 than at 37 C. Keatinge (7) fund that the respnse f islated vascular strips t epinephrine prgressively decreased when the temperature was lwered frm 36 C, being cmpletely ablished between 12.5 and 10 C. In using a similar preparatin, Sams and Winkelmann fund a decrease in catechlamine-induced cntractin and in additin a changed respnse pattern when the temperature was lwered (8). On the ther hand, hypthermia has been reprted t ptentiate certain smth-muscle effects f catechlamines. Thus, the cutaneus veins f dgs cntract mre strngly in respnse t NE when the temperature is lwered frm 37 dwn t 17 C (9). In additin, epinephrine has been shwn t inhibit the spntaneus activity f the islated rabbit small intestine mre effectively at 30 than at 38 C (10). An increased respnse during hypthermia has als been fund in the islated guinea pig vas deferens by Delia Bella et al. (11). They fund that the cntractin n nerve stimulatin increased 5 t 10 times when the temperature was lwered frm 32 t 20 C. Methds Cats f bth sexes weighing 2.5 t 4.5 kg were used fr the study. The experimental animals were anesthetied with sdium pentbarbital (30 mg/kg ip) and heparine (1000 IU/kg iv). The abdmen was pened by a midline incisin and the spleen tgether with its vascular and nervus supply was islated. The rgan was transferred t a perfusin chamber and perfused with a physilgical salt slutin (in MM: NaCl, 103.4; KC1, 4.8; MgSO, 7 HO, 1.2; CaCl 2, 2.5; Na-pyruvate, 4.8; Na-1-glutamate 2.2; KH 2 PO 4, 1.0; Na HCO 3> 25; dextrse, 11.5; ascrbic acid, 0.11) aerated with 5% CO 2 in O 2. The flw rate was kept at 7.5 ml/min by a cnstant rate perfusin pump. Perfusin pressure was registered with a Statham pressure transducer cnnected t a Grass Mdel 7 plygraph. The effluent frm the spleen was divided int 10 ml prtins by means f an autmatic fractin cllectr. Supramaximal electrical stimulatin f the splenic nerves was carried ut with platinum electrdes, using a Grass S4 stimulatr, perated at a frequency f 10 cps, a duratin f 2 msec, and a stimulatin strength f 10 t 15 vlts, fr stimulatin perids f 20 secnds, separated by an interval f 10 minutes. The temperature was measured in the perfusing medium, in the perfusin chamber, and n the surface f the spleen. The experiment was started by an intra-arterial infusin f 30^ic f tritium-labeled dl-ne (specific activity 5 mc/ynmle, New England Nuclear Crp.). After washing fr 30 minutes by perfusin with NE-free Rrebs-Henseleit's slutin, the spleen was expsed either t nerve stimulatin r t intra-arterial injectin f NE (0.3 t 0.4 /j.g), phenethylamine (20 ug) r tyramine (20 /i.g). The temperature f the system was varied stepwise between 8 and 37 C, bth increasing and decreasing the temperature. The nerve stimulatin r injectin f NE, phenethylamine, r tyramine was carried ut at each step. The time curse and prfile f the NE utput frm the sympathetic nerves in the cat spleen was mnitred by measuring the amunt f radiactivity in each 10-ml fractin f the effluent frm the perfused rgan. The radiactivity f the different samples was determined by cunting 0.5-ml aliquts in a Packard Liquid Spectrmeter using a 7:3 tluene-abslute ethanl slutin cntaining 4 g f 2-5-diphenylxale and 100 mg f l-4-bis-2 (4-methyl-5-phenyIxaIyI) benene per liter f tluene. Quenching was mnitred by internal standards f 3 H-dl-NE. Results Accrding t previus evidence, almst all f the radiactive material released in respnse t nerve stimulatin r t injectin f tyramine in the preparatin used cnsists f intact NE (12). Reductin f the temperature f the spleen depressed the efflux f NE and the pressr effects caused by electrical nerve stimulatin as well as by phenethylamine r tyramine (Figs. 1, 2). Hwever, the tyramineinduced verflw f NE was clearly mre temperature dependent (Fig. 2). Similar results were btained with phenethylamine. The relative fall in the NE-releasing effect f tyramine was significantly mre marked than that f electrical nerve stimulatin, bth at 25 and 15 C. The average resting perfusin pressure was CircMlttin Ruurtb, Vl. XXVII, July 1970

