Release and Vasoactive Actions of Catecholamines During Inhibition of Prostaglandin Synthesis in Normal Man

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1 Release and Vasoactive Actions of Catecholamines During Inhibition of Prostaglandin Synthesis in Normal Man H. VIERHAPPER, M.D., BEATRIX GRUBECK-LOEBENSTEIN, M.D., ADRIENNE KORN, M.D. AND W. WALDHAUSL, M.D. SUMMARY To assess the effect of prostaglandin inhibition upon the vasoactive actions of endogenous and exogenous catecholamines in healthy man, indomethacin (150 mg/day for 3 days) was administered to six healthy men in the sodium-repleted state. Pretreatment with indomethacin did not interfere with the response of blood pressure and pulse rate to orthostasis ( minutes), a cold pressor test ( minutes), and the intravenous (i.v.) administration of norepinephrine (NE) (50,0, and 00 ng kg' 1 miir 1 ). plasma concentrations of epinephrine (E) and NE as well as the concentrations of E during orthostasis and cold pressor test remained uninfluenced by pretreatment with indomethacin. While the release of NE during orthostasis appeared to be suppressed in the indomethacin-treated state, it was unchanged during the cold pressor test. These results indicate that inhibition of endogenous prostaglandin synthesis may suppress the release of NE, but does not have a major impact on the vasoactive actions of endogenous and exogenous catecholamines in normal man. (Hypertension 4: , 19) KEY WORDS indomethacin prostaglandins epinephrine norepinephrine THE inhibition of prostaglandin synthesis with indomethacin 1 has been shown to interfere with the vascular sensitivity to various vasoconstrictor stimuli in vitro. In vivo, indomethacin enhances the pressor action of angiotensin II (All) in healthy man, 3 ' 4 and an enhanced pressor response to All and NE has been described in some indomethacin-treated patients with Bartter's syndrome. 6 Studies of the influence of prostaglandin inhibition by indomethacin on the release of endogenous catecholamines in man have indicated an inhibitory effect on the release of NE, 9 although other authors have failed to confirm this observation. To examine the influence of prostaglandin inhibition upon the vasoactive effects of both exogenous and endogenous catecholamines, we studied the pressor From the I. Medizinische Universitatsklinik, Division of Clinical Endocrinology and Diabetes Mellitus, Vienna, Austria. This study was presented in part at the 14th Meeting of the European Society for Clinical Investigation, Salzburg, April, 190, and at the Meeting of the Eastern Section of the American Federation for Clinical Research, Boston, Massachusetts, October, 19O. M Supported by the Medizinisch wissenschaftlicher Fonds des Burgermeisters der Bundeshauptstadt Wien. Received October 14, 190; revision accepted June 1, 191. Address for reprints: Dr. H. Vierhapper, I. Medizinische Universitatsklinik, Division of Clinical Endocrinology and Diabetes Mellitus, Lazarettgasse 14, A-90, Wien, Austria. 11 action of exogenous NE as well as the changes in blood pressure, pulse rate, and plasma catecholamines during orthostasis and cold pressor test in healthy subjects prior to and following the administration of indomethacin. Experimental Protocol Subjects Six healthy male volunteers aged 0 to 6 years (mean, 3 ± 1 years) whose weight was 90% to 1% of ideal body weight (based on Metropolitan Life insurance tables, 1959) were included in the study. The purpose and potential risks of the study were carefully explained to all subjects, and written, informed consent was obtained before their participation. No medication was permitted for at least 6 weeks before the study. All subjects fasted overnight and abstained from alcoholic beverages, coffee, and cigarette smoking for at least 1 hours prior to each test. They ate their regular diet on an outpatient basis, with 6 g of supplementary salt ( g t.i.d.) for 3 days. On the third day, a 4-hour urine collection was made to determine urinary sodium (Na + ) and potassium (K + ) concentrations. The subjects were in the supine position for hours before each test, and 60 minutes before the beginning of the experimental protocol they had indwell-

