The Pivotal Role of Adrenomedullin in Producing Hyperdynamic Circulation During the Early Stage of Sepsis

PAPER The Pivotal Role of Adrenomedullin in Producing Hyperdynamic Circulation During the Early Stage of Sepsis

Ping Wang, MD; Zheng F. Ba; William G. Cioffi, MD; Kirby I. Bland, MD; Irshad H. Chaudry, PhD

Background: Initial cardiovascular responses during sep- Results: Cardiac output, stroke volume, and microvas- sis are characterized by hyperdynamic circulation. Al- cular blood flow in the liver, small intestine, kidney, and though studies have shown that a novel potent vasodi- spleen increased, and total peripheral resistance de- latory peptide, adrenomedullin (ADM), is up-regulated creased 0 and 30 minutes after ADM infusion. In addi- under such conditions, it remains unknown whether ADM tion, plasma levels of ADM increased from the preinfu- is responsible for initiating the hyperdynamic response. sion level of 92.7 ± 5.3 to 691.1 ± 28.2 pg/mL 30 minutes after ADM infusion, which was similar to ADM levels ob- Objective: To determine whether increased ADM reserved during early sepsis. Moreover, 5 hours after the lease during early sepsis plays any major role in produc- onset of sepsis, cardiac output, stroke volume, and mi- ing hyperdynamic circulation. crovascular blood flow in various organs increased and total peripheral resistance decreased. Administration of Design, Intervention, and Main Outcome Measure: anti-ADM antibodies, however, prevented the occur- Synthetic rat ADM (8.5 µg/kg of body weight) was infused rence of the hyperdynamic response. intravenously in normal rats for 15 minutes at a constant rate.Cardiacoutput,strokevolume,andmicrovascularblood Conclusions: The results suggest that increased ADM flow in various organs were determined immediately as well production and/or release plays a major role in produc- as 30 minutes after ADM infusion. At 30 minutes after in- ing hyperdynamic responses during early sepsis. Since fusion, plasma ADM level was also measured. In additional our previous studies have shown that vascular respon- groups, rats were subjected to sepsis by cecal ligation and siveness to ADM decreases in late sepsis, maintenance puncture. At 1.5 hours after cecal ligation and puncture, of ADM vascular responsiveness by pharmacological specific anti–rat ADM antibodies were infused, which com- agents during the course of sepsis may prevent transi- pletely neutralized the circulating ADM. Various hemody- tion from the hyperdynamic to the hypodynamic state. namic variables were measured 5 hours after cecal ligation and puncture (ie, the early stage of sepsis). Arch Surg. 1998;133:1298-1304

ESPITE ADVANCES in the leadstoaprogressivedeteriorationofcelland treatmentofsepticpatients, organfunctionsandeventualmortality.Thus, a large number of such pa- it is important to identify the mediator(s) re- tients subsequently die of sponsible for producing hyperdynamic cir- ensuing septic shock and culation during the early stage of sepsis. multiple-organD failure. Septic shock and Adrenomedullin (ADM) is a potent va- multiple-organfailurecontinuetobethemost sodilatory peptide (52–amino acid resi- common causes of death in surgical inten- dues in the human and 50–amino acid resi- sive care units, and the incidence of sepsis dues in the rat) that was first purified in 1993 has increased substantially during the past from human pheochromocytomas.4 The va- 2 decades.1,2 Polymicrobial sepsis is charac- sodilatory effect of ADM is thought to be pri- terized by an initial hyperdynamic phase fol- marily mediated through specific ADM re- lowed by a late hypodynamic phase.2,3 How- ceptors,5 which are functionally coupled to ever, the mechanism responsible for produc- adenylate cyclase via G proteins.6 Adreno- ing the transition from the hyperdynamic to From the Center for Surgical the hypodynamic state remains unknown. Research and Department of It is possible that the factor(s) responsible for thetransitionofhemodynamicresponsesdur- This article is also available on our Surgery, Brown University Web site: www.ama-assn.org/surgery. School of Medicine and Rhode ing sepsis is not fully understood and con- Island Hospital, Providence, RI. sequently not prevented, and such factor(s)