3 SYMPATHETIC NERVE FUNCTION IN HYPOTHERMIA 41 I* cr > ir in UJ? I mm Hg 65 cpm t 10 t 25 T 37 t TEMP C FIGURE 1 Typical experiment shwing the effect f hypthermia n utflw f radiactivity and pressr respnse t 20 secnds f nerve stimulatin in the islated cat spleen. UJ % f 2 cntrl 100 Q. uj I/) -> UJ Z Q: < < 50 q > r 0 L v TEMP. C FIGURE 2 Effect f hypthermia n the utflw f radiactivity frm islated cat spleens in respnse t nerve stimulatin r tyramine injectins. Vertical bars = means ± SE (n = 5) in percent f cntrl values btained at 37 C. Nerve stimulatin and tyramine injectin values statistically separated at 25 C (P < 0.01) and at 15 C (P < 0.02). TA = tyramine. abut 25 mm Hg. During hypthermia this pressure rse t 30 mm Hg in sme f the Oremltiicm R,lurch, Vl. XXVII, J*I) 1970 experiments. At 37 C the pressr respnse t nerve stimulatin was 20 t 30 mm Hg. This

4 42 WENNMALM, HEDQVIST, STJARNE a. hi. I/) UJ a mm Hg TEMP. 'C FIGURE Regressin line shwing the effect f graded hypthermia n the pressr res^jnse t nerve stimulatin in three islated cat spleens. Id t HI a.) UJ a. mm Hg 75 r TEMP.'C FIGURE Regressin line shwing the effect f graded hypthermia n the pressr respnse t injectin f NE in three islated cat spleens. respnse prgressively decreased with the fall in temperature (Fig. 3) t be almst cmpletely ablished at temperatures belw 15 C. Virtually the same effect was btained when nerve stimulatin was replaced by injectin f cnstant amunts f NE (Fig. 4). The pressr respnses t tyramine and phenethylamine were abut 5 mm Hg at 37 C in all experiments. This respnse rapidly decreased when the temperature was lwered, and was cmpletely lst at arund 25 C. Discussin Hypthermia was fund t depress the NE efflux and the pressr respnses bth t deplariatin and t tyramine r phenethylamine, althugh t a different extent. The reduced NE verflw n nerve stimulatin might be due t direct effects f hypthermia n nerve cnductin, n release f NE frm the nerves, r n remval r enymatic inactivatin f the NE released. Nerve cnductin is blcked between 1 and 7.1 C (2, 13). Thus, impaired nerve cnductin might have influenced the verflw f NE t sme extent in the lwer temperature range f the present study. Hwever, at mderate hypthermia impaired cnductin was prbably nt f any majr imprtance. Release f NE frm the nerves in respnse t nerve stimulatin has previusly been shwn t be depressed by hypthermia (3). This is in accrdance with the present results. The prcess f spntaneus release f NE frm islated bvine splenic nerve granules is highly temperature dependent. The basic release rate at 37 is 8 t 10 times higher than that at 20 C (4, 5). Hwever, the utflw f NE in respnse t nerve stimulatin in the present series was far less temperature dependent, the rati f NE efflux at 37 and that at 20 C being abut 1.5. Thus it seems unlikely that the release f NE frm the nerves in respnse t deplariatin is immediately dependent n the same mechanisms as thse determining spntaneus release f NE frm islated nerve granules in the temperature range mentined. Reductin f temperature belw 20 C prduced a prnunced depressin f the verflw f NE in respnse t nerve stimulatin. During such extreme hypthermia the temperature dependence f the utflw respnse t nerve stimulatin was clse t that fund fr tyramine and phenethy- CircuUtin Rtuircb, Vl. XXVII, July 1970