2 CATECHOLAMINE ACTION DURING PROSTAGLANDIN INHIBITION/Vierhapper et al. 113 ing catheters inserted in each antecubital vein, one for infusion and the other for blood sampling. The sequence of the subsequent functional studies was: orthostasis, cold pressor test, i.v. NE. Functional Studies Orthostasis Blood pressure (BP) and pulse rate (PR) measurements were taken supine at 5-minute intervals for an initial observation period of 30 minutes. Then the subjects assumed an upright position for minutes, for BP and PR determinations at -minute intervals; they then reassumed the supine position for BP and PR control every 5 minutes for 30 minutes. Blood samples were drawn at 15,0,,, and minutes to determine E and NE. In addition, blood samples were obtained at 15 and 0 minutes for the determination of plasma aldosterone and the serum concentrations of Na + and K +. Cold Pressor Test Following an observation period of 15 minutes during which BP and PR were measured at 5-minute intervals, the subjects were asked to immerse their hands and forearms in ice water for minutes. BP and PR were measured at 1 and minutes during immersion as well as 3, 4, 5,, and 15 minutes after the start of immersion. Blood samples for the determination of E and NE were drawn at -15, 0, 1, and minutes. Administration of Norepinephrine Following an initial observation period of 15 minutes with BP and PR measurements at 5-minute intervals, NE (Arterenol, Hoechst AC, Frankfurt/ Main, Federal Republic of Germany, diluted in 0.9% saline) was administered by constant (1 ml/min) infusion at fixed rates of 50, 0, and 00 ng kg 1 min 1 ; t = 15 minutes each). BP and PR measurements were continued at 5-minute intervals. Blood samples for the determination of E and NE were obtained at -15, 0, and at 14, 9, and 44 minutes (i.e.: 1 minute before the end of the infusion of each single dose of NE). Equilibration periods of 60 minutes were observed between the various functional studies. The tests were repeated under otherwise identical conditions after oral ingestion of 3 X 50 mg indomethacin (Indocin, Merck, Sharp and Dohme, Haarlem)/day for 3 days. The last dose of indomethacin was given hours before starting the procedures according to the experimental protocol. The sequence of the indomethacin and control studies, which were 1 week apart, was randomized. Methods All BP readings were taken by the same person using a cuff sphygmomanometer. Mean blood pressure (BP m ) was calculated as diastolic BP plus one-third of pulse height. Concentrations of Na + and K + in urine and serum were determined by flame photometry. Aldosterone was determined radioimmunologically following extraction by dichloromethane and thin layer chromatography (cyclohexane - ethyl acetate 0:0)." Plasma concentrations of NE and E were determined by radioenzymatic analysis based on the conversion of the catecholamines into their o-methylated analogs by catechol-o-methyltransferase in the presence of S- adenosyl-methionine- 3 H. 1 The intra- and interassay coefficients of variation for plasma aldosterone, E, and NE were < %. Data in text and figures are presented as x ± SE. Student's t test for matched pairs and analysis of variance were used for statistical evaluation. Results Following administration of indomethacin in the sodium-repleted state, no change was observed in body weight (75. ± 5.5 kg in the untreated vs 76.3 ± 5.3 kg in the indomethacin-treated subjects), in urinary volume (basal: 1470 ± 9 ml untreated vs 137 ± 14 ml treated), in Na + -excretion (49.0 ± 1.3 mmole/day untreated vs 19.5 ± 16.7 mmole/ day treated), in K + -excretion (131.3 ± 15.0 mmole/ day untreated vs ±. mmole/day treated), as well as in serum sodium (14.3 ± 0. mmole/liter untreated vs 14.5 ± 1. mmole/liter treated) and potassium (4.4 ± 0.07 mmole/liter untreated vs 4.6 ± 0. mmole/liter treated. Following administration of indomethacin, plasma aldosterone was suppressed (without indomethacin, 5.3 ± 0.6 ng/0 ml; after indomethacin, 3.7 ± 0. ng/0 ml; p < 0.05). Changes in Blood Pressure and Pulse Rate During Orthostasis and Cold Pressor Test Orthostasis induced an initial rise in diastolic BP from 77.3 ± 3.0 to 4. ±.7 mm Hg (p < 0.01) without indomethacin and from 0.7 ±. mm Hg to 5.0 ± 4.3 mm Hg (n.s.) after the application of indomethacin. This was followed by a fall in diastolic BP to 75. ± 6. mm Hg without indomethacin (n.s.) and to 70. ± 7. mm Hg following indomethacin (n.s.). Systolic BP fell from 11. ± 1. mm Hg to.3 ± 4. mm Hg (before indomethacin; p < 0.05) and from ± 3. mm Hg to 6.7 ±. mm Hg (after indomethacin; n.s.). Thus, BP m fell from 91.0 ±.4 mm Hg to 6.7 ± 6. mm Hg (n.s.) and from 93.3 ± 1.9 mm Hg to.3 ± 4. mm Hg (n.s.) respectively (fig. 1). In subjects without medication, cold pressor test induced a rise in diastolic BP (0.3 ±.3 to 0.0 ± 3. mm Hg,/? < ), systolic BP (.0 ± 1.4 to 144. ± 7.6 mm Hg, p < 0.05) and BP m (93.5 ± 1.4 to 114. ± 4.5 mm Hg, p < 0.005). Following indomethacin, the respective changes in BP were: 1.7 ±.4 mm Hg to.0 ± 3. mm Hg (diastolic,/? < ); ± 3.0 mm Hg to 14.5 ± 4.6 mm Hg (systolic, p < 0.005); and 94.0 ±.3 mm Hg to ± 3.3 mm Hg (BP m, p < ).