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©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 MATERIALS AND METHODS of incision was bathed with 1% lidocaine hydrochloride to provide analgesia throughout the study period. Sham- operated-on animals underwent the same surgical proce- The experiments described herein were performed in ad- dure except that the cecum was neither ligated nor punc- herence to the National Institutes of Health guidelines for tured. Plasma ADM was neutralized by intravenous the use of experimental animals. This project was ap- administration of specific anti-ADM (rat) antisera (ie, poly- proved by the Institutional Animal Care and Use Commit- clonal antibodies; host: rabbit; Peninsula Laboratories) 1.5 tee of Rhode Island Hospital, Providence. hours after CLP. A total of 0.5 mL of isotonic sodium chloride solution containing 8 µL of undiluted anti-ADM anti- INTRAVENOUS INFUSION sera (according to Peninsula Laboratories information, this OF SYNTHETIC RAT ADM dose of anti-ADM antibodies is adequate to neutralize plasma ADM during early sepsis) was given and various hemody- Male Sprague-Dawley rats (Charles River Laboratory, Wilm- namic measures were determined 5 hours after CLP (n = 5 ington, Mass), weighing 275 to 325 g, were fasted for 16 hours per group). It should be noted that 5 hours after CLP rep- before the experiment but were allowed water ad libitum. resents a typical time point of the early, hyperdynamic stage The animals were anesthetized with methoxyflurane inha- of polymicrobial sepsis.3 Vehicle-treated septic animals relation and the left femoral artery was cannulated with poly- ceived 0.5 mL of isotonic sodium chloride solution. ethylene 50 tubing (Becton Dickinson, Sparks, Md) for measurement of mean arterial pressure and heart rate or DETERMINATION OF CARDIAC OUTPUT withdrawal of blood samples for ADM assays. The right fem- AND MICROVASCULAR BLOOD FLOW oral vein was cannulated with polyethylene 10 tubing for ADM administration. Subsequent anesthesia was main- Cardiac output (CO) was measured by an indocyanine green tained by intravenous injection of pentobarbital sodium (ap- dilution technique with a 2.4 F fiberoptic catheter and in proximately 30 mg/kg of body weight). After 30 to 60 min- vivo hemoreflectometer, as we described previously.11 The utes of stabilization, synthetic rat ADM 1-50 (Peninsula CO was calculated according to the principle of dye dilu- Laboratories, Belmont, Calif) at a dose of 8.5 µg/kg of body tion. Stroke volume and total peripheral resistance (TPR) weight in 0.5 mL of isotonic sodium chloride solution con- were then calculated. Microvascular blood flow on the sur- taining 0.2% bovine serum albumin or 0.5 mL of vehicle (ie, face of the liver, small intestine, kidney, and spleen was de- isotonic sodium chloride solution with 0.2% bovine serum termined by laser Doppler flowmetry (Laserflo, Model BPM albumin) was infused via the femoral venous catheter dur- 403A; TSI Inc, St Paul, Minn).11 The measured microvas- ing 15 minutes at a constant infusion rate (n = 5 per group). cular blood flow was the microvascular red blood cell flux Various hemodynamic variables were measured immedi- in approximately 1 mm3 on the surface of each organ with ately as well as 30 minutes after the completion of ADM or a unit of milliliters per minute per 100 g of tissue, as indi- vehicle infusion. The mean arterial pressure and heart rate cated by the manufacturer. Although this is a reliable tech- were monitored by a blood pressure analyzer (Micro-Med, nique for determining the alterations in organ surface per- Louisville, Ky). The animals were then killed at the end of fusion, the flow unit suggested by the manufacturer should the experiment by an overdose of pentobarbital sodium. be considered as arbitrary rather than absolute units.

ANIMAL MODEL OF EARLY SEPSIS DETERMINATION OF PLASMA ADM AND ADMINISTRATION OF ANTI-ADM ANTIBODIES By means of a radioimmunoassay kit specific for rat ADM (Peninsula Laboratories), plasma levels of ADM were as- Polymicrobial sepsis was induced in male Sprague- sayed according to the procedure provided by the manu- Dawley rats (275-325 g) by CLP according to the method facturer. Briefly, 1.5-mL blood samples were collected into of Chaudry et al.10 Briefly, rats were fasted overnight be- a polypropylene tube containing EDTA (1 mg/mL) and apro- fore the induction of sepsis but allowed water ad libitum. tinin (500 kU/mL) at various time points after CLP and The animals were anesthetized with methoxyflurane inha- plasma was separated immediately. The rat ADM assay does lation, and a 2-cm ventral midline incision was made. The not have any cross-reactivity with human ADM, amykin, cecum was then exposed, ligated just distal to the ileoce- or endothelin 1. cal valve to avoid intestinal obstruction, punctured twice with an 18-gauge needle, and returned to the abdominal STATISTICAL ANALYSIS cavity. The abdominal incision was then closed in layers, and the animal received isotonic sodium chloride solu- One-way analysis of variance and Tukey test or Student t tion, 3 mL per 100 g of body weight, subcutaneously im- test were used, and the differences were considered sig- mediately after CLP (ie, fluid resuscitation). The area nificant at PՅ.05. Results are presented as mean ± SEM.