5 SYMPATHETIC NERVE FUNCTION IN HYPOTHERMIA 43 lamine in the 37 t 25 C temperature range. On the ther hand, the temperature dependence f the phenethylamine- r tyramineinduced release f NE frm the nerves was mre clsely related t that f spntaneus release f NE frm islated nerve granules. This may be f particular significance since the NE-releasing effect f tyramine n the nerves appears t be exerted in part by direct actin n the granules (14). At this level tyramine may act bth by displacement f granule-bund NE (15) and by inhibitin f reuptake int the granules f the NE released, spntaneusly r by tyramine. Thus the bserved similarity in temperature dependence between spntaneus release f NE frm islated granules in vitr and tyramineinduced release f NE frm the whle neurn, may indicate that spntaneus release f NE frm granules in situ may be rate limiting fr the effect f tyramine n the nerves. Hypthermia may in additin interfere with the effects f tyramine at ther levels, such as n the uptake f tyramine int the nerves (12) and n the tyramine-induced inhibitin f reuptake int nerves f the NE released (16,17). The vascnstrictr effect f adrenaline has been shwn t be reduced by lw temperature (6, 7). Hwever, it has recently been shwn that the respnse t NE is markedly increased in dg's cutaneus veins (9). In the present study the pressr effect in respnse t nerve stimulatin gradually decreased when the temperature was lwered. Nt nly was the amplitude f the respnse altered, but als the pattern f the curve. At 37 C there was a sharp initial rise at the beginning f the nerve stimulatin r NE injectin, but during hypthermia this sharp rise became gradually less prnunced, giving the curve a mre flattened prfile. In cnclusin, hypthermia depresses several functins f the sympathetic neureffectr system f the islated perfused cat spleen, apparently by reducing NE release frm the nerves as well as the sensitivity f the smth muscle t NE. Hwever, there is a marked Circulatin Ruurcb, Vl. XXVll, J*LJ 1970 difference in the influence f hypthermia n the effect f the tw stimuli used fr inducing NE release, in the 37 t 20 C temperature range. Thus, the "physilgical" release f NE induced by nerve impulses is much mre resistant t hypthermia than the "pharmaclgical" release induced by sympathmimetic amines. This supprts the cncept that these tw types f NE release depend n distinctly different mechanisms. It als indicates that "physilgical" release, i.e., secretin n deplariatin, is nt immediately dependent n spntaneus release f NE frm nerve granules, while "pharmaclgical" release may well be s (cf. 18). References 1. LEDUC, J.: Catechlamine prductin and release in expsure and acclimatin t cld. Acta Physil Scand 53 (suppl.): 183, FRANZ, D. N., ANT) Icc, A.: Cnductin failure in myelinated and nnmyelinated axns at lw temperatures. J Physil (Lndn) 199: 319, KIBPEKAR, S. M., PRAT, J. C, AND YAMAMOTO, H.: Dependence f nrepinephrine (NE) release frm spleen n metablic energy. Fed Prc 28: 415, EULER, U. S. v., AND LISHAJKO, F.: Catechlamine release and uptake in islated adrenergic nerve granules. Acta Physil Scand 57: 468, EULER, U. S. V., AND LISHAJKO, F.: Reuptake and net uptake f nradrenaline in adrenergic nerve granules with a nte n the affinity fr 1- and d-ismers. Acta Physil Scand 71: 151, SMITH, D. J.: Cnstrictin f islated arteries and their vasa vasrum prduced by lw temperature. Amer J Physil 171: 528, KEATINCE, W. R.: Effect f lw temperatures n the respnses f arteries t cnstrictr drugs. I Physil (Lndn) 142: 395, SAMS, W. M., JB., AND WINKELMANN, R. K.: Temperature effects n islated resistance vessels f skin and mesentery. Amer J Physil 216: 112, WEBB-PEPLOE, M. M., AND SHEPHERD, J. T.: Peripheral mechanism invlved in respnse f dgs' cutaneus veins t lcal temperature change. Circ Res 23: 701, RYDIN, H.: Influence f different temperatures n the actin f drugs n autnmic effectr cells. Acta Physil Scand 11: 270, DELLA BELLA, D., GANDDVI, A., AND PRETI, M.: Influence f temperature n the respnses f

6 44 WENNMALM, HEDQVIST, STJARNE the guinea-pig hypgastric nerve-vas deferens 15. preparatin. J Pharm Pharmacl 17: 265, HEDQVIST, P., OLTVERIO, A., AND STJARNE, L.: Inhibitin by phenxybenamine f the nr- 16. adrenaline releasing effect f tyramine. Acta Physil Scand 72: 385, PAINTAL, A. S.: Cmparisn f the nerve 17. impulses f mammalian nnmedullated nerve fibres with thse f the smallest diameter medullated fibres. J Physil (Lndn) 193: 523, EULER, U. S. v., AND LISHAJKO, F.: Effects f directly and indirectly acting sympathmimetic amines n adrenergic transmitter Acta Physil Scand 73: 78, granules. SCHUMANN, H. J., AND PHUJPPU, A.: Untersuchungen um Mechanismus der Freisetung vn Brencatechinaminen durch Tyramin. Arch Exp Path Pharmakl 241: 273, IVERSEN, L. L.: Inhibitin f nradrenaline uptake by sympathmimetic amines. J Pharm Pharmacl 16: 435, LrNDMAR, R., AND MuSCHOLL, E.: Die Verstarkung der Nradrenalin-Wirkung durch Tyramin. Arch Exp Path Pharmakl 252: 122, KOPIN, I. J., BREESE, G. R., KRAUSS, K. R., AND WEISE, V. K.: Selective release f newly synthesied nrepinephrine frm the cat spleen during sympathetic nerve stimulatin. J Pharmacl Exp Ther 161: 279, CircmLai* Ruurcb, Vl. XXVII, July 1970

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

Effect of Prolonged Cold Storage on the Contractile Response of Strips of Rabbit Aorta to Various Agents 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 http://ahajurnals.rg