3 114 HYPERTENSION VOL 4, No 1, JANUARY-FEBRUARY 19 blood pressure (diastolic, systolic, and mean) as well as the observed maximal and overall changes (as judged by analysis of variance) in diastolic BP, systolic BP, and BP m both during orthostasis and during cold pressor test were not influenced by pretreatment with indomethacin. A significant (p < 0.05 to < 0.005)-increase in PR was seen both during orthostasis and cold pressor test prior to as well as following administration of indomethacin. PR was lower following treatment with indomethacin than without indomethacin both prior to orthostasis (63 ± 5 vs 67 ± 5 beats/min;/? < 0.005) and prior to cold pressor test (64 ± 5 vs 69 ± 6 beats/min; p < 0.05). Although the overall response of PR both during orthostasis and cold pressor test was not influenced by treatment with indomethacin (analysis of variance, p > 0.05), a lower PR was observed intermittently both during orthostasis and cold pressor test (p < 0.05). Plasma Catecholamines During Orthostasis and During Cold Pressor Test Plasma concentrations of NE during orthostasis and cold pressor test are shown in table 1. Administration of indomethacin did not influence basal NE prior to orthostasis and prior to cold pressor test. An increase in plasma NE concentrations above basal levels during orthostasis was observed both before {p < TABLE 1. Plasma Concentrations of Norepmephrme (pg/mt) m Six Normal Men During Orthostasis and Cold Pressor Test Prior to and Following Treatment with Indomethacin Time Subject number (min) Orthostasis: without indomethacin Orthostasis: after indomethacin X±SE 179. ± ± ± ± ± ± ± ± ) and after indomethacin administration (p < ). A rise in plasma NE was also to be seen during cold pressor test both without indomethacin (p < 0.05) and following indomethacin (p < 0.05) treatment. Maximum concentrations of plasma NE were higher during orthostasis than during cold pressor test without indomethacin as well as after indomethacin administration (p < 0.01 and < 0.05 respectively). Indomethacin did not induce any differences in the individual maximal concentrations of NE during orthostasis (377.5 ± 19. pg/ml vs 50.3 ± 47.4 pg/ml without indomethacin, n.s.) or during cold pressor test (93. ± 33.4 pg/ml vs ± 51.1 pg/ml without indomethacin, n.s.). However, analysis of variance revealed a smaller overall response of plasma NE to orthostasis after indomethacin when compared to the untreated state (p < 0.05). The overall response of NE to cold pressor test did not appear to be influenced by indomethacin, as calculated by analysis of variance (p > 0.05). Plasma concentrations of E during orthostasis and cold pressor test are shown in table. Treatment with indomethacin did not influence basal E prior to orthostasis or to cold pressor test. Neither orthostasis nor cold pressor test induced a significant rise in plasma E above basal concentrations. Maximal plasma E concentrations during orthostasis were not different from those seen during cold pressor test both in the untreated state and following treatment with indomethacin., TABLE Plasma Concentrations of Epinephnne (pg/mt) in Six Normal Men During Orthostasis and Cold Pressor Test Prior to and Following Treatment with Indomethacin Time (min) 1 Orthostasis. Subject number without indomethacin Orthostasis. after indomethacin X ±SE 4.3 ± ± ±.9.0 ±9 3.7 ± ± ± ± 16.5 Cold pressor test without indomethacin ± ± ± 53.7 Cold pressor test without indomethacin ± ± ± 7.6 Cold pressor test after indomethacin ± ± ±33.4 Cold pressor test after indomethacin ± ± ±.4