medullin also stimulates Ca2+ mobilization in vascular endothelium-derived NO release) lead to vascular relax- dothelial cells, resulting in an activation of constitutive ni- ation under normal conditions.5 Studies have indicated that tric oxide (NO) synthase.5 Thus, these 2 events (ie, a direct circulating levels of ADM are elevated in patients with sep- effect on vascular smooth muscle cells to increase intra- tic shock.7,8 In this regard, recent studies by Wang et al9 cellular cyclic adenosine monophosphate by stimulating have demonstrated that plasma levels of ADM and its gene adenylate cyclase activity via ADM receptors, and an alter- expression in various tissues increased significantly dur- native effect on vascular endothelial cells by increasing en- ing both the early and late stages of sepsis induced by ce-

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40 50 · 100 g –1

20 25 CO, mL · min

0 0 ∗ 160 B ∗ 150 B ∗ ∗

–1 120 100 · 100 g –1 80 L · Beat

µ 50 40 SV, SV,

0 0

4 C 120 C ∗ ∗ 3 ∗ ∗ 80 · min 100 g Microvascular Blood Flow, Arbitrary Units Microvascular Blood Flow, –1 2

40 1 TPR, mm Hg · mL

0 0 Before 0 min 30 min

Figure 1. Alterations in cardiac output (CO) (A), stroke volume (SV) (B), and 60 D ∗ total peripheral resistance (TPR) (C) before, immediately after (0 minutes), and 30 minutes after adrenomedullin infusion (n = 5). Data are presented as ∗ mean ± SEM and compared by 1-way analysis of variance and Tukey test. Asterisk indicates PϽ.05 vs the values before adrenomedullin infusion. 40

cal ligation and puncture (CLP). In addition, up-reg- 20 ulation of ADM production and the onset of the hyperdynamic state occur simultaneously after CLP.3,9 Our hypothesis, therefore, is that ADM plays a major role in 0 producing hyperdynamic circulation during the early stage Before 0 min 30 min of polymicrobial sepsis. To test this hypothesis, the pres- Figure 2. Alterations in microvascular blood flow in the liver (A), small ent study was carried out to determine whether infusion intestine (B), kidney (C), and spleen (D) before, immediately after (0 of synthetic rat ADM in normal animals produces hyper- minutes), and 30 minutes after adrenomedullin infusion (n = 5). Data are dynamic responses and, if so, whether inhibition of ADM presented as mean ± SEM and compared by 1-way analysis of variance and Tukey test. Asterisk indicates PϽ.05 vs the values before adrenomedullin early after the onset of sepsis prevents the occurrence of infusion. the hyperdynamic state.

RESULTS administration of ADM did not produce any significant alterations in heart rate (data not shown). The values of mean arterial pressure decreased by an av- Administration of ADM at a dose of 8.5 µg/kg in nor- erage of 19 mm Hg 15 minutes after the beginning of the mal animals increased CO by 20% and 41% (P = .02) at synthetic ADM infusion (from an average of 125 to 106 0 and 30 minutes, respectively, after ADM infusion mm Hg). However, this decrease in mean arterial pres- (Figure 1, A). Similarly, stroke volume increased by 21% sure was not statistically different from the preinfusion to 27% (P = .02) at both time points (Figure 1, B). In con- level (by 1-way analysis of variance). Within 30 min- trast, TPR values decreased by 22% to 28% (P = .01) 0 utes after the completion of ADM infusion, mean arte- and 30 minutes, respectively, after the completion of ADM rial pressure increased to 125.0 ± 4.4 mm Hg. Similarly, infusion (Figure 1, C).

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† 40 40 † · 100 g –1

20 20 CO, mL · min

0 0

160 B ∗ 150 B ∗

–1 120 † 100 † · 100 g –1 80 L · Beat

µ 50 40 SV, SV,

0 0

4 C † 120 C ∗

3 ∗ 80 † · min 100 g Microvascular Blood Flow, Arbitrary Units Microvascular Blood Flow, –1 2

40 1 TPR, mm Hg · mL

0 0 Sham Vehicle ADM-Abs

Figure 3. Alterations in cardiac output (CO) (A), stroke volume (SV) (B), and 60 D ∗ total peripheral resistance (TPR) (C) in sham-operated-on animals, animals treated with isotonic sodium chloride solution (vehicle), and animals treated with antiadrenomedullin antibodies (ADM-Abs) 5 hours after cecal ligation and puncture (n = 5 per group). Data are presented as mean ± SEM and 40 compared by 1-way analysis of variance and Tukey test. Asterisk indicates † PϽ.05 vs sham; dagger, PϽ.05 vs vehicle.

