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Opioid Peptide Modulation of Circulatory and Endocrine Response to Mental Stress in Humans
Peptides, Vol. 18, No. 2, pp. 169–175, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 / .00
PII S0196-9781(96)00319-1
Opioid Peptide Modulation of Circulatory and Endocrine Response to Mental Stress in Humans FIORELLA FONTANA,* 1 PASQUALE BERNARDI,* EMILIO MERLO PICH,† STEFANO BOSCHI,‡ ROSARIA DE IASIO,‡ SANTI SPAMPINATO§ AND GABRIELE GROSSIH *Istituto di Patologia Speciale Medica e Metodologia Clinica, Ospedale S. Orsola, Via Massarenti 9, 40138 Bologna, Italy †Glaxo Institute for Molecular Biology, 14 Chemin des Aulx, 1228 Plan-les Ouates, Ginevra, Switzerland ‡Servizio di Farmacologia Clinica, Ospedale S. Orsola, Via Massarenti 9, 40138 Bologna, Italy §Dipartimento di Farmacologia, Via Irnerio 48, 40126 Bologna, Italy HLaboratorio Centralizzato, Ospedale S. Orsola, Via Massarenti 9, 40138 Bologna, Italy Received 10 May 1996; Accepted 22 October 1996 FONTANA, F., P. BERNARDI, E. MERLO PICH, S. BOSCHI, R. DE IASIO, S. SPAMPINATO AND G. GROSSI. Opioid peptide modulation of circulatory and endocrine response to mental stress in humans. PEPTIDES 18 ( 2 ) 169 – 175, 1997. — Healthy subjects were classified according to their percent increase in systolic blood pressure ( SBP ) after mental arithmetic test ( MAT ) as low ( DSBP 9.3 – 15.1%, n Å 15 ) and high ( DSBP 35.1 – 45.4%, n Å 15 ) responders. During MAT, low responders showed significantly ( p õ 0.01 ) increased plasma levels of b-endorphin, cortisol, catecholamines, and atrial natriuretic factor ( ANF ) and decreased levels of endothelin-1, whereas high responders showed increased ( p õ 0.01 ) levels of Metenkephalin, dynorphin B, and catecholamines. Pretreatment with naloxone hydrochloride enhanced ( p õ 0.01 ) SBP, heart rate, noradrenaline, cortisol, and endothelin-1 levels, and reduced ( p õ 0.01 ) ANF in low responders in response to MAT, whereas it decreased ( p õ 0.01 ) hemodynamic parameters, noradrenaline, and endothelin-1 in high responders. The individual differences in hemodynamic and endocrine responses to MAT may depend on a different activation of the endogenous opioid system. q 1997 Elsevier Science Inc. Mental arithmetic test Atrial natriuretic factor
b-Endorphin Dynorphin B Cortisol Endothelin-1
Met-enkephalin
SOME reports have demonstrated that different forms of stress activate the endogenous opioid peptide system, which, in turn, contributes to changes in systemic cardiovascular function and endocrine response occurring under these conditions ( 21,29,32 ) . Mental arithmetic test ( MAT ) is one of the most useful procedures for eliciting reliable stress-induced cardiovascular responses in humans ( 36 ) . Several studies have examined the effects of this psychological stressor on systemic and coronary hemodynamics ( 28 – 30,36 ) , whereas relatively few studies have investigated the concomitant effects on the neuroendocrine system and, in particular, the role of the endogenous opioid peptide system ( 28,30,36 ) . In the only available investigation on an opioid peptide participation in the cardiovascular response to MAT, an opioid peptide antagonist ( naloxone ) produced a significant blood pressure ( BP ) increase in hypotensive subjects who responded moderately to MAT ( 28 ) . Interestingly, when subjects with borderline hypertension were tested, naloxone did not affect their exaggerated cardiovascular response to MAT ( 28 ) . It is still unknown
1
Naloxone
Catecholamines
whether the individual differences in the magnitude of circulatory responses to psychological stress in normotensive subjects are related to a different activation of the opioid peptide system ( 37 ) . The first aim of the present study was to characterize the profile of circulating levels of the three main opioid peptides [i.e. b-endorphin (BE), Met-enkephalin (ME), and dynorphin B (DYN B)] after MAT in normal healthy volunteers divided into two groups (low responders and high responders) according to their systolic blood pressure (SBP) increase in response to MAT. The second aim was to study the functional relationships between endogenous opioid peptide, sympathetic–adrenal medullary and pituitary–adrenal cortical systems after MAT, by evaluating plasma catecholamine and cortisol concentrations after MAT with and without pretreatment with naloxone hydrochloride (NARCAN). In addition, we measured plasma concentrations of atrial natriuretic factor (ANF) and endothelin-1 (ET), two other hormones mainly released in response to acute stress (20,24) and involved in cardiovascular control (31,33).
Requests for reprints should be addressed to Dr. Fiorella Fontana.
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FONTANA ET AL. METHOD
Subjects Thirty subjects (12 females and 18 males, mean age 29.2 { 0.7 years) were selected out of a population of 67 normotensive subjects with a high education and normal verbal ability. These subjects, selected among young physicians and medical students at our University, were tested with MAT. In this population the percent increments in SBP in response to MAT varied between 9.3% and 45.4% (mean { SD Å 25.1 { 10.0%) with respect to basal, pretest values. We selected two groups using a boundary defined by the SD: low responders and high responders. Low responders (n Å 15, 6 females and 9 males, mean age 29.6 { 1.0 years) were defined as those individuals whose increase in SBP to MAT was less than 15.1% (population mean 0 1 SD), whereas high responders (n Å 15, 6 females and 9 males, mean age 28.7 { 1.2 years) were those individuals whose increase in SBP was more than 35.1% (population mean / 1 SD). Subjects with intermediate increases (15.2–35.1%) in SBP in response to MAT were excluded from this study. All selected subjects had resting SBP values õ 130 mmHg and diastolic blood pressure (DBP) values õ 85 mmHg. BP was recorded using a fully automatic recorder set to take a measurement every 30 min throughout 24 h. The BP values of both parents of each subjects were also measured, and subjects were excluded if one of the parents was found to be hypertensive. In all subjects physical examination, chest radiogram, electrocardiogram, echocardiogram, and biochemical findings were normal. They were not taking any medication. All ate a 3–4 g/day sodium diet for 4–5 days before the study and for 24 h they did not consume tea, coffee, or cigarettes. None of the subjects was dependent on any drugs. Written informed consent was obtained from all subjects, and the protocol was approved by the Reaserch Committee of S. Orsola Hospital. Experimental Procedure The subjects were studied in the morning (between 0800 and 0900 h) after an overnight fast. MAT was performed by the subjects in supine position during placebo infusion (1.5 ml/min saline) following a 30-min period of rest. MAT consisted of asking the subjects to subtract the number 17 serially from 1,013 until 10 was reached and then repeat the procedure again for 3 min. They were urged to perform as quickly and accurately as possible, and reproached for lack of effort if the answer was not correct. Periodically, the subjects were urged to improve his/her performance. A week later 14 of these subjects, 7 low responders (3 females and 4 males, mean age 29.1 { 1.5 years) and 7 high responders (3 females and 4 males, mean age 28.8 { 1.6 years), underwent a second MAT during naloxone infusion (9.5 mg/kg/min). The dose of naloxone was chosen on the basis of a previous study on patients with congestive heart failure in whom 8 mg of naloxone produced maximal inhibition of hormonal and hemodynamic opioid peptide effects in the absence of any side effect (15). A catheter was inserted in the left antecubital vein; after a rest period, placebo or naloxone was infused 10 min before starting and during the MAT. In all subjects electrocardiogram leads were placed on the chest and a sphygmomanometer cuff was placed on the right arm. Blood samples for the determination of catecholamines, cortisol, ANF, and ET were taken through a needle placed in the left forearm vein before and immediately after MAT during placebo and naloxone infusion; BE, ME, and DYN B were assessed only before and after MAT with placebo.