4 CATECHOLAMINE ACTION DURING PROSTAGLANDIN INHIBITION/Kitr/iapper et al. 115 CRT. 1J0 p < 0 05 in p<0.0os 90 t^ 70 -s H'l i i 01>tS MINUTES» 15 > o T 1. Mean blood pressure (BP^ mm Hg) and pulse rate (PR X min~l) in healthy, male subjects (n = o) and after (») treatment with indo6) during orthostasis and cold pressor test (CPT) prior to (o methacin (x ± SE). FIGURE Indomethacin also failed to induce any differences in the individual maximal concentrations of E during orthostasis (6.7 ± 16. pg/ml following indomethacin vs ± 6.4 pg/ml in the untreated state; n.s.) or during cold pressor test (5.7 ±13.1 pg/ml vs ± 9.6 pg/ml in the untreated state; n.s.). No difference was found in the overall response of E to orthostasis or cold pressor test between the indomethacin-treated and the untreated state (p > 0.05). Effect of Exogenous Noreplnephrine The pressor response observed in the six subjects during the i.v. administration of 50, 0, and 00 ng NE kg^min"1 (fig. ) was unaltered by pretreatment with indomethacin as calculated by analysis of variance. Pulse rate during the infusion of norepinephrine was similar in the untreated state and after indomethacin administration. Plasma concentrations of NE were identical (table 3) at the end of the respective infusion periods of NE for the untreated and the indomethacin-treated state. Plasma concentrations of E did not change while NE was infused. TABLE 3 Plasma Concentrations of Norepinephrine (pg/ml) m Six Normal Men (x ± SE) Following the Intravenous Administration of Various Doses of Norepinephrine Prior to and After Treatment with Indomethacin Plasma norepinephrine concentration (pg/ml) Intravenous After norepinephrine Without indomethacin (ng kg"1 min"1) indomethacin ± ± ± ± ± ± Discussion The functional capacity of the sympathetic nervous system has been tested in humans by various stimuli including orthostasis and the cold pressor test. Both maneuvers result in an increase of plasma catecholamine (predominantly NE) concentrations.1*"1 The expected changes in BP and pulse rate during orthostasis and cold pressor test were observed in the

5 116 HYPERTENSION VOL 4, No 1, JANUARY-FEBRUARY P< t 0 i 5 70 MOflEPINEPHRINE (ng hq -1 mln-<) » M <.5 MINUTES FIGURE. Mean blood pressure (BP^ mm Hg) and pulse rate (PR X min~ l ) in healthy, male subjects (n = 6) during i v administration of norepinephrine (50, 0, and 00 ng kg' 1 min~ l ) prior to (o o) and after (,' treatment with indomethacin (x ± SE). six healthy subjects in this study. Orthostasis proved to be a more powerful stimulus for NE secretion than the cold pressor test, while changes in plasma E during both maneuvers were not significant. Little information is available about the impact of endogenous prostaglandins on both the release and the vasoactive effects of catecholamines in vivo. The six subjects presented in this study were given a dose of indomethacin that has been shown to decrease urinary PG excretion by 71%." Thus, although concentrations of prostaglandins were not determined in this study, inhibition of endogenous prostaglandin synthesis can be assumed. This may also explain the observed suppression in plasma aldosterone following indomethacin when compared to the untreated yet sodium-repleted state, as decreased concentrations of aldosterone have previously been observed in healthy subjects following treatment with indomethacin. 4 Treatment with indomethacin neither modified basal BP nor the changes in BP during orthostasis and cold pressor test. The observed decrease in pulse rate following treatment with indomethacin is in accordance with observations by others 17 and could be due to the constant, albeit not significant, elevation of BP during treatment with indomethacin. Whereas basal plasma concentrations of E and NE as well as the concentrations of E during orthostasis and cold pressor test were uninfluenced by pretreatment with indomethacin, plasma concentrations of NE during orthostasis were lower in the indomethacin-treated state when compared to the untreated state by analysis of variance. It is conceivable that indomethacin was not equally effective in inhibiting prostaglandin synthesis in the adrenal medulla and in sympathetic neuronal junctions, thus accounting for the different behavior of E and NE. Possibly due to the large range in the observed concentrations of NE no significant differences were found between the individual maximal concentrations of NE during orthostasis in the untreated vs the indomethacin-treated state. Since sympathetic nervous activity in normal subjects decreases with sodium loading, 1 the high sodium-uptake in the subjects presented in this study may have blunted the release of NE during orthostasis. This may account for the less clearcut suppression of NE release by indomethacin in comparison to data recently published by GQllner et al., 9 who found decreased concentrations of NE (basal and after orthostasis) following treatment with indomethacin in normal subjects consuming 59 mmoles Na + /day. Emotional factors affecting catecholamine release 1 ' are not likely to account for the discrepancies between our data and those of Gflllner et al., since both studies were performed in a randomized setup. A recent report on unaltered plasma catecholamines in indomethacin-treated normal men prior to and following orthostasis is difficult to evaluate in this context, since no data on sodium uptake are given.

6 CATECHOLAMINE ACTION DURING PROSTAGLANDIN INHIBITION/Vierhapper et al. 117 Following pretreatment with indomethacin, the pressor response upon the infusion of various doses of NE was unchanged in the six subjects presented in this study. These data fail to confirm an enhanced pressor response in indomethacin-treated healthy men as described in abstract form by Guthrie et al. w However, that study may not have been performed in a randomized set up. Our data are not necessarily in conflict with results obtained in vitro which indicate a modulatory influence of prostaglandins upon the vasoactive actions of NE, since these interactions vary considerably between different prostaglandins 31 and different vascular beds. Thus, the pressor action of NE is potentiated by PGEj in some in vitro systems, while it is attenuated in others." Moreover, the inhibition of endogenous prostaglandins has failed to modify vasoconstrictor responses in some in vitro experiments." Our results suggest that in man there is no overall effect of prostaglandin inhibition upon the pressor action of exogenous NE under physiological conditions. This effect may be of more importance when prostaglandin synthesis is stimulated under basal conditions, thus explaining the increased pressor response to NE in some indomethacin-treated patients with Bartter's syndrome. 6 '' The enhanced pressor action of All observed in indomethacin-treated healthy subjects*' * may be due in part to decreased concentrations of endogenous All, resulting from a direct and/or indirect suppression of renin. Decreased concentrations of endogenous All render the vascular All receptors more sensitive to the actions of exogenous All." From our results it appears that the enhanced pressor response of All in indomethacin-treated healthy subjects is a phenomenon that doesn't necessarily apply to other vasoconstrictor agents. In conclusion, our data suggest that a suppression of NE release occurs following treatment with indomethacin in healthy subjects. However it appears that the inhibition of prostaglandin synthesis by indomethacin does not have any impact on the release of E or on the pressor response to exogenous NE in healthy man. Although these results do not exclude interactions of endogenous prostaglandins and catecholamines in local circulatory control, they indicate that the inhibition of prostaglandin synthesis does not have a major impact on the vasoactive actions of catecholamines in normal man. Acknowledgments The technical assistance of Ms. R. MOller, Ms E. Nowotny, Mr. P. Nowotny and Dipl. Ing. M Komjati is gratefully acknowledged References 1 Vane JR' Inhibition of prostaglandin synthesis as a mechanism of action for aspinn-like drugs. Nature 31: 3, 1971 Messina EJ, Weiner R, Kaley G: Prostaglandins and local circulatory control Fed Proc 35: 367, Negus P, Tannen RL, Dunn MJ. Indomethacin potentiates the vasoconstrictor actions of angiotensin II in normal man Prostaglandins 1: 175, Vierhapper H, Waldhausl W, Nowotny P. Effect of indomethacin upon angiotensin-induced changes in blood pressure and plasma aldosterone in normal man Eur J Clin Invest 11: Gill JR Jr. Bartter's syndrome Ann Rev Med 31: 405, Bartter FC, Gill JR Jr, FrOhch JC, Bowden RE, Hollifield JW, Radfar N, Keiser HR, Oates JA, Seyberth H, Taylor AA. Prostaglandins are overproduced by the kidneys and mediate hyperreninemia in Bartter's syndrome. Trans Assoc Am Physicians 9: 77, Silverberg AB, Mennes PA, Cryer PE: Resistance to endogenous norepinephnne in Bartter's syndrome. Reversion during indomethacin administration. Am J Med 64: 31, 197. Vierhapper H, Waldhfiusl W- Effect of indomethacin upon the renin-angiotensin system in patients with Bartter's syndrome. Eur J Clin Invest : 119, GOllner HG, Lake CR, Bartter FC, Kafka MS. Effect of inhibition of prostaglandin synthesis on sympathetic nervous system function in man J Clin Endocnnol Metab 49: 55, 1979 Rubin P, Blaschke T- Plasma catecholamines in man are not influenced by the inhibition of prostaglandin synthesis Prostaglandins 17: 51, Herkner K, Nowotny P, WaldhSusl W: Die Bestimmung von Aldosteron in Harn und Plasma J Chromatogr 146: 73, Da Prada M, ZOrcher G: Simultaneous radioenzymatic determination of plasma and tissue adrenaline, noradrenaline and dopamine within the fentomole range. Life Sciences 19: 1161, Cryer PE, Santiago J V, Shah SD' Measurement of norepinephnne and epinephnne in small volumes of human plasma by a single isotope derivative method response to the upright posture. J Clin Endocnnol Metab 39: 5, Lake CR, Ziegler MG, Coleman MD, Kopin IJ. Age-adjusted plasma norepinephnne levels are similar in normotensive and hypertensive subjects. N Engl J Med 96: 0, Robertson D, Johnson GA, Robertson RM, Nies AS, Shand DG, Oates JA Comparative assessment of stimuli that release neuronal and adrenomedullary catecholamines in man. Circulation 59: 637, Rane A, Oelz O, FrOhch JC, Seyberth HW, Sweetman BJ, Watson JT, Wilkinson GR, Oates JA Relation between plasma concentrations of indomethacin and its effect on prostaglandin synthesis and platelet aggregation in man. Clin Pharmacol Ther 3: 65, Boyer TD, Zia P, Reynolds TB: Effect of indomethacin and prostaglandin A, on renal function and plasma renin activity in alcoholic liver disease Gastroenterology 77: 15, Luft FC, Rankin LI, Henry DP, Bloch R, Grim CE, Weyman AE, Murray RH, Weinberger MH- Plasma and unnary norcpinephnne values at extremes of sodium intake in normal man. Hypertension 1: 61, Dimsdale JE, Moss J' Plasma catecholamines in stress and exercise. JAMA 43: 340, Guthne GP, Messerli FH, Kuchel O, Genest J. Enhanced sensitivity to pressor agents by indomethacin in normal man (abstr). Clin Res 4: A, Okuno T, Kondo K, Suzuki H, Saruta T Effects of prostaglandins E,, I,, and F^,, arachidomc acid and indomethacin on pressor responses to norepinephnne in conscious rats Prostaglandins 19: 55, 190 Kondo K, Okuno T, Suzuki H, Saruta T- Effects of prostaglandins E, and I It and arachidomc acid on vascular reactivity to norepinephnne in isolated rat mesenteric artery, hind limb and splenic artery. Prostaglandins Med 4: 1, Gottlieb AL, Lippton HL, Parey SE, Paustian PW, Kadowitz PJ. Blockade of vasoconstnetor responses by prostacychn (PGI,), PGE, and PGE[ in the rabbit hindquarters vascular bed Prostaglandins Med 4: 1, Hollenberg NK, Chenitz WR, Adams DF, Williams GH. Reciprocal influence of salt intake on adrenal glomerulosa and renal vascular responses to angiotensin II in normal man. J Clin Invest 54: 34, Vierhapper H, Grubeck-Loebenstein B, Korn A., Waldhflusl W. 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