20 Immediately after ADM infusion, microvascular blood flow in the liver increased significantly (by 56%) and remained elevated 30 minutes after infusion 0 (P= .01; Figure 2, A). Similarly, microvascular blood Sham Vehicle ADM-Abs flow increased by 30% to 45% in the small intestine Figure 4. Alterations in microvascular blood flow in the liver (A), small (P = .004; Figure 2, B), by 34% to 37% in the kidney intestine (B), kidney (C), and spleen (D) in sham-operated-on animals, (P = .02; Figure 2, C), and by 77% to 114% in the spleen animals treated with isotonic sodium chloride solution (vehicle), and animals (PϽ.05; Figure 2, D) 0 and 30 minutes, respectively, treated with antiadrenomedullin antibodies (ADM-Abs) 5 hours after cecal ligation and puncture (n = 5 per group). Data are presented as mean ± SEM after ADM infusion. and compared by 1-way analysis of variance and Tukey test. Asterisk Before the infusion of ADM, plasma ADM level was indicates PϽ.05 vs sham; dagger, PϽ.05 vs vehicle. found to be 92.7 ± 5.3 pg/mL. Thirty minutes after the completion of ADM administration, plasma levels of ADM increased by approximately 6.5-fold to 691.1 ± 28.2 pg/mL mals (Figure 3, A and B). In addition, TPR decreased (PϽ.001). by 26% 5 hours after CLP (P = .02), and administration Five hours after the onset of sepsis, CO and stroke of anti-ADM antibodies prevented the reduction in TPR volume increased by 33% (P = .02; Figure 3, A) and 41% (Figure 3, C). (P = .001; Figure 3, B), respectively. Administration of anti- As illustrated in Figure 4, microvascular blood flow ADM antibodies 1.5 hours after CLP, however, pre- increased by 41% in the liver (P = .003), 33% in the small vented the elevation of both measures. There was no dif- intestine (PϽ.001), 24% in the kidney (P = .02), and 85% ference in CO and stroke volume between animals that in the spleen (P = .008) 5 hours after the onset of sepsis. received anti-ADM antibodies and sham-operated-on ani- Administration of anti-ADM antibodies 1.5 hours after

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©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 CLP, however, prevented the increase of microvascular istration of anti-ADM antibodies at a later time point, such blood flow in the tested organs (Figure 4). as 5 hours after CLP, reverses hyperdynamic and hyper- cardiovascular responses. COMMENT Studies have indicated that, in human subjects, ADM production and/or release increases after septic shock7,8 Studies have suggested that the potent vasodilatory me- as well as other pathophysiological conditions, such as diator NO (most likely derived from inducible NO syn- ischemic injury and organ failure.19,20 In addition, the in- thase [iNOS]) may be responsible for producing hypo- crease in ADM gene expression and its production can tension during septic shock or after administration of be observed after administration of bacterial endotox- endotoxin.12,13 Nitric oxide has also been found to me- in21 and proinflammatory cytokines, such as tumor ne- diate the redistribution of intrarenal blood flow during crosis factor and interleukin 1.22 Using the CLP model bacteremia.14 However, it remains unclear whether this of sepsis, we recently demonstrated that plasma levels of molecule and/or other vasodilatory agents are respon- ADM increased as early as 2 hours after CLP and were sible for producing the hyperdynamic state observed dur- sustained even 30 hours after the onset of sepsis.9 More- ing the early stage of polymicrobial sepsis. In this re- over, the ADM levels in the heart and aorta were found gard, our recent studies have indicated that the release to be elevated during sepsis.23 Studies by other investi- of endothelium-derived NO decreased during both early gators have shown that ADM gene expression is up- and late stages of sepsis.15,16 In addition, it has been dem- regulated in the small intestine, liver, lungs, aorta, heart, onstrated that plasma levels of nitrate or nitrite (stable and skeletal muscle after endotoxin administration.21,24 products of NO, reflecting iNOS activity) increased sev- In this regard, we have shown that ADM gene expres- eral hours after the onset of hyperdynamic circulation sion increased in the small intestine, left ventricle, and in the CLP model of sepsis.3,17 In view of these findings, thoracic aorta, but not in the liver and kidney, during it is unlikely that the alteration in NO production is re- the early stage of sepsis.9 Although endotoxin plays a role sponsible for initiating the hyperdynamic circulation dur- in producing various cardiovascular responses during sep- ing the early stage of sepsis. Nor is it likely that NO is sis, the observed plasma levels of endotoxin under such responsible for producing the transition from hyperdy- conditions are much lower than those encountered af- namic circulation to hypodynamic circulation during sep- ter endotoxemia or endotoxic shock.25 Thus, it appears sis. Although circulating levels of prostacyclin increase unlikely that endotoxin is the sole factor responsible for significantly during early sepsis, administration of the cy- ADM production during sepsis. Since endotoxin and pro- clo-oxygenase inhibitor indomethacin or the specific pros- inflammatory cytokines synergistically stimulate the pro- tacyclin synthase inhibitor tranylcypromine does not pre- duction of ADM,22 and since circulating levels of tumor vent the occurrence of hyperdynamic circulation under necrosis factor, interleukin 1, and interleukin 6 in- such conditions.18 Thus, it is likely that mediator(s) other crease significantly during sepsis26-28 or after endotoxin than NO and prostacyclin are responsible for producing administration,29 it appears that the increased circulat- hyperdynamic responses during early sepsis. The pres- ing levels of proinflammatory cytokines and endotoxin ent study, therefore, was undertaken to determine whether are responsible for the up-regulated ADM production dur- the novel vasodilatory peptide ADM plays any signifi- ing sepsis. cant role in producing hyperdynamic circulation dur- Parkes30 reported that administration of ADM in con- ing the early stage of sepsis. scious sheep increased CO and coronary blood flow and The results indicate that administration of syn- decreased peripheral vascular resistance. Moreover, ad- thetic rat ADM at a dose similar to that observed during ministration of ADM in rats increased CO and blood flow early sepsis,9 which did not produce significant alter- in the heart, lungs, kidney, and small intestine while de- ations in mean arterial pressure or heart rate, markedly creasing vascular resistance.31 However, an increase in the increased CO and stroke volume while decreasing TPR. dosage of ADM and/or its infusion rate may induce sys- In addition, infusion of ADM increased microvascular temic hypotension.30,31 In the present study, a relatively small blood flow in the liver, small intestine, kidney, and spleen. dose of ADM was administered to normal rats, in contrast Furthermore, CO, stroke volume, and microvascular to the study by He et al,31 in which ADM infusion pro- blood flow in various organs increased while TPR de- duced hypotension. It should be noted that the effects of creased significantly 5 hours after the onset of sepsis. Ad- ADM on the cardiovascular system were determined only ministration of specific anti-ADM antibodies 1.5 hours 0 and 30 minutes after the completion of ADM infusion. after CLP, however, prevented the occurrence of the hy- Thus, it remains unknown how long ADM-induced hyperdynamic response under such conditions. These find- percardiovascular responses persist after ADM adminis- ings, therefore, suggest that increased ADM production tration and whether an elevated plasma level of ADM is re- or release plays a major role in producing hyperdy- quired to maintain the hyperdynamic circulation. namic circulation during the early stage of sepsis. It should Although we have not determined ventricular per- be noted that anti-ADM antibodies were administered 1.5 formance after ADM infusion in normal animals or in hours after the onset of sepsis. This time point was se- septic animals after administration of anti-ADM anti- lected because our recent studies have shown that plasma bodies in the present study, Zhou et al32 recently levels of ADM increased 2 hours but not 1.5 hours after reported that ventricular contractility (as indicated by CLP.9 Anti-ADM antibodies were administered just be- the maximum rate of left ventricular pressure increase fore the increase in circulating ADM to neutralize ADM and decrease) increased significantly during the early activity. However, it remains unknown whether admin- stage of sepsis. In addition to its effects on the vascula-