Heart rate (HR) and cardiac rhythm were monitored continuously by electrocardiography, and BP determinations were made at 1-min intervals. Electrocardiogram was recorded by a Cardiostat 3 Siemens electrocardiograph; BP was measured by an automatic Hewlett–Packard 78354A sphygmomanometer. BP throughout 24 h was recorded using a fully automatic recorder (Profilomat; Disetronic Medical Systems, Burgdorf, Switzerland). Hormone Assay Determination of peptide hormones: radioimmunoassay. Plasma levels of immunoreactive b-endorphin-like material (IRBE), IR-Met-enkephalin (IR-ME), and IR-dynorphin B (IRDYN B) were evaluated by radioimmunoassay after extraction on Sep-Pak C18 cartridges (Water Associates). IR-BE was extracted from 10 ml of blood adopting a method described previously (8). Blood was collected into chilled tubes containing ethylendiaminetetraacetic acid (EDTA; 15% w/v) and plasma separated from the blood cells by centrifugation (3000 rpm/15 min, 47C). The plasma was collected, 1 N HCl (100 ml/ml) added, fibrin removed by centrifugation ( 12,000 rpm/ 10 min, 47C ) , and IR-BE was extracted on activated Sep-Pak cartridges. The samples were lyophilized, dissolved in radioimmunoassay buffer, and evaluated according to the protocol suggested by Amersham ( Little Chalfan, England ) using ( 3[ 125I ] iodotyrosyl 27 ) BE ( human ) as tracer (obtained from Amersham). The antiserum was purchased from Amersham (code N0. 1621); it was raised in rabbits against porcine BE and it completely cross-reacts on a molar basis with human b-lipotropin. This antiserum gave no cross-reactivity with ACTH, aMSH, Met- and Leu-enkephalin, and DYN-related peptides. To validate the plasma extraction procedure, the recovery of radiolabeled BE added directly to blood samples was determined and found to be between 84% and 86%. Reverse-phase high performance liquid chromatography (RP-HPLC) analysis of the recovered radioactivity, carried out as later described, did not display any significant degradation of the radiolabeled peptide (data not shown). Plasma extraction of IR-DYN B was carried out as described (35). Blood (10 ml) was collected into chilled tubes containing appropriate enzyme inhibitors and 7.3 mmol EDTA/ml. Samples were placed on ice, centrifuged (3000 rpm/15 min, 47C), and processed as described previously (35) to extract IR-DYN A using Sep-Pak cartridges. IR-DYN B was assayed using a commercially available antiserum (Peninsula Laboratories, Belmont, CA), which cross-reacts with DYN A(1–32) (70%; this peptide contains DYN B in its sequence), DYN B(1–29) (30%) whereas the cross-reactivity with DYN A(1–17), DYN A(1– 8), and Leu-enkephalin was less than 1%. The tracer, [ 125I]DYN B, was purchased from Peninsula Laboratories and radioimmunoassay procedure was carried out as described previously (35). Blood samples (10 ml) for IR-ME analysis were collected into cold tubes containing heparin (14 units/ml) and a mixture of ice-cold aprotinin (5%) and 17% citric acid (20 ml/ml) (2); then IR-ME was extracted (23) and assayed by radioimmunoassay procedure using a commercially available antiserum (Peninsula Laboratories) and [ 125I][Tyr 1-Met(O)]enkephalin (DuPont NEN, Cologno Monzese, Italy). The ME antiserum cross-reacts equally with ME-sulphoxide, to a small extent (3%) with Leu-enkephalin, and only minimally ( õ1%) with ME-ArgPhe or BE. Radioimmunoassay procedure was carried out according to the manufacturer’s directions (Peninsula Laboratories). Recovery of radiolabeled DYN B and ME directly added to blood samples was between 78% and 81%. RP-HPLC analysis
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OPIOID SYSTEM AND MENTAL ARITHMETIC TEST (carried out as later described) of the recovered radioactivity confirmed that both peptides were not degraded during the extraction procedure. In the radioimmunoassay procedure inhibition of tracer binding by 15% was considered the detection limit, and for the serially diluted extracts each tracer followed curves parallel to the standard curve. Intra- and interassay variations were less than 5%. Antibody dilution was chosen for 30% binding of counts added, and the limits of detection were: IR-DYN B 10 fmol/ml [mean { SD inhibitory concentration (IC50 ) 113 { 18 fmol/ml]; IR-BE 3 fmol/ml (IC50 15 { 6 fmol/ml); and IR-ME 12 fmol/ml (IC50 33 { 16 fmol/ml). ANF was determined, after chromatographic preextraction, by a radioimmunoassay procedure according to a method fully detailed previously (14). The detection limit for ANF was 0.6 fmol/ml. The intra-assay variation was 5.2% and the interassay variation 11.8%. The recovery of synthetic a-human-ANF added to blood samples was 85 { 2% with a coefficient of variation of 18%. Blood samples for ET determination were placed in cold tubes containing EDTA (1 mg/ml) and aprotinin (500 KIU/ml). Blood was centrifuged at 3000 rpm for 15 min at 07C. Plasma was stored at 0807C until assay. Plasma acidified (2 ml) with 0.1% trifluoracetic acid (TFA; 2 ml) and centrifuged at 3000 rpm for 15 min at 07C was applied to 200 mg C18 Sep column (Peninsula Laboratories Inc., Belmont, CA) preactivated with 4 ml of 0.1% TFA and 20 ml of 60% acetonitrile in 0.1% of TFA. The column was then washed with 20 ml 0.1% TFA. ET-like material was eluted with 3 ml of 60% acetonitrile in 0.1% TFA. The eluant was dried under nitrogen in a water bath at 407C and reconstituted in buffer. ET was then measured with the Peninsula radioimmunoassay kit. The recovery [ 125I]ET added directly to plasma samples was 81 { 3%. The effective range of the standard curve was between 0.4 and 51.3 fmol of ET per assay tube; the ED50 was 4.4 fmol of ET per assay tube. The intra-assay variation was 7.2% and the interassay variation 11.2%. Determination of peptide hormones: high performance liquid chromatography. Validation of the authenticity of IR-BE, IRDYN B, IR-ME, IR-ANF, and IR-ET in plasma extracts was obtained by RP-HPLC. For BE assay, a procedure described previously (38) was adopted. A mBondapak column (3.9 1 300 mm) was used. Elution was carried out with 0.225% TFA in water (solvent A) and 0.225% TFA in acetonitrile (solvent B); starting composition: 35% B; gradient linear to 40% B over 7.5 min. The flow rate was 1 ml/min. Fractions were collected for radioimmunoassay procedure every 30 s (0.5 ml), lyophilized, and redissolved in radioimmunoassay buffer. Two major peaks of immunoreactivity were found. These showed elution patterns identical to those of standard human BE and porcine b-lipotrophin (obtained from Peninsula Laboratories). Radiolabelled BE eluted after cold reference BE (data not shown). For IR-DYN B assay, the procedure described previously (35) was adopted. Pooled blood samples (20 ml obtained from healthy volunteers) were processed as described previously (35) and lyophilized. Samples were reconstituted in 350 ml of 5 mM TFA, injected on to a mBondapak C18 column, and eluted with a linear gradient of 19–65% acetonitrile in 5 mM TFA (60 min, 1.5 ml/min, 0.9 min/fraction). Fractions were collected and lyophilized for radioimmunoassay procedure. Two immunoreactive peaks were detected. One component had the same retention time as authentic DYN B whereas a second component showed the same retention time as DYN A(1–32) (data not shown). For ME assay, a procedure described previously was adopted (23) and a mBondapak C18 column was used. The flow rate of
171 the mobile phase (22% acetonitrile in 0.1% TFA) was 1.2 ml/ min. Fractions (1.2 ml each) were collected, lyophilized, redissolved in radioimmunoassay buffer, and assayed as previously described. Pooled blood samples (20 ml; obtained from healthy volunteers) were processed as previously reported, lyophilized, and reconstituted with 350 ml of mobile phase before being injected on to the column. Most of IR-ME eluted in the same position of standard ME (obtained from Peninsula Laboratories) and ME-sulphoxide (obtained from Sigma) (23) (data not shown). For the ANF assay, plasma extracts from Bond Elut columns were injected into the cromatograph equipped with mBondapack C18 column. Elution was carried out using a gradient of 0.1% TFA in CH3CN (solvent A) and 0.1% TFA in water (solvent B) as follows: starting 5% A/B for 5 min, linear gradient to 50% A/B from 5 to 30 min, then to 90% from 30 to 40 min. Flow rate was 1 ml/min. For the ET assay, the conditions were similar to that described for ANF except for the gradient shape, which was from 10% to 60% A/B in 60 min. Plasma chromatographed for ANF and ET each provided a major immunoreactive peak, which was shown to be identical to the standard synthetic peptide (data not shown). Determination of catecholamines and cortisol. Noradrenaline and adrenaline were determined by column switching high performance liquid chromatography with electrochemical detection as detailed previously (16). The detection limits for noradrenaline and adrenaline were 11.8 and 16.3 fmol/ml, respectively. The intra-assay variations of noradrenaline and adrenaline were 3.0% and 4.2%, respectively, and the interassay variations were 5.1% and 3.5%. The recovery of radiolabeled noradrenaline and adrenaline added to blood samples was above 90%. The quantitative measurement of cortisol was determined by a chemiluminescent immunoassay using the Immulite cortisol procedure (3). Immulite combines simple, well-established solid phase technology, 1/4 polystyrene beads, with an efficient and easily automated separation scheme. The solid phase is coated with a polyclonal rabbit antibody specific for cortisol. The patient samples, placed in cold tubes containing EDTA (1 mg/ml) and aprotinin (500 KIU/ml), were introduced into the test unit together with alkaline phosphatase-conjugated cortisol and incubated for approximately 30 min at 377C. After removal of the unbound enzyme conjugate by centrifugation, substrate was added and the test unit was incubated for a further 10 min. The detection limit of the assay was 0.5 nmol/dl. The intra-assay variation was 6.8% and the interassay variation 9.9%. The spiking recovery of cortisol added to blood samples was 92 { 3% with a coefficient of variation of 12%. Statistical Analysis Two-way analysis of variance (ANOVA) was used to compare hormonal and hemodynamic values before and after MAT with placebo in the two groups of subjects. Three-way ANOVA with one between factor (naloxone or placebo treatment) and two within factors (groups of subjects, and before and after MAT) was used to compare hormonal and hemodynamic values in the subjects who underwent the MAT during naloxone infusion. Individual means were then compared using post hoc Duncan’s test. All analyses were performed on original data, expressed in the figure as percent of basal values. The associations between opioid peptides and ANF vs. all other parameters as well as the association between catecholamines, ET, or cortisol and hemodynamic parameters were analyzed by Pearson’s r correlation coefficient and regression analysis. Values are expressed as mean { SEM, and p õ 0.05 was considered statistically significant.
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FONTANA ET AL. TABLE 1 HEMODYNAMIC AND HORMONAL PARAMETERS BEFORE (A) AND AFTER (B) MAT DURING PLACEBO INFUSION (1.5 ml/min SALINE) IN LOW AND HIGH RESPONDERS High Responders
Low Responders A
SBP (mmHg) DBP (mmHg) HR (bpm) BE (fmol/ml) ME (fmol/ml) DYN B (fmol/ml) A (fmol/ml) NA (fmol/ml) Cortisol (pmol/ml) ANF (fmol/ml) ET (fmol/ml)
116.8 { 76.8 { 68.2 { 8.1 { 9.0 { 21.4 { 221.6 { 936.3 { 146.0 { 3.0 { 2.8 {
B
1.6 1.1 1.4 0.3 0.7 1.0 19.1 30.1 10.4 0.1 0.1
130.0 { 91.0 { 84.4 { 14.6 { 9.4 { 24.3 { 332.9 { 1481.3 { 226.2 { 5.6 { 1.9 {
A
1.7* 1.0* 1.8* 1.5* 0.5 2.8 26.2* 69.1* 13.5* 0.3* 0.2*
115.3 { 79.8 { 67.6 { 8.9 { 9.0 { 20.8 { 222.1 { 929.8 { 132.4 { 3.0 { 2.4 {
B
1.5 1.5 1.1 0.2 0.3 1.4 15.8 26.6 10.7 0.2 0.1
160.8 { 98.4 { 92.0 { 10.4 { 20.7 { 35.5 { 337.8 { 1893.3 { 136.8 { 3.2 { 2.2 {
1.7*† 1.5*† 1.8*† 0.5† 1.1*† 1.2*† 21.8* 52.0*† 9.9† 0.2† 0.1
Values are expressed as means { SEM. Low responders (n Å 15), subjects with 9.3–15.1% systolic blood pressure increase in response to MAT; high responders (n Å 15), subjects with 35.1–45.4% systolic blood pressure increase in response to MAT. MAT, mental arithmetic test; SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate; BE, b-endorphin; ME, Met-enkephalin; DYN B, dynorphin B; A, adrenaline; NA, noradrenaline; ANF, atrial natriuretic factor; ET, endothelin. * p õ 0.01 vs. respective value before MAT; † p õ 0.01 vs. respective value of low responders. RESULTS
Table 1 summarizes hemodynamic and hormonal results obtained in the two groups of subjects in response to MAT during placebo infusion. Under basal, pretest conditions low responders and high responders did not significantly differ in any of the measured variables. In low responders MAT significantly (p õ 0.01) increased SBP, F (3, 56) Å 170.07, DBP, F (3, 56) Å 56.23, HR, F (3, 56) Å 59.31, plasma BE, F(3, 56) Å 22.01, adrenaline, F (3, 56) Å 9.54, noradrenaline, F(3, 56) Å 91.54, cortisol, F (3, 56) Å 14.48, and ANF, F(3, 56) Å 22.98, levels and significantly (p
õ 0.01) decreased plasma ET, F (3, 56) Å 4.53, concentration. In high responders MAT significantly (p õ 0.01) increased SBP, F(3, 56) Å 29.80, DBP, F (3, 56) Å 14.57, HR, F (3, 56) Å 14.42, plasma ME, F (3, 56) Å 29.96, DYN B, F (3, 56) Å 18.95, adrenaline, F(3, 56) Å 15.21, and noradrenaline, F(3, 56) Å 13.17, levels. SBP, F (1, 28) Å 151.21, DBP, F (1, 28) Å 16.21, HR, F (1, 28) Å 8.53, plasma ME, F (1, 28) Å 89.01, DYN B, F(1, 28) Å 89.16, and noradrenaline, F (1, 28) Å 22.66, concentrations were significantly (p õ 0.01) higher in high responders after MAT than the respective values of low responders, whereas plasma BE, F(1, 28) Å 9.01, cortisol, F (1, 28) Å 28.19, and ANF, F (1, 28) Å 31.02, concentrations were significantly (p õ 0.01) lower. No difference was observed in adrenaline and ET.