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Despite major advances in the treatment of trauma vic- 1. Baue AE. Multiple organ failure, multiple organ dysfunction syndrome, and sys- tims, a large number of those patients subsequently die temic inflammatory response syndrome: why no magic bullets? Arch Surg. 1997; of sepsis, septic shock, and the ensuing multiple-organ 132:703-707. failure. It is encouraging, however, that the complex 2. Deitch EA. Animal models of sepsis and shock: a review and lessons learned. pathobiology of sepsis is becoming better understood as Shock. 1998;9:1-11. more investigations are being reported. Through such 3. Wang P, Chaudry IH. Mechanism of hepatocellular dysfunction during hyperdy- studies, information may be forthcoming that will lead namic sepsis. Am J Physiol. 1996;270:R927-R938. 4. Kitamura K, Kangawa K, Kawamoto M, et al. Adrenomedullin: a novel hypoten- to better treatment of septic patients. Polymicrobial sep- sive peptide isolated from human pheochromocytoma. Biochem Biophys Res sis is characterized by an early, hyperdynamic phase fol- Commun. 1993;192:553-560. lowed by a late, hypodynamic phase. It is, therefore, im- 5. Shimekake Y, Nagata K, Ohta S, et al. Adrenomedullin stimulated two signal trans- portant to determine the mechanism responsible for duction pathways, cAMP accumulation and Ca2+ mobilization, in bovine aortic producing the hyperdynamic response during early sep- endothelial cells. J Biol Chem. 1995;270:4412-4417. sis and the factor(s) contributing to the transition from 6. Eguchi S, Hirata Y, Iwasaki H, et al. Structure-activity relationship of adreno- the hyperdynamic to the hypodynamic stage of sepsis. medullin, a novel vasodilatory peptide, in cultured rat vascular smooth muscle In this regard, the present study indicates that the in- cells. Endocrinology. 1994;135:2454-2458. creased ADM production or release plays a major role 7. Hirata Y, Mitaka C, Sato K, et al. Increased circulating adrenomedullin, a novel in producing hyperdynamic circulation during the early vasodilatory peptide, in sepsis. J Clin Endocrinol Metab. 1996;81:1449-1453. 8. Nishio K, Akai Y, Murao Y, et al. Increased plasma concentrations of adreno- stage of sepsis. In addition, recent studies have demon- medullin correlate with relaxation of vascular tone in patients with septic shock. strated that the vascular responsiveness to ADM de- Crit Care Med. 1997;25:953-957. creased significantly during the late, hypodynamic phase 9. Wang P, Zhou M, Ba ZF, Cioffi WG, Chaudry IH. Up-regulation of a novel potent of sepsis, whereas ADM-induced vascular relaxation re- vasodilatory peptide adrenomedullin during polymicrobial sepsis. Shock. 1998; mained unaltered during the early, hyperdynamic phase.33 10:118-122. It is therefore possible that a reduced vascular respon- 10. Chaudry IH, Wichterman KA, Baue AE. Effect of sepsis on tissue adenine nucleo- siveness to ADM (most likely via down-regulation of ADM tide levels. Surgery. 1979;85:205-211. receptors) is responsible for producing the transition from 11. Wang P, Ba ZF, Chaudry IH. Hepatic extraction of indocyanine green is de- the hyperdynamic to the hypodynamic stage of sepsis. pressed early in sepsis despite increased hepatic blood flow and cardiac output. Arch Surg. 1991;126:219-224. In view of this, we propose that maintenance of the vas- 12. Szabo C. Alterations in nitric oxide production in various forms of circulatory cular responsiveness to ADM by the administration of shock. New Horizons. 1995;3:2-32. pharmacological agents during the course of sepsis may 13. Cobb JP, Danner RL. Nitric oxide and septic shock. JAMA. 1996;275:1192-1196. prevent the transition from the hyperdynamic to the hy- 14. Garrison RN, Wilson MA, Matheson PJ, Spain DA. Nitric oxide mediates redis- podynamic state of sepsis, thereby maintaining hemo- tribution of intrarenal blood flow during bacteremia. J Trauma. 1995;39:90-97. dynamic stability during sepsis and preventing the on- 15. Wang P, Ba ZF, Chaudry IH. Endothelium-dependent relaxation is depressed at set of septic shock and multiple-organ failure. the macro- and microcirculatory levels during sepsis. Am J Physiol. 1995;269: R988-R994. 16. Zhou M, Wang P, Chaudry IH. Endothelial nitric oxide synthase is downregu- lated during hyperdynamic sepsis. Biochim Biophys Acta. 1997;1335:182-190. 17. Meguid RA, Wang P, Chaudry IH. Does endothelin or nitric oxide play any major role in producing hyperdynamic circulation during sepsis [abstract]? Shock. 1997; ture, ADM may also contribute to producing the hyper- 7(suppl):65.

dynamic circulation during early sepsis by increasing 18. Wang P, Ba ZF, Cioffi WG, Bland KI, Chaudry IH. Is prostacyclin (PGI2) respon- ventricular performance. sible for producing the hyperdynamic state during early sepsis [abstract]? Shock. In summary, our results indicate that administra- 1998;9(suppl):37. 19. Wang X, Yue TL, Barone FC, et al. Discovery of adrenomedullin in rat ischemic tion of synthetic rat ADM in normal animals increased cortex and evidence for its role in exacerbating focal brain ischemic damage. Proc CO, stroke volume, and microvascular blood flow in the Natl Acad SciUSA. 1995;92:11480-11484. liver, small intestine, kidney, and spleen and decreased 20. Ishimitsu T, Nishikimi T, Saito Y, et al. Plasma levels of adrenomedullin, a newly TPR. Moreover, various cardiovascular variables in- identified hypotensive peptide, in patients with hypertension and renal failure. creased and peripheral vascular resistance decreased 5 J Clin Invest. 1994;94:2158-2161. 21. Shoji H, Minamina N, Kangawa K, Matsuo H. Endotoxin markedly elevates plasma hours after CLP (ie, at an early stage of sepsis). Admin- concentration and gene transcription of adrenomedullin in rat. Biochem Bio- istration of anti-ADM antibodies, however, prevented the phys Res Commun. 1995;215:531-537. occurrence of hyperdynamic circulation under such con- 22. Sugo S, Minamino N, Shoji H, et al. Interleukin-1, tumor necrosis factor and li- ditions. These findings lead us to conclude that in- popolysaccharide additively stimulate production of adrenomedullin in vascular creased ADM production and/or release plays a major role smooth muscle cells. Biochem Biophys Res Commun. 1995;207:25-32. 23. Zhou M, Chaudry IH, Wang P. Upregulation of adrenomedullin in the heart and in producing hyperdynamic circulation observed dur- aorta in polymicrobial sepsis [abstract]. Shock. 1998;9(suppl):38. ing early sepsis. 24. So S, Hattori Y, Kasai K, Shimoda SI, Gross SS. Up-regulation of rat adrenomedullin gene expression by endotoxin: relation to nitric oxide synthesis. Life This investigation was supported by grant RO1 GM 57468 Sci. 1996;58:PL309-PL315. 25. Ayala A, Deol ZK, Lehman DL, Herdon CD, Chaudry IH. Does endotoxin play a (Dr Wang) from the National Institutes of Health, Bethesda, major role in inducing the depression of macrophage function during polymi- Md. Dr Wang is the recipient of National Institutes of Health crobial sepsis? Arch Surg. 1995;130:1178-1185. Independent Scientist Award KO2 AI 01461. 26. Ertel W, Morrison MH, Wang P, Ba ZF, Ayala A, Chaudry IH. The complex pat- Presented at the 18th Annual Meeting of the Surgical tern of cytokines in sepsis: association between prostaglandins, cachectin and Infection Society, New York, NY, April 30, 1998. interleukins. Ann Surg. 1991;214:141-148. 27. Wang P, Ba ZF, Chaudry IH. Mechanism of hepatocellular dysfunction during early Reprints: Ping Wang, MD, Center for Surgical Re- sepsis: key role of increased gene expression and release of proinflammatory cy- search, Rhode Island Hospital, Middle House II, 593 Eddy tokines tumor necrosis factor and interleukin-6. Arch Surg. 1997;132:364-370. St, Providence, RI 02903 (e-mail:[email protected]). 28. Meyer TA, Wang J, Tiao GM, Ogle CK, Fischer JE, Hasselgren PO. Sepsis and

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©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 endotoxemia stimulate intestinal interleukin-6 production. Surgery. 1995;118: induced, iNOS-derived nitric oxide may not be the reason for 336-342. the occurrence of hyperdynamic circulation. 29. West MA, Bennet T, Clair L. Reprogrammed macrophage tumor necrosis factor You also asked the time course of ADM, whether or not and interleukin-1 release with inflammatory pretreatment: differential regulation at a zero time the cardiac output increased; however, that in- by endotoxin and zymosan. J Trauma. 1995;39:404-410. 30. Parkes DG. Cardiovascular actions of adrenomedullin in conscious sheep. Am J crease was not a significant difference. Physiol. 1995;268:H2574-H2578. Another question you mentioned is the administration of 31. He H, Bessho H, Fujisawa Y, et al. Effects of a synthetic rat adrenomedullin on antibody in the ADM-infused animal, whether or not that ad- regional hemodynamics in rats. Eur J Pharmacol. 1995;273:209-214. ministration of ADM antibody can prevent the occurrence of 32. Zhou M, Wang P, Chaudry IH. Cardiac contractility and structure are not signifi- hyperdynamic circulation. We have not conducted such stud- cantly compromised even during the late, hypodynamic stage of sepsis. Shock. ies; however, the sepsis studies indicate that when you give an- 1998;9:352-358. tibody at the early stage of sepsis, you prevent the hyperdy- 33. Wang P, Ba ZF, Cioffi WG, Chaudry IH. Factors responsible for producing the namic response. Since ADM is responsible for the increase in transition from the hyperdynamic to hypodynamic stage of sepsis: role of a novel vasodilatory peptide, adrenomedullin. Surg Forum. 1997;48:122-124. cardiac output, stroke volume, and blood flow, we would expect that if you give antibody at the time of perfused ADM, you may see no increase in cardiac output and blood flow. In other DISCUSSION words, the antibody in that setting may prevent the occurrence of hyperdynamic response. Michael A. West, PhD, MD, Minneapolis, Minn: What the au- The time course we did is zero time and 30 minutes. We thors have shown us is that possibly this, at least for many of did not look at the median time points, for instance, 15 minus, new molecule, the ADM, may in fact have an important he- utes. I would expect that at 15 minutes after ADM infusion, modynamic role. These data presented here are very tantaliz- the cardiac output and the blood flow probably increased as ing, but there are a number of important controls that I think we showed at 30 minutes. are missing, and certainly I’m sure that the laboratory is going Declan Carey, MD, Cardiff, Wales: Dr Wang, you stated to be pursuing further studies in this. in one of your method slides that your anti–rat ADM antibody Some of the specific questions that I would like to ask re- completely blocked the ADM response and then very clearly mea- late to the fact that you saw, at least in your infusion of ADM, sured in your cecal ligation and puncture model the levels of ADM; some very significant effects, even at time zero, and I want to however, you didn’t provide us proof in the treated animals that kind of concentrate on that. I am wondering what the mecha- you were able to block the ADM levels by measuring the levels nism for this is. in the treated animals. Why did you not do that? You showed a decrease in peripheral resistance, an in- Dr Wang: The reason we did not measure ADM levels in crease in stroke volume, and a borderline increase, as far as sta- ADM antibody–treated animals is because the measurement was tistical significance, in terms of cardiac output, even at time zero, radioimmunoassay. One way to look at it is to look at the physi- the same that you saw at 30 minutes, and I am wondering how ological response of the blood sample from dead animals. One of that ADM response might be mediated. In your manuscript the the experiments we should do is take a blood sample from the authors talk about the possibility that some of the mecha- ADM antibody–treated septic animals in order to stimulate blood nisms for ADM may involve increases in cyclic AMP within the vessels to see whether or not we will see a vascular relaxation. cells and may in fact involve increase in calcium in the cells Richard J. Howard, MD, Gainesville, Fla: Let me ask a and activation of a constitutive nitric oxide synthase. question that seems too obvious: is that why it is probably not My next question is, if nitric oxide synthase is involved, a good question? Does ADM, as the name suggests, adreno- have you looked at whether there are any associated increased medullin, come from the adrenal gland or does it come from levels of nitrate or nitrite in those cells? other sites? If it comes only from the adrenal gland, the obvi- You showed us the ADM levels at 30 minutes after the in- ous experiment is what happens to adrenalectomized animals, fusion. You didn’t show us any ADM levels at time zero. And and have you studied those? the other control that I would like to see, and perhaps you have Dr Wang: You are right; initially it was isolated from the some data on it, is in terms of your ADM infusion. Did you try tumor cell line from the adrenal gland; however, we have done administering your antibody to show the same sort of speci- several studies to look at the tissue levels of adrenal level pro- ficity that is implied in your CLP model? Because you could tein as well as the gene expression. It basically expressed in most block your CLP effects with the antibody, could you prevent organs and initially in other organs except macrophage. How- at time zero and 30 minutes your ADM infusion effects with ever, recently, even in this cell population, we still can see ad- the antibody, or is there something else going on? renomedullin. Finally, in terms of the time course that we saw, again, with R. Neal Garrison, MD, Louisville, Ky: I am curious as to the infusion, you showed us 30 minutes. Do you have any in- how you define or how you measure peripheral vascular resis- termediate time points to suggest how quickly this response tance. The main peripheral vascular resistant bed is skeletal comes in? It is a very interesting hypothesis and interesting data. muscle, which accounts for about 90% of vascular resistance Dr Wang: Dr West, in terms of the control group, you are beds; the liver and the kidney and the splanchnic organs are right, we have a long way to look at the effect of ADM in terms organ-specific perfusion beds. Did you measure the effect of of its work during sepsis. The mechanism of ADM, as you point this compound on perfusion to skeletal muscle beds? out, is through an increase of cyclic AMP and an increase in Dr Wang: How did we measure total peripheral resis- nitric oxide release, and we have not measured nitrate and ni- tance? We used a classic physiological measure: peripheral re- trite in this particular study; however, previously we have shown sistance is equal to mean cardiac output divided by blood pres- that nitrate and nitrite levels in the circulation do not increase sure minus central venous pressure. at 2 and 5 hours; however, they increased significantly at 10 In terms of your second question, we have not specifi- and 20 hours. Since hyperdynamic circulation occurs as early cally measured organ resistance. That can be calculated by the as 2 hours, the increased nitrate and nitrite at 10 hours do not blood flow to that organ as well as the blood pressure to that affect the time course. In other words, the increased iNOS- specific organ.

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