FIG. 1. Relationship between plasma b-endorphin and atrial natriuretic factor levels after mental arithmetic test during placebo in low responders (n Å 15) ( r Å 0.81, p õ 0.01).
FIG. 2. Relationship between plasma Met-enkephalin and noradrenaline levels after mental arithmetic test during placebo in high responders (n Å 15) (r Å 0.77, p õ 0.01).
Effects of MAT on Low and High Responders Under Placebo Infusion
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Significant positive correlations after MAT were found between BE and ANF values in low responders (Fig. 1) as well as between ME and noradrenaline in high responders (Fig. 2). In the latter group ME and systolic blood pressure (r Å 0.65, p õ 0.01), and noradrenaline and systolic blood pressure (r Å 0.62, p õ 0.01) were also correlated. No significant correlation was found among all the other paired parameters. Effects of MAT on Low and High Responders Under Naloxone Infusion Basal hemodynamic and hormonal parameters measured during naloxone infusion before MAT did not significantly differ from those previously measured during placebo before MAT in the same individuals of both groups (see legend of Fig. 3). During naloxone infusion in low responders, MAT significantly (p õ 0.01) increased SBP, F(7, 48) Å 67.25, DBP, F (7, 48) Å 38.69, HR, F (7, 48) Å 34.23, plasma adrenaline, F(7, 48) Å 5.18, noradrenaline, F(7, 48) Å 43.84, and cortisol, F (7, 48) Å 36.69, levels, but not ANF and ET when compared to basal values. Comparison with the effects during placebo indicates significant (p õ 0.01) increases in SBP, F (1, 1, 1, 52) Å 15.52, HR, F (1, 1, 1, 52) Å 6.91, plasma noradrenaline, F(1, 1, 1, 52) Å 6.18, cortisol, F(1, 1, 1, 52) Å 24.10, ET levels, F (1, 1, 1, 52) Å 6.14, and a significant (p õ 0.01) decrease in plasma ANF, F (1, 1, 1, 52) Å 7.04, concentration (Fig. 3). During naloxone infusion in high responders, MAT significantly (p õ 0.01) increased SBP, F(7, 48) Å 122.59, DBP, F (7, 48) Å 115.54, HR, F (7, 48) Å 15.52, plasma adrenaline, F(7, 48) Å 34.98, noradrenaline, F(7, 48) Å 138.41, levels, and significantly (p õ 0.01) decreased plasma ET, F (7, 48) Å 4.35, concentration, whereas no effect was observed for cortisol and ANF. Comparison with the effects during placebo indicates significant (p õ 0.01) decreases in SBP, F (1, 1, 1, 52) Å 15.52, DBP, F(1, 1, 1, 52) Å 27.35, HR, F(1, 1, 1, 52) Å 89.71, circulating levels of noradrenaline, F(1, 1, 1, 52) Å 95.42, and ET, F(1, 1, 1, 52) Å 7.76 (Fig. 3). Comparisons of the MAT responses between the two groups during naloxone infusion revealed that HR, F(1, 12) Å 32.25, values, plasma noradrenaline, F (1, 12) Å 22.09, cortisol, F (1, 12) Å 112.16, and ET, F (1, 12) Å 25.88, levels were significantly higher (p õ 0.01) in low responders, whereas no difference was observed in all the other parameters (Fig. 3). Finally, no relevant effects of gender on hormonal and hemodynamic parameters were observed during MAT with or without naloxone. DISCUSSION
This study showed that healthy subjects, selected according to the intensity of SBP response to MAT, displayed a different endocrine profile after testing. As far as opioid peptides are concerned, the subjects with a small percent increment in SBP produced by MAT (low responders) showed an increase in circulating levels of BE and no change in ME and DYN B. On the contrary, the subjects with a large percent increment in SBP after MAT (high responders) showed an increase in plasma ME and DYN B concentrations and no change on BE. Both groups showed an increase in plasma catecholamine levels that, only in low responders, was accompanied by an increase in plasma cortisol, ANF levels and a decrease in plasma ET concentration. As expected, plasma noradrenaline concentration as well as DBP and HR values after MAT were higher in high responders than in low responders. Evidence on the effects of emotional stressors on hemodynamic response (28–30,36) shows a surprisingly wide range
FIG. 3. Relative changes in hemodynamic and hormonal parameters after mental arithmetic test (MAT) during placebo (1.5 ml/min saline) and naloxone hydrochloride (9.5 mg/kg/min) infusion in low responders (n Å 7) (upper panel) and high responders (n Å 7) (lower panel), expressed as a percentage of the respective basal values. Basal values of these parameters before MAT during placebo and naloxone were: systolic blood pressure (SBP) 113.0 { 1.9 and 114.6 { 1.3 mmHg, diastolic blood pressure (DBP) 73.0 { 2.0 and 75.0 { 2.2 mmHg, heart rate (HR) 67.2 { 3.1 and 67.6 { 2.0 bpm, adrenaline (A) 233.6 { 26.1 and 195.9 { 27.2 fmol/ml, noradrenaline (NA) 958.2 { 29.5 and 937.4 { 33.6 fmol/ml, cortisol 144.0 { 8.2 and 136.8 { 8.2 pmol/ml, atrial natriuretic factor (ANF) 2.6 { 0.2 and 2.5 { 0.1 fmol/ml, endothelin-1 (ET) 2.8 { 0.3 and 2.8 { 0.2 fmol/ml in low responders; SBP 118.0 { 2.0 and 117.0 { 1.2 mmHg, DBP 82.4 { 1.8 and 80.0 { 0.8 mmHg, HR 67.4 { 1.4 and 66.6 { 1.4 bpm, A 198.6 { 10.9 and 193.2 { 20.7 fmol/ml, NA 889.6 { 28.3 and 864.7 { 29.7 fmol/ml, cortisol 119.7 { 9.1 and 132.9 { 21.7 pmol/ml, ANF 2.7 { 0.5 and 2.6 { 0.3 fmol/ml, ET 2.7 { 0.4 and 2.7 { 0.2 fmol/ml in high responders. Values are expressed as means { SEM. Duncan’s test: * p õ 0.05, †p õ 0.01 vs. MAT during placebo; ‡p õ 0.01 vs. high responders.
even in normal subjects (37). However, previous studies regarding measurements of plasma BE, catecholamine, and glucocorticoid levels after MAT (1,10,29) did not indicate a relationship between changes of these hormones and hemodynamic parameters, possibly because the hormonal pattern in response to psychological stress was not investigated in subjects selected for a distinct hemodynamic profile. In low responders, the increase in circulating levels of BE and cortisol suggests a predominant activation of the pituitary–adrenal cortical pathway. It is known that BE and adrenocorticotropin hormone, originating from the same pituitary precursor, are cosecreted from the anterior pituitary (12). On the other hand, in high responders the increase in plasma ME, DYN B, but not in BE and cortisol levels, associated with a larger increment in plasma noradrenaline concentration, suggests a prevalent activation of the sympathetic–adrenal medullary pathway, with a less important involvement of the pituitary–adrenal cortical axis. In agreement with these results it has been found that in animals restraint stress elevates plasma catecholamines and ME levels as well as both blood pressure and heart rate values (4). It is well known that the major stores of ME and DYN are sympathetic nerve endings, sympathetic ganglia, and, to a lesser extent, the adrenal medulla and numerous cell groups located in the central nervous system (22,25,34,36,41). Consequently, a strong increase in sympathetic activity can be expected to be associated with an increase in circulating levels of these peptides
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(18,19,39). Recent data suggest that an increase in brain opioids can stimulate the sympathetic outflow during stress (9,27). Moreover, it has been demonstrated in animals that ME reduces the vagal partecipation in the control of heart rate, inducing a shift in the autonomic balance in favor of sympathetic control (7). Therefore, in high responders a contribution of the MEinduced decrease in vagal tone to the observed hemodynamic changes during MAT cannot be ruled out. The blockade of opioid receptors by naloxone in low responders altered the MAT response by increasing SBP, HR, plasma noradrenaline, cortisol, and ET, and by decreasing plasma ANF levels. On the contrary, naloxone decreased BP, HR, plasma noradrenaline, and ET concentrations in high responders. The almost reciprocal pattern of hemodynamic changes induced by naloxone in the two groups confirms the complex modulatory role of endogenous opioid peptide system on cardiovascular functions. In low responders the naloxone-induced increase in noradrenaline, SBP, and HR in response to MAT reflects a suppression of the inhibitory effects of endogenous opioids on sympatho– adrenergic function. These data are consistent with inhibitory effects of endogenous opioids on noradrenaline release from sympathetic nerve endings (11) and with their central inhibitory inputs on the pressor area involved in regulating sympatho–adrenergic function (9). The further increase in plasma cortisol concentration after MAT with naloxone with respect to placebo may be due to the suppression of a hypothalamic inhibitory opioidergic effect on the release of corticotropin-releasing factor (6). The strong suppression by naloxone of the MAT-induced increase in plasma ANF levels suggests that BE may trigger ANF release during stress in these subjects. The direct correlation observed in these subjects after MAT between ANF and BE, but not ME and DYN B, supports this hypothesis. Previous evidence indicated that BE mediates ANF release during physical exercise in man (26). In high responders, the naloxone-induced attenuation of the increase in plasma noradrenaline levels and hemodynamic values in response to MAT is consistent with the hypothesis that ME and DYN B may stimulate sympatho–adrenergic activity and, in turn, affect BP response to MAT. This interpretation is supported by the significant direct correlation between ME and noradrenaline levels and between noradrenaline levels and SBP observed in these subjects after MAT. However, the effects of naloxone on the hemodynamic parameters during MAT may be attributable, at least in part, to a blockade of the inhibitory effects of ME on the vagal tone (7). Our findings are in agreement with recent studies in animals showing a distinct hemodynamic profile in response to
psychological stress ( 30 ) . The administration of an opioid antagonist to animals with low hemodynamic response to stress increases SBP reactivity, whereas the opioid antagonist reduces circulatory reactivity in animals showing excessive hemodynamic response ( 30 ) . The inhibitory action of the endogenous opioid system on the sympathetic nervous system after acute emotional stress has been reported in recent studies on young hypotensive subjects with low hemodynamic response to MAT (28,29). The stimulatory effects of opioid peptides on sympatho–adrenergic activity in response to MAT are compatible with functional studies showing that the role of the opioid peptides in the central control of BP depends not only on distinct inhibitory inputs to a pressor area, but also on inhibitory inputs to a depressor area (9) regulating sympatho–adrenergic activity. Biochemical and pharmacological evidence suggests the involvement of the central opioid system in the development of spontaneous and experimental hypertension in animals (13,42). Pressor effects of opioid peptides in man have been previously described in patients with cerebrovascular disease during an acute increase in BP in whom naloxone administration significantly decreased BP values (17). It is difficult to establish to what extent ET is involved in the hemodynamic response to MAT. ET is secreted from endothelial cells and the signals participating in its control are likely to come from the circulation (40) or from the autonomic nervous system (40). In low responders the decrease in circulating levels of ET may contribute to the attenuated pressor response to the test. The suppression of this effect by naloxone suggests that BE may reduce ET secretion during the enhanced cardiac function induced by acute stress. In high responders with elevated reactivity to MAT opioid peptide blockade decreased plasma ET concentration, suggesting stimulating effects of ME and/or DYN B. However, it cannot be excluded that other factors under opioid peptide control may be involved in the effect of naloxone on ET release. In conclusion, the present study indicates that individual variability of the hemodynamic and endocrine responses to MAT depend on a different activation of the endogenous opioid system. In subjects with moderate hemodynamic response to MAT the increase in circulating levels of BE may attenuate sympatho– adrenergic hyperactivity and enhance ANF secretion. In subjects with elevated hemodynamic reactivity to MAT the increase in circulating levels of ME and DYN B seems to enhance sympatho–adrenergic activity typical of this group of patients. Naloxone infusion reduced the differences in the cardiovascular and endocrine responses to MAT between low and high responders, supporting a bidirectional regulatory role for these peptides in adaptation to stress.
REFERENCES 1. al’Absi, M.; Lovallo, W. R.; McKey, B. S.; Pincomb, G. A. Borderline hypertensives produce exaggerated adrenocortical responses to mental stress. Psychosom. Med. 56:245–250; 1994. 2. Aloyo, V.; Mousa, S. A.; Van Loon, G.R. Stabilization of methionine-enkephalin in human and rat blood. Life Sci. 39:21 – 28; 1986. 3. Babson, A. L. The cirrus Immulite automated immunoassay system. J. Clin. Immunoassay 14:83–88; 1991. 4. Barron, B. A.; Van Loon, G. R. Role of sympathoadrenomedullary system in cardiovascular response to stress in rats. J. Autonom. Nerv. Syst. 28:179–188; 1989. 5. Bernardi, P.; Fontana, F.; Merlo Pich, E.; et al. Plasma endogenous opioid levels in acute myocardial infarction patients, with and without pain. Eur. Heart J. 13:1074–1079; 1992. 6. Buckingham, J. C.; Cooper, T. A. Influence of opioid substances on hypothalamo–pituitary–adrenocortical activity in the rat. In: Deli-
7. 8.
9.
10. 11.
tala, G.; Motta, M.; Serio, M., Eds. Opioid modulation of endocrine function. New York: Raven Press; 1984:81–87. Caffrey, J. L.; Mateo, Z.; Napier, L. D.; Gaugl, J. F.; Barron, B. A. Intrinsic cardiac enkephalins inhibit vagal bradycardia in the dog. Am. J. Physiol. Heart Circ. Physiol. 268(37):H848–H855; 1995. Cahill, C. A.; Matthews, J. D.; Akil, H. Human plasma b-endorphinlike peptides: A rapid, high recovery extraction technique and validation of radioimmunoassay. J. Clin. Endocrinol. Metab. 56:992– 997; 1983. Chalmers, J.; Pilowsky, P. Brainstem and bulbospinal neurotransmitter systems in the control of blood pressure. J. Hypertens. 9:675– 694; 1991. Dimsdale, J. E.; Moss, J. Plasma catecholamines in stress and exercise. JAMA 243:340–342; 1980. Dubocovich, M. L.; Langer, S. Z. Pharmacological differentiation of presynaptic inhibitory a-adrenoceptors and opiate recep-
/ 2x11 2315 Mp 174 Sunday Apr 06 09:19 PM EL–PEP (v.18#2) 2315
OPIOID SYSTEM AND MENTAL ARITHMETIC TEST
12.
13. 14. 15.
16.
17. 18. 19.
20. 21. 22. 23. 24. 25. 26.
tors in the cat nictitating membrane. Br. J. Pharmacol. 70:383 – 388; 1980. Farrell, P. A.; Gates, W. K.; Maksud, M. G.; Morgan, W. P. Increases in plasma b-endorphin/ b-lipotrophin immunoreactivity after treadmill running in humans. J. Appl. Physiol. 52:1245–1249; 1982. Feuerstein, G.; Siren, A. L. The opioid peptides: A role in hypertension? Hypertension 9:561–565; 1987. Fontana, F.; Bernardi, P.; Spagnolo, N.; Capelli, M. Plasma atrial natriuretic factor in low output heart failure syndromes. Klin. Wochenschr. 68:313–319; 1990. Fontana, F.; Bernardi, P.; Merlo Pich, E.; et al. Relationship between plasma atrial natriuretic factor and opioid peptide levels in healthy subjects and in patients with acute congestive heart failure. Eur. Heart J. 14:219–225; 1993. Grossi, G.; Bargossi, A. M.; Lucarelli, C.; Paradisi, R.; Sprovieri, C.; Sprovieri, G. Improvements in automated analysis of catecholamine and related metabolites in biological samples by column switching high-performamce liquid chromatography. J. Chromatogr. 541:273–284; 1991. Guardigli, G.; Fontana, F.; Ventura, C.; et al. Endogenous opioids and hypertensive crisis in patients with acute cerebrovascular accident. J. Endocrinol. Invest. 12(Suppl.4):9–12; 1989. Hanbauer, I.; Kelly, G. D.; Saiani, L.; Yang, H. Y. T. [Met 5]-enkephalin-like peptides of the adrenal medulla: release by nerve stimulation and functional implications. Peptides 3:469–473; 1982. Hexum, T. D.; Hanbauer, I.; Govoni, S.; Yang, H. Y. T.; Costa, E. Secretion of enkephalin-like peptides from canine adrenal gland following splanchnic nerve stimulation. Neuropeptides 1:137–142; 1980. Hirata, Y.; Itoh, K. I.; Ando, K.; Endo, M.; Marumo, F. Plasma endothelin levels during surgery. N. Engl. J. Med. 321:1686; 1989. Kapusta, D. R.; Jones, S. Y.; DiBona, G. F. Opioids in the systemic hemodynamic and renal responses to stress in conscious spontaneously hypertensive rats. Hypertension 13:808–816; 1989. Klein, R.; Wilson S.; Dzielak, D. Opioids and norepinephrine appear to be co-stored in large dense-core vescicles of sympathetic nerve. Fed. Proc. 41:1076; 1981. Kolta, M. G.; Pierzchala, K.; Houdi, A. A.; Van Loon, G. R. Effects of diabetes on the levels of two forms of Met-enkephalin in plasma and peripheral tissues of the rat. Neuropeptides 21:55–63; 1992. Leino, T.; Leppaluoto, J.; Huttunen, P.; Ruokonen, A.; Kuronen, P. Neuroendocrine responses to real and simulated BA Hawk MK 51 flight. Aviat. Space Environ. Med. 66:108–113; 1995. Livett, B. G.; Day, R.; Elde, R. P.; Howe, P. R. C. Co-storage of enkephalins and adrenaline in bovine adrenal medulla. Neuroscience 7:1323–1332; 1982. Louisy, F.; Guezennec, C. Y.; Lartigue, M.; Aldigier, J. C.; Galen, F. X. Influence of endogenous opioids on atrial natriuretic factor
175
27. 28. 29. 30.
31. 32. 33. 34. 35. 36. 37. 38.
39. 40. 41. 42.
release during exercise in man. Eur. J. Appl. Physiol. 59:34–38; 1989. Marson, L.; Kiritsy–Roy, J. A.; Van Loon, G. R. m-Opioid peptide modulation of cardiovascular and sympathoadrenal responses to stress. Am. J. Physiol. 257:R901–R908; 1989. McCubbin, J. A.; Surwit, R. S.; Williams, R. B. Endogenous opiate peptides, stress reactivity, and risk for hypertension. Hypertension 7:808–811; 1985. McCubbin, J. A.; Surwit, R. S.; Williams, R. B.; Nemeroff, C. B.; McNeilly, M. Altered pituitary hormone response to naloxone in hypertension development. Hypertension 14:636–644; 1989. McCubbin, J. A.; Kaplan, J. R.; Manuck, S. B.; Adams, M. R. Opioidergic inhibition of circulatory and endocrine stress responses in cynomolgus monkeys: A preliminary study. Psychosom. Med. 55:23–28; 1993. Miller, W. L.; Redfield, M. M.; Burnett, J. C., Jr. Integrated cardiac, renal, and endocrine actions of endothelin. J. Clin. Invest. 83:317– 320; 1989. Naranjo, J. R.; Urdin, M. C.; Borrell, J.; Fuentes, J. A. Evidence for a central but not adrenal, opioid mediation in hypertension induced by brief isolation. Life Sci. 38:1923– 1930; 1986. Roy, L. F.; Ogilva, R. I.; Larochelle, P.; Hamet, P.; Leene, F. H. H. Cardiac and vascular effects of atrial natriuretic factor and sodium nitroprusside in healthy men. Circulation 79:383–392; 1989. Spampinato, S.; Goldstein, A. Immunoreactive dynorphin in rat tissues and plasma. Neuropeptides 3:193–212; 1983. Spampinato, S.; Paradisi, R.; Canossa, M.; et al. Immunoreactive dynorphin A-like material in extracted human hypothalamic–hypophysial plasma. Life Sci. 52:223–230; 1993. Specchia, G.; Falcone, C.; Traversi, E.; et al. Mental stress as a provocative test in patients with various clinical syndromes of coronary heart disease. Circulation 83(Suppl II):II-108–114; 1991. Ushiyama, K.; Ogawa, T.; Ishii, M.; Ajisaka, R.; Sugishita, Y.; Ito, I. Physiologic neuroendocrine arousal by mental arithmetic stress test in healthy subjects. Am. J. Cardiol. 67:101–103; 1991. Venn, R. F. Combined high-performance liquid chromatographicradioimmunoassay method for the analysis of endorphins, enkephalins and other neurotransmitter peptides. J. Liq. Chromatogr. 423:93–104; 1987. Viveros, O.; Diliberto, E., Jr.; Hazum, E.; Chang, K. J. Opiate-like materials in the adrenal medulla: Evidence for storage and secretion with catecholamines. Mol. Pharmacol. 16:1101–1108; 1979. Yanagisawa, M.; Kurihara, H.; Kimura, S.; et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332:411–415; 1988. Yang, H. Y. T.; Hong, J. S.; Costa, E. Regional distribution of Leu- and Met-enkephalin in rat brain. Neuropharmacology 16:303–307; 1977. Zamir, N.; Simantov, R.; Segal, M. Pain sensitivity and opioid activity in genetically and experimetally hypertensive rats. Brain Res. 184:299–310; 1980.
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PII S0196-9781(96)00319-1
Opioid Peptide Modulation of Circulatory and Endocrine Response to Mental Stress in Humans FIORELLA FONTANA,* 1 PASQUALE BERNARDI,* EMILIO MERLO PICH,† STEFANO BOSCHI,‡ ROSARIA DE IASIO,‡ SANTI SPAMPINATO§ AND GABRIELE GROSSIH *Istituto di Patologia Speciale Medica e Metodologia Clinica, Ospedale S. Orsola, Via Massarenti 9, 40138 Bologna, Italy †Glaxo Institute for Molecular Biology, 14 Chemin des Aulx, 1228 Plan-les Ouates, Ginevra, Switzerland ‡Servizio di Farmacologia Clinica, Ospedale S. Orsola, Via Massarenti 9, 40138 Bologna, Italy §Dipartimento di Farmacologia, Via Irnerio 48, 40126 Bologna, Italy HLaboratorio Centralizzato, Ospedale S. Orsola, Via Massarenti 9, 40138 Bologna, Italy Received 10 May 1996; Accepted 22 October 1996 FONTANA, F., P. BERNARDI, E. MERLO PICH, S. BOSCHI, R. DE IASIO, S. SPAMPINATO AND G. GROSSI. Opioid peptide modulation of circulatory and endocrine response to mental stress in humans. PEPTIDES 18 ( 2 ) 169 – 175, 1997. — Healthy subjects were classified according to their percent increase in systolic blood pressure ( SBP ) after mental arithmetic test ( MAT ) as low ( DSBP 9.3 – 15.1%, n Å 15 ) and high ( DSBP 35.1 – 45.4%, n Å 15 ) responders. During MAT, low responders showed significantly ( p õ 0.01 ) increased plasma levels of b-endorphin, cortisol, catecholamines, and atrial natriuretic factor ( ANF ) and decreased levels of endothelin-1, whereas high responders showed increased ( p õ 0.01 ) levels of Metenkephalin, dynorphin B, and catecholamines. Pretreatment with naloxone hydrochloride enhanced ( p õ 0.01 ) SBP, heart rate, noradrenaline, cortisol, and endothelin-1 levels, and reduced ( p õ 0.01 ) ANF in low responders in response to MAT, whereas it decreased ( p õ 0.01 ) hemodynamic parameters, noradrenaline, and endothelin-1 in high responders. The individual differences in hemodynamic and endocrine responses to MAT may depend on a different activation of the endogenous opioid system. q 1997 Elsevier Science Inc. Mental arithmetic test Atrial natriuretic factor
b-Endorphin Dynorphin B Cortisol Endothelin-1
Met-enkephalin
SOME reports have demonstrated that different forms of stress activate the endogenous opioid peptide system, which, in turn, contributes to changes in systemic cardiovascular function and endocrine response occurring under these conditions ( 21,29,32 ) . Mental arithmetic test ( MAT ) is one of the most useful procedures for eliciting reliable stress-induced cardiovascular responses in humans ( 36 ) . Several studies have examined the effects of this psychological stressor on systemic and coronary hemodynamics ( 28 – 30,36 ) , whereas relatively few studies have investigated the concomitant effects on the neuroendocrine system and, in particular, the role of the endogenous opioid peptide system ( 28,30,36 ) . In the only available investigation on an opioid peptide participation in the cardiovascular response to MAT, an opioid peptide antagonist ( naloxone ) produced a significant blood pressure ( BP ) increase in hypotensive subjects who responded moderately to MAT ( 28 ) . Interestingly, when subjects with borderline hypertension were tested, naloxone did not affect their exaggerated cardiovascular response to MAT ( 28 ) . It is still unknown
1
Naloxone
Catecholamines
whether the individual differences in the magnitude of circulatory responses to psychological stress in normotensive subjects are related to a different activation of the opioid peptide system ( 37 ) . The first aim of the present study was to characterize the profile of circulating levels of the three main opioid peptides [i.e. b-endorphin (BE), Met-enkephalin (ME), and dynorphin B (DYN B)] after MAT in normal healthy volunteers divided into two groups (low responders and high responders) according to their systolic blood pressure (SBP) increase in response to MAT. The second aim was to study the functional relationships between endogenous opioid peptide, sympathetic–adrenal medullary and pituitary–adrenal cortical systems after MAT, by evaluating plasma catecholamine and cortisol concentrations after MAT with and without pretreatment with naloxone hydrochloride (NARCAN). In addition, we measured plasma concentrations of atrial natriuretic factor (ANF) and endothelin-1 (ET), two other hormones mainly released in response to acute stress (20,24) and involved in cardiovascular control (31,33).
Requests for reprints should be addressed to Dr. Fiorella Fontana.
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Subjects Thirty subjects (12 females and 18 males, mean age 29.2 { 0.7 years) were selected out of a population of 67 normotensive subjects with a high education and normal verbal ability. These subjects, selected among young physicians and medical students at our University, were tested with MAT. In this population the percent increments in SBP in response to MAT varied between 9.3% and 45.4% (mean { SD Å 25.1 { 10.0%) with respect to basal, pretest values. We selected two groups using a boundary defined by the SD: low responders and high responders. Low responders (n Å 15, 6 females and 9 males, mean age 29.6 { 1.0 years) were defined as those individuals whose increase in SBP to MAT was less than 15.1% (population mean 0 1 SD), whereas high responders (n Å 15, 6 females and 9 males, mean age 28.7 { 1.2 years) were those individuals whose increase in SBP was more than 35.1% (population mean / 1 SD). Subjects with intermediate increases (15.2–35.1%) in SBP in response to MAT were excluded from this study. All selected subjects had resting SBP values õ 130 mmHg and diastolic blood pressure (DBP) values õ 85 mmHg. BP was recorded using a fully automatic recorder set to take a measurement every 30 min throughout 24 h. The BP values of both parents of each subjects were also measured, and subjects were excluded if one of the parents was found to be hypertensive. In all subjects physical examination, chest radiogram, electrocardiogram, echocardiogram, and biochemical findings were normal. They were not taking any medication. All ate a 3–4 g/day sodium diet for 4–5 days before the study and for 24 h they did not consume tea, coffee, or cigarettes. None of the subjects was dependent on any drugs. Written informed consent was obtained from all subjects, and the protocol was approved by the Reaserch Committee of S. Orsola Hospital. Experimental Procedure The subjects were studied in the morning (between 0800 and 0900 h) after an overnight fast. MAT was performed by the subjects in supine position during placebo infusion (1.5 ml/min saline) following a 30-min period of rest. MAT consisted of asking the subjects to subtract the number 17 serially from 1,013 until 10 was reached and then repeat the procedure again for 3 min. They were urged to perform as quickly and accurately as possible, and reproached for lack of effort if the answer was not correct. Periodically, the subjects were urged to improve his/her performance. A week later 14 of these subjects, 7 low responders (3 females and 4 males, mean age 29.1 { 1.5 years) and 7 high responders (3 females and 4 males, mean age 28.8 { 1.6 years), underwent a second MAT during naloxone infusion (9.5 mg/kg/min). The dose of naloxone was chosen on the basis of a previous study on patients with congestive heart failure in whom 8 mg of naloxone produced maximal inhibition of hormonal and hemodynamic opioid peptide effects in the absence of any side effect (15). A catheter was inserted in the left antecubital vein; after a rest period, placebo or naloxone was infused 10 min before starting and during the MAT. In all subjects electrocardiogram leads were placed on the chest and a sphygmomanometer cuff was placed on the right arm. Blood samples for the determination of catecholamines, cortisol, ANF, and ET were taken through a needle placed in the left forearm vein before and immediately after MAT during placebo and naloxone infusion; BE, ME, and DYN B were assessed only before and after MAT with placebo.
Heart rate (HR) and cardiac rhythm were monitored continuously by electrocardiography, and BP determinations were made at 1-min intervals. Electrocardiogram was recorded by a Cardiostat 3 Siemens electrocardiograph; BP was measured by an automatic Hewlett–Packard 78354A sphygmomanometer. BP throughout 24 h was recorded using a fully automatic recorder (Profilomat; Disetronic Medical Systems, Burgdorf, Switzerland). Hormone Assay Determination of peptide hormones: radioimmunoassay. Plasma levels of immunoreactive b-endorphin-like material (IRBE), IR-Met-enkephalin (IR-ME), and IR-dynorphin B (IRDYN B) were evaluated by radioimmunoassay after extraction on Sep-Pak C18 cartridges (Water Associates). IR-BE was extracted from 10 ml of blood adopting a method described previously (8). Blood was collected into chilled tubes containing ethylendiaminetetraacetic acid (EDTA; 15% w/v) and plasma separated from the blood cells by centrifugation (3000 rpm/15 min, 47C). The plasma was collected, 1 N HCl (100 ml/ml) added, fibrin removed by centrifugation ( 12,000 rpm/ 10 min, 47C ) , and IR-BE was extracted on activated Sep-Pak cartridges. The samples were lyophilized, dissolved in radioimmunoassay buffer, and evaluated according to the protocol suggested by Amersham ( Little Chalfan, England ) using ( 3[ 125I ] iodotyrosyl 27 ) BE ( human ) as tracer (obtained from Amersham). The antiserum was purchased from Amersham (code N0. 1621); it was raised in rabbits against porcine BE and it completely cross-reacts on a molar basis with human b-lipotropin. This antiserum gave no cross-reactivity with ACTH, aMSH, Met- and Leu-enkephalin, and DYN-related peptides. To validate the plasma extraction procedure, the recovery of radiolabeled BE added directly to blood samples was determined and found to be between 84% and 86%. Reverse-phase high performance liquid chromatography (RP-HPLC) analysis of the recovered radioactivity, carried out as later described, did not display any significant degradation of the radiolabeled peptide (data not shown). Plasma extraction of IR-DYN B was carried out as described (35). Blood (10 ml) was collected into chilled tubes containing appropriate enzyme inhibitors and 7.3 mmol EDTA/ml. Samples were placed on ice, centrifuged (3000 rpm/15 min, 47C), and processed as described previously (35) to extract IR-DYN A using Sep-Pak cartridges. IR-DYN B was assayed using a commercially available antiserum (Peninsula Laboratories, Belmont, CA), which cross-reacts with DYN A(1–32) (70%; this peptide contains DYN B in its sequence), DYN B(1–29) (30%) whereas the cross-reactivity with DYN A(1–17), DYN A(1– 8), and Leu-enkephalin was less than 1%. The tracer, [ 125I]DYN B, was purchased from Peninsula Laboratories and radioimmunoassay procedure was carried out as described previously (35). Blood samples (10 ml) for IR-ME analysis were collected into cold tubes containing heparin (14 units/ml) and a mixture of ice-cold aprotinin (5%) and 17% citric acid (20 ml/ml) (2); then IR-ME was extracted (23) and assayed by radioimmunoassay procedure using a commercially available antiserum (Peninsula Laboratories) and [ 125I][Tyr 1-Met(O)]enkephalin (DuPont NEN, Cologno Monzese, Italy). The ME antiserum cross-reacts equally with ME-sulphoxide, to a small extent (3%) with Leu-enkephalin, and only minimally ( õ1%) with ME-ArgPhe or BE. Radioimmunoassay procedure was carried out according to the manufacturer’s directions (Peninsula Laboratories). Recovery of radiolabeled DYN B and ME directly added to blood samples was between 78% and 81%. RP-HPLC analysis
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OPIOID SYSTEM AND MENTAL ARITHMETIC TEST (carried out as later described) of the recovered radioactivity confirmed that both peptides were not degraded during the extraction procedure. In the radioimmunoassay procedure inhibition of tracer binding by 15% was considered the detection limit, and for the serially diluted extracts each tracer followed curves parallel to the standard curve. Intra- and interassay variations were less than 5%. Antibody dilution was chosen for 30% binding of counts added, and the limits of detection were: IR-DYN B 10 fmol/ml [mean { SD inhibitory concentration (IC50 ) 113 { 18 fmol/ml]; IR-BE 3 fmol/ml (IC50 15 { 6 fmol/ml); and IR-ME 12 fmol/ml (IC50 33 { 16 fmol/ml). ANF was determined, after chromatographic preextraction, by a radioimmunoassay procedure according to a method fully detailed previously (14). The detection limit for ANF was 0.6 fmol/ml. The intra-assay variation was 5.2% and the interassay variation 11.8%. The recovery of synthetic a-human-ANF added to blood samples was 85 { 2% with a coefficient of variation of 18%. Blood samples for ET determination were placed in cold tubes containing EDTA (1 mg/ml) and aprotinin (500 KIU/ml). Blood was centrifuged at 3000 rpm for 15 min at 07C. Plasma was stored at 0807C until assay. Plasma acidified (2 ml) with 0.1% trifluoracetic acid (TFA; 2 ml) and centrifuged at 3000 rpm for 15 min at 07C was applied to 200 mg C18 Sep column (Peninsula Laboratories Inc., Belmont, CA) preactivated with 4 ml of 0.1% TFA and 20 ml of 60% acetonitrile in 0.1% of TFA. The column was then washed with 20 ml 0.1% TFA. ET-like material was eluted with 3 ml of 60% acetonitrile in 0.1% TFA. The eluant was dried under nitrogen in a water bath at 407C and reconstituted in buffer. ET was then measured with the Peninsula radioimmunoassay kit. The recovery [ 125I]ET added directly to plasma samples was 81 { 3%. The effective range of the standard curve was between 0.4 and 51.3 fmol of ET per assay tube; the ED50 was 4.4 fmol of ET per assay tube. The intra-assay variation was 7.2% and the interassay variation 11.2%. Determination of peptide hormones: high performance liquid chromatography. Validation of the authenticity of IR-BE, IRDYN B, IR-ME, IR-ANF, and IR-ET in plasma extracts was obtained by RP-HPLC. For BE assay, a procedure described previously (38) was adopted. A mBondapak column (3.9 1 300 mm) was used. Elution was carried out with 0.225% TFA in water (solvent A) and 0.225% TFA in acetonitrile (solvent B); starting composition: 35% B; gradient linear to 40% B over 7.5 min. The flow rate was 1 ml/min. Fractions were collected for radioimmunoassay procedure every 30 s (0.5 ml), lyophilized, and redissolved in radioimmunoassay buffer. Two major peaks of immunoreactivity were found. These showed elution patterns identical to those of standard human BE and porcine b-lipotrophin (obtained from Peninsula Laboratories). Radiolabelled BE eluted after cold reference BE (data not shown). For IR-DYN B assay, the procedure described previously (35) was adopted. Pooled blood samples (20 ml obtained from healthy volunteers) were processed as described previously (35) and lyophilized. Samples were reconstituted in 350 ml of 5 mM TFA, injected on to a mBondapak C18 column, and eluted with a linear gradient of 19–65% acetonitrile in 5 mM TFA (60 min, 1.5 ml/min, 0.9 min/fraction). Fractions were collected and lyophilized for radioimmunoassay procedure. Two immunoreactive peaks were detected. One component had the same retention time as authentic DYN B whereas a second component showed the same retention time as DYN A(1–32) (data not shown). For ME assay, a procedure described previously was adopted (23) and a mBondapak C18 column was used. The flow rate of
171 the mobile phase (22% acetonitrile in 0.1% TFA) was 1.2 ml/ min. Fractions (1.2 ml each) were collected, lyophilized, redissolved in radioimmunoassay buffer, and assayed as previously described. Pooled blood samples (20 ml; obtained from healthy volunteers) were processed as previously reported, lyophilized, and reconstituted with 350 ml of mobile phase before being injected on to the column. Most of IR-ME eluted in the same position of standard ME (obtained from Peninsula Laboratories) and ME-sulphoxide (obtained from Sigma) (23) (data not shown). For the ANF assay, plasma extracts from Bond Elut columns were injected into the cromatograph equipped with mBondapack C18 column. Elution was carried out using a gradient of 0.1% TFA in CH3CN (solvent A) and 0.1% TFA in water (solvent B) as follows: starting 5% A/B for 5 min, linear gradient to 50% A/B from 5 to 30 min, then to 90% from 30 to 40 min. Flow rate was 1 ml/min. For the ET assay, the conditions were similar to that described for ANF except for the gradient shape, which was from 10% to 60% A/B in 60 min. Plasma chromatographed for ANF and ET each provided a major immunoreactive peak, which was shown to be identical to the standard synthetic peptide (data not shown). Determination of catecholamines and cortisol. Noradrenaline and adrenaline were determined by column switching high performance liquid chromatography with electrochemical detection as detailed previously (16). The detection limits for noradrenaline and adrenaline were 11.8 and 16.3 fmol/ml, respectively. The intra-assay variations of noradrenaline and adrenaline were 3.0% and 4.2%, respectively, and the interassay variations were 5.1% and 3.5%. The recovery of radiolabeled noradrenaline and adrenaline added to blood samples was above 90%. The quantitative measurement of cortisol was determined by a chemiluminescent immunoassay using the Immulite cortisol procedure (3). Immulite combines simple, well-established solid phase technology, 1/4 polystyrene beads, with an efficient and easily automated separation scheme. The solid phase is coated with a polyclonal rabbit antibody specific for cortisol. The patient samples, placed in cold tubes containing EDTA (1 mg/ml) and aprotinin (500 KIU/ml), were introduced into the test unit together with alkaline phosphatase-conjugated cortisol and incubated for approximately 30 min at 377C. After removal of the unbound enzyme conjugate by centrifugation, substrate was added and the test unit was incubated for a further 10 min. The detection limit of the assay was 0.5 nmol/dl. The intra-assay variation was 6.8% and the interassay variation 9.9%. The spiking recovery of cortisol added to blood samples was 92 { 3% with a coefficient of variation of 12%. Statistical Analysis Two-way analysis of variance (ANOVA) was used to compare hormonal and hemodynamic values before and after MAT with placebo in the two groups of subjects. Three-way ANOVA with one between factor (naloxone or placebo treatment) and two within factors (groups of subjects, and before and after MAT) was used to compare hormonal and hemodynamic values in the subjects who underwent the MAT during naloxone infusion. Individual means were then compared using post hoc Duncan’s test. All analyses were performed on original data, expressed in the figure as percent of basal values. The associations between opioid peptides and ANF vs. all other parameters as well as the association between catecholamines, ET, or cortisol and hemodynamic parameters were analyzed by Pearson’s r correlation coefficient and regression analysis. Values are expressed as mean { SEM, and p õ 0.05 was considered statistically significant.
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FONTANA ET AL. TABLE 1 HEMODYNAMIC AND HORMONAL PARAMETERS BEFORE (A) AND AFTER (B) MAT DURING PLACEBO INFUSION (1.5 ml/min SALINE) IN LOW AND HIGH RESPONDERS High Responders
Low Responders A
SBP (mmHg) DBP (mmHg) HR (bpm) BE (fmol/ml) ME (fmol/ml) DYN B (fmol/ml) A (fmol/ml) NA (fmol/ml) Cortisol (pmol/ml) ANF (fmol/ml) ET (fmol/ml)
116.8 { 76.8 { 68.2 { 8.1 { 9.0 { 21.4 { 221.6 { 936.3 { 146.0 { 3.0 { 2.8 {
B
1.6 1.1 1.4 0.3 0.7 1.0 19.1 30.1 10.4 0.1 0.1
130.0 { 91.0 { 84.4 { 14.6 { 9.4 { 24.3 { 332.9 { 1481.3 { 226.2 { 5.6 { 1.9 {
A
1.7* 1.0* 1.8* 1.5* 0.5 2.8 26.2* 69.1* 13.5* 0.3* 0.2*
115.3 { 79.8 { 67.6 { 8.9 { 9.0 { 20.8 { 222.1 { 929.8 { 132.4 { 3.0 { 2.4 {
B
1.5 1.5 1.1 0.2 0.3 1.4 15.8 26.6 10.7 0.2 0.1
160.8 { 98.4 { 92.0 { 10.4 { 20.7 { 35.5 { 337.8 { 1893.3 { 136.8 { 3.2 { 2.2 {
1.7*† 1.5*† 1.8*† 0.5† 1.1*† 1.2*† 21.8* 52.0*† 9.9† 0.2† 0.1
Values are expressed as means { SEM. Low responders (n Å 15), subjects with 9.3–15.1% systolic blood pressure increase in response to MAT; high responders (n Å 15), subjects with 35.1–45.4% systolic blood pressure increase in response to MAT. MAT, mental arithmetic test; SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate; BE, b-endorphin; ME, Met-enkephalin; DYN B, dynorphin B; A, adrenaline; NA, noradrenaline; ANF, atrial natriuretic factor; ET, endothelin. * p õ 0.01 vs. respective value before MAT; † p õ 0.01 vs. respective value of low responders. RESULTS
Table 1 summarizes hemodynamic and hormonal results obtained in the two groups of subjects in response to MAT during placebo infusion. Under basal, pretest conditions low responders and high responders did not significantly differ in any of the measured variables. In low responders MAT significantly (p õ 0.01) increased SBP, F (3, 56) Å 170.07, DBP, F (3, 56) Å 56.23, HR, F (3, 56) Å 59.31, plasma BE, F(3, 56) Å 22.01, adrenaline, F (3, 56) Å 9.54, noradrenaline, F(3, 56) Å 91.54, cortisol, F (3, 56) Å 14.48, and ANF, F(3, 56) Å 22.98, levels and significantly (p
õ 0.01) decreased plasma ET, F (3, 56) Å 4.53, concentration. In high responders MAT significantly (p õ 0.01) increased SBP, F(3, 56) Å 29.80, DBP, F (3, 56) Å 14.57, HR, F (3, 56) Å 14.42, plasma ME, F (3, 56) Å 29.96, DYN B, F (3, 56) Å 18.95, adrenaline, F(3, 56) Å 15.21, and noradrenaline, F(3, 56) Å 13.17, levels. SBP, F (1, 28) Å 151.21, DBP, F (1, 28) Å 16.21, HR, F (1, 28) Å 8.53, plasma ME, F (1, 28) Å 89.01, DYN B, F(1, 28) Å 89.16, and noradrenaline, F (1, 28) Å 22.66, concentrations were significantly (p õ 0.01) higher in high responders after MAT than the respective values of low responders, whereas plasma BE, F(1, 28) Å 9.01, cortisol, F (1, 28) Å 28.19, and ANF, F (1, 28) Å 31.02, concentrations were significantly (p õ 0.01) lower. No difference was observed in adrenaline and ET.
FIG. 1. Relationship between plasma b-endorphin and atrial natriuretic factor levels after mental arithmetic test during placebo in low responders (n Å 15) ( r Å 0.81, p õ 0.01).
FIG. 2. Relationship between plasma Met-enkephalin and noradrenaline levels after mental arithmetic test during placebo in high responders (n Å 15) (r Å 0.77, p õ 0.01).
Effects of MAT on Low and High Responders Under Placebo Infusion
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Significant positive correlations after MAT were found between BE and ANF values in low responders (Fig. 1) as well as between ME and noradrenaline in high responders (Fig. 2). In the latter group ME and systolic blood pressure (r Å 0.65, p õ 0.01), and noradrenaline and systolic blood pressure (r Å 0.62, p õ 0.01) were also correlated. No significant correlation was found among all the other paired parameters. Effects of MAT on Low and High Responders Under Naloxone Infusion Basal hemodynamic and hormonal parameters measured during naloxone infusion before MAT did not significantly differ from those previously measured during placebo before MAT in the same individuals of both groups (see legend of Fig. 3). During naloxone infusion in low responders, MAT significantly (p õ 0.01) increased SBP, F(7, 48) Å 67.25, DBP, F (7, 48) Å 38.69, HR, F (7, 48) Å 34.23, plasma adrenaline, F(7, 48) Å 5.18, noradrenaline, F(7, 48) Å 43.84, and cortisol, F (7, 48) Å 36.69, levels, but not ANF and ET when compared to basal values. Comparison with the effects during placebo indicates significant (p õ 0.01) increases in SBP, F (1, 1, 1, 52) Å 15.52, HR, F (1, 1, 1, 52) Å 6.91, plasma noradrenaline, F(1, 1, 1, 52) Å 6.18, cortisol, F(1, 1, 1, 52) Å 24.10, ET levels, F (1, 1, 1, 52) Å 6.14, and a significant (p õ 0.01) decrease in plasma ANF, F (1, 1, 1, 52) Å 7.04, concentration (Fig. 3). During naloxone infusion in high responders, MAT significantly (p õ 0.01) increased SBP, F(7, 48) Å 122.59, DBP, F (7, 48) Å 115.54, HR, F (7, 48) Å 15.52, plasma adrenaline, F(7, 48) Å 34.98, noradrenaline, F(7, 48) Å 138.41, levels, and significantly (p õ 0.01) decreased plasma ET, F (7, 48) Å 4.35, concentration, whereas no effect was observed for cortisol and ANF. Comparison with the effects during placebo indicates significant (p õ 0.01) decreases in SBP, F (1, 1, 1, 52) Å 15.52, DBP, F(1, 1, 1, 52) Å 27.35, HR, F(1, 1, 1, 52) Å 89.71, circulating levels of noradrenaline, F(1, 1, 1, 52) Å 95.42, and ET, F(1, 1, 1, 52) Å 7.76 (Fig. 3). Comparisons of the MAT responses between the two groups during naloxone infusion revealed that HR, F(1, 12) Å 32.25, values, plasma noradrenaline, F (1, 12) Å 22.09, cortisol, F (1, 12) Å 112.16, and ET, F (1, 12) Å 25.88, levels were significantly higher (p õ 0.01) in low responders, whereas no difference was observed in all the other parameters (Fig. 3). Finally, no relevant effects of gender on hormonal and hemodynamic parameters were observed during MAT with or without naloxone. DISCUSSION
This study showed that healthy subjects, selected according to the intensity of SBP response to MAT, displayed a different endocrine profile after testing. As far as opioid peptides are concerned, the subjects with a small percent increment in SBP produced by MAT (low responders) showed an increase in circulating levels of BE and no change in ME and DYN B. On the contrary, the subjects with a large percent increment in SBP after MAT (high responders) showed an increase in plasma ME and DYN B concentrations and no change on BE. Both groups showed an increase in plasma catecholamine levels that, only in low responders, was accompanied by an increase in plasma cortisol, ANF levels and a decrease in plasma ET concentration. As expected, plasma noradrenaline concentration as well as DBP and HR values after MAT were higher in high responders than in low responders. Evidence on the effects of emotional stressors on hemodynamic response (28–30,36) shows a surprisingly wide range
FIG. 3. Relative changes in hemodynamic and hormonal parameters after mental arithmetic test (MAT) during placebo (1.5 ml/min saline) and naloxone hydrochloride (9.5 mg/kg/min) infusion in low responders (n Å 7) (upper panel) and high responders (n Å 7) (lower panel), expressed as a percentage of the respective basal values. Basal values of these parameters before MAT during placebo and naloxone were: systolic blood pressure (SBP) 113.0 { 1.9 and 114.6 { 1.3 mmHg, diastolic blood pressure (DBP) 73.0 { 2.0 and 75.0 { 2.2 mmHg, heart rate (HR) 67.2 { 3.1 and 67.6 { 2.0 bpm, adrenaline (A) 233.6 { 26.1 and 195.9 { 27.2 fmol/ml, noradrenaline (NA) 958.2 { 29.5 and 937.4 { 33.6 fmol/ml, cortisol 144.0 { 8.2 and 136.8 { 8.2 pmol/ml, atrial natriuretic factor (ANF) 2.6 { 0.2 and 2.5 { 0.1 fmol/ml, endothelin-1 (ET) 2.8 { 0.3 and 2.8 { 0.2 fmol/ml in low responders; SBP 118.0 { 2.0 and 117.0 { 1.2 mmHg, DBP 82.4 { 1.8 and 80.0 { 0.8 mmHg, HR 67.4 { 1.4 and 66.6 { 1.4 bpm, A 198.6 { 10.9 and 193.2 { 20.7 fmol/ml, NA 889.6 { 28.3 and 864.7 { 29.7 fmol/ml, cortisol 119.7 { 9.1 and 132.9 { 21.7 pmol/ml, ANF 2.7 { 0.5 and 2.6 { 0.3 fmol/ml, ET 2.7 { 0.4 and 2.7 { 0.2 fmol/ml in high responders. Values are expressed as means { SEM. Duncan’s test: * p õ 0.05, †p õ 0.01 vs. MAT during placebo; ‡p õ 0.01 vs. high responders.
even in normal subjects (37). However, previous studies regarding measurements of plasma BE, catecholamine, and glucocorticoid levels after MAT (1,10,29) did not indicate a relationship between changes of these hormones and hemodynamic parameters, possibly because the hormonal pattern in response to psychological stress was not investigated in subjects selected for a distinct hemodynamic profile. In low responders, the increase in circulating levels of BE and cortisol suggests a predominant activation of the pituitary–adrenal cortical pathway. It is known that BE and adrenocorticotropin hormone, originating from the same pituitary precursor, are cosecreted from the anterior pituitary (12). On the other hand, in high responders the increase in plasma ME, DYN B, but not in BE and cortisol levels, associated with a larger increment in plasma noradrenaline concentration, suggests a prevalent activation of the sympathetic–adrenal medullary pathway, with a less important involvement of the pituitary–adrenal cortical axis. In agreement with these results it has been found that in animals restraint stress elevates plasma catecholamines and ME levels as well as both blood pressure and heart rate values (4). It is well known that the major stores of ME and DYN are sympathetic nerve endings, sympathetic ganglia, and, to a lesser extent, the adrenal medulla and numerous cell groups located in the central nervous system (22,25,34,36,41). Consequently, a strong increase in sympathetic activity can be expected to be associated with an increase in circulating levels of these peptides
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(18,19,39). Recent data suggest that an increase in brain opioids can stimulate the sympathetic outflow during stress (9,27). Moreover, it has been demonstrated in animals that ME reduces the vagal partecipation in the control of heart rate, inducing a shift in the autonomic balance in favor of sympathetic control (7). Therefore, in high responders a contribution of the MEinduced decrease in vagal tone to the observed hemodynamic changes during MAT cannot be ruled out. The blockade of opioid receptors by naloxone in low responders altered the MAT response by increasing SBP, HR, plasma noradrenaline, cortisol, and ET, and by decreasing plasma ANF levels. On the contrary, naloxone decreased BP, HR, plasma noradrenaline, and ET concentrations in high responders. The almost reciprocal pattern of hemodynamic changes induced by naloxone in the two groups confirms the complex modulatory role of endogenous opioid peptide system on cardiovascular functions. In low responders the naloxone-induced increase in noradrenaline, SBP, and HR in response to MAT reflects a suppression of the inhibitory effects of endogenous opioids on sympatho– adrenergic function. These data are consistent with inhibitory effects of endogenous opioids on noradrenaline release from sympathetic nerve endings (11) and with their central inhibitory inputs on the pressor area involved in regulating sympatho–adrenergic function (9). The further increase in plasma cortisol concentration after MAT with naloxone with respect to placebo may be due to the suppression of a hypothalamic inhibitory opioidergic effect on the release of corticotropin-releasing factor (6). The strong suppression by naloxone of the MAT-induced increase in plasma ANF levels suggests that BE may trigger ANF release during stress in these subjects. The direct correlation observed in these subjects after MAT between ANF and BE, but not ME and DYN B, supports this hypothesis. Previous evidence indicated that BE mediates ANF release during physical exercise in man (26). In high responders, the naloxone-induced attenuation of the increase in plasma noradrenaline levels and hemodynamic values in response to MAT is consistent with the hypothesis that ME and DYN B may stimulate sympatho–adrenergic activity and, in turn, affect BP response to MAT. This interpretation is supported by the significant direct correlation between ME and noradrenaline levels and between noradrenaline levels and SBP observed in these subjects after MAT. However, the effects of naloxone on the hemodynamic parameters during MAT may be attributable, at least in part, to a blockade of the inhibitory effects of ME on the vagal tone (7). Our findings are in agreement with recent studies in animals showing a distinct hemodynamic profile in response to
psychological stress ( 30 ) . The administration of an opioid antagonist to animals with low hemodynamic response to stress increases SBP reactivity, whereas the opioid antagonist reduces circulatory reactivity in animals showing excessive hemodynamic response ( 30 ) . The inhibitory action of the endogenous opioid system on the sympathetic nervous system after acute emotional stress has been reported in recent studies on young hypotensive subjects with low hemodynamic response to MAT (28,29). The stimulatory effects of opioid peptides on sympatho–adrenergic activity in response to MAT are compatible with functional studies showing that the role of the opioid peptides in the central control of BP depends not only on distinct inhibitory inputs to a pressor area, but also on inhibitory inputs to a depressor area (9) regulating sympatho–adrenergic activity. Biochemical and pharmacological evidence suggests the involvement of the central opioid system in the development of spontaneous and experimental hypertension in animals (13,42). Pressor effects of opioid peptides in man have been previously described in patients with cerebrovascular disease during an acute increase in BP in whom naloxone administration significantly decreased BP values (17). It is difficult to establish to what extent ET is involved in the hemodynamic response to MAT. ET is secreted from endothelial cells and the signals participating in its control are likely to come from the circulation (40) or from the autonomic nervous system (40). In low responders the decrease in circulating levels of ET may contribute to the attenuated pressor response to the test. The suppression of this effect by naloxone suggests that BE may reduce ET secretion during the enhanced cardiac function induced by acute stress. In high responders with elevated reactivity to MAT opioid peptide blockade decreased plasma ET concentration, suggesting stimulating effects of ME and/or DYN B. However, it cannot be excluded that other factors under opioid peptide control may be involved in the effect of naloxone on ET release. In conclusion, the present study indicates that individual variability of the hemodynamic and endocrine responses to MAT depend on a different activation of the endogenous opioid system. In subjects with moderate hemodynamic response to MAT the increase in circulating levels of BE may attenuate sympatho– adrenergic hyperactivity and enhance ANF secretion. In subjects with elevated hemodynamic reactivity to MAT the increase in circulating levels of ME and DYN B seems to enhance sympatho–adrenergic activity typical of this group of patients. Naloxone infusion reduced the differences in the cardiovascular and endocrine responses to MAT between low and high responders, supporting a bidirectional regulatory role for these peptides in adaptation to stress.
REFERENCES 1. al’Absi, M.; Lovallo, W. R.; McKey, B. S.; Pincomb, G. A. Borderline hypertensives produce exaggerated adrenocortical responses to mental stress. Psychosom. Med. 56:245–250; 1994. 2. Aloyo, V.; Mousa, S. A.; Van Loon, G.R. Stabilization of methionine-enkephalin in human and rat blood. Life Sci. 39:21 – 28; 1986. 3. Babson, A. L. The cirrus Immulite automated immunoassay system. J. Clin. Immunoassay 14:83–88; 1991. 4. Barron, B. A.; Van Loon, G. R. Role of sympathoadrenomedullary system in cardiovascular response to stress in rats. J. Autonom. Nerv. Syst. 28:179–188; 1989. 5. Bernardi, P.; Fontana, F.; Merlo Pich, E.; et al. Plasma endogenous opioid levels in acute myocardial infarction patients, with and without pain. Eur. Heart J. 13:1074–1079; 1992. 6. Buckingham, J. C.; Cooper, T. A. Influence of opioid substances on hypothalamo–pituitary–adrenocortical activity in the rat. In: Deli-
7. 8.
9.
10. 11.
tala, G.; Motta, M.; Serio, M., Eds. Opioid modulation of endocrine function. New York: Raven Press; 1984:81–87. Caffrey, J. L.; Mateo, Z.; Napier, L. D.; Gaugl, J. F.; Barron, B. A. Intrinsic cardiac enkephalins inhibit vagal bradycardia in the dog. Am. J. Physiol. Heart Circ. Physiol. 268(37):H848–H855; 1995. Cahill, C. A.; Matthews, J. D.; Akil, H. Human plasma b-endorphinlike peptides: A rapid, high recovery extraction technique and validation of radioimmunoassay. J. Clin. Endocrinol. Metab. 56:992– 997; 1983. Chalmers, J.; Pilowsky, P. Brainstem and bulbospinal neurotransmitter systems in the control of blood pressure. J. Hypertens. 9:675– 694; 1991. Dimsdale, J. E.; Moss, J. Plasma catecholamines in stress and exercise. JAMA 243:340–342; 1980. Dubocovich, M. L.; Langer, S. Z. Pharmacological differentiation of presynaptic inhibitory a-adrenoceptors and opiate recep-
/ 2x11 2315 Mp 174 Sunday Apr 06 09:19 PM EL–PEP (v.18#2) 2315
OPIOID SYSTEM AND MENTAL ARITHMETIC TEST
12.
13. 14. 15.
16.
17. 18. 19.
20. 21. 22. 23. 24. 25. 26.
tors in the cat nictitating membrane. Br. J. Pharmacol. 70:383 – 388; 1980. Farrell, P. A.; Gates, W. K.; Maksud, M. G.; Morgan, W. P. Increases in plasma b-endorphin/ b-lipotrophin immunoreactivity after treadmill running in humans. J. Appl. Physiol. 52:1245–1249; 1982. Feuerstein, G.; Siren, A. L. The opioid peptides: A role in hypertension? Hypertension 9:561–565; 1987. Fontana, F.; Bernardi, P.; Spagnolo, N.; Capelli, M. Plasma atrial natriuretic factor in low output heart failure syndromes. Klin. Wochenschr. 68:313–319; 1990. Fontana, F.; Bernardi, P.; Merlo Pich, E.; et al. Relationship between plasma atrial natriuretic factor and opioid peptide levels in healthy subjects and in patients with acute congestive heart failure. Eur. Heart J. 14:219–225; 1993. Grossi, G.; Bargossi, A. M.; Lucarelli, C.; Paradisi, R.; Sprovieri, C.; Sprovieri, G. Improvements in automated analysis of catecholamine and related metabolites in biological samples by column switching high-performamce liquid chromatography. J. Chromatogr. 541:273–284; 1991. Guardigli, G.; Fontana, F.; Ventura, C.; et al. Endogenous opioids and hypertensive crisis in patients with acute cerebrovascular accident. J. Endocrinol. Invest. 12(Suppl.4):9–12; 1989. Hanbauer, I.; Kelly, G. D.; Saiani, L.; Yang, H. Y. T. [Met 5]-enkephalin-like peptides of the adrenal medulla: release by nerve stimulation and functional implications. Peptides 3:469–473; 1982. Hexum, T. D.; Hanbauer, I.; Govoni, S.; Yang, H. Y. T.; Costa, E. Secretion of enkephalin-like peptides from canine adrenal gland following splanchnic nerve stimulation. Neuropeptides 1:137–142; 1980. Hirata, Y.; Itoh, K. I.; Ando, K.; Endo, M.; Marumo, F. Plasma endothelin levels during surgery. N. Engl. J. Med. 321:1686; 1989. Kapusta, D. R.; Jones, S. Y.; DiBona, G. F. Opioids in the systemic hemodynamic and renal responses to stress in conscious spontaneously hypertensive rats. Hypertension 13:808–816; 1989. Klein, R.; Wilson S.; Dzielak, D. Opioids and norepinephrine appear to be co-stored in large dense-core vescicles of sympathetic nerve. Fed. Proc. 41:1076; 1981. Kolta, M. G.; Pierzchala, K.; Houdi, A. A.; Van Loon, G. R. Effects of diabetes on the levels of two forms of Met-enkephalin in plasma and peripheral tissues of the rat. Neuropeptides 21:55–63; 1992. Leino, T.; Leppaluoto, J.; Huttunen, P.; Ruokonen, A.; Kuronen, P. Neuroendocrine responses to real and simulated BA Hawk MK 51 flight. Aviat. Space Environ. Med. 66:108–113; 1995. Livett, B. G.; Day, R.; Elde, R. P.; Howe, P. R. C. Co-storage of enkephalins and adrenaline in bovine adrenal medulla. Neuroscience 7:1323–1332; 1982. Louisy, F.; Guezennec, C. Y.; Lartigue, M.; Aldigier, J. C.; Galen, F. X. Influence of endogenous opioids on atrial natriuretic factor
175
27. 28. 29. 30.
31. 32. 33. 34. 35. 36. 37. 38.
39. 40. 41. 42.
release during exercise in man. Eur. J. Appl. Physiol. 59:34–38; 1989. Marson, L.; Kiritsy–Roy, J. A.; Van Loon, G. R. m-Opioid peptide modulation of cardiovascular and sympathoadrenal responses to stress. Am. J. Physiol. 257:R901–R908; 1989. McCubbin, J. A.; Surwit, R. S.; Williams, R. B. Endogenous opiate peptides, stress reactivity, and risk for hypertension. Hypertension 7:808–811; 1985. McCubbin, J. A.; Surwit, R. S.; Williams, R. B.; Nemeroff, C. B.; McNeilly, M. Altered pituitary hormone response to naloxone in hypertension development. Hypertension 14:636–644; 1989. McCubbin, J. A.; Kaplan, J. R.; Manuck, S. B.; Adams, M. R. Opioidergic inhibition of circulatory and endocrine stress responses in cynomolgus monkeys: A preliminary study. Psychosom. Med. 55:23–28; 1993. Miller, W. L.; Redfield, M. M.; Burnett, J. C., Jr. Integrated cardiac, renal, and endocrine actions of endothelin. J. Clin. Invest. 83:317– 320; 1989. Naranjo, J. R.; Urdin, M. C.; Borrell, J.; Fuentes, J. A. Evidence for a central but not adrenal, opioid mediation in hypertension induced by brief isolation. Life Sci. 38:1923– 1930; 1986. Roy, L. F.; Ogilva, R. I.; Larochelle, P.; Hamet, P.; Leene, F. H. H. Cardiac and vascular effects of atrial natriuretic factor and sodium nitroprusside in healthy men. Circulation 79:383–392; 1989. Spampinato, S.; Goldstein, A. Immunoreactive dynorphin in rat tissues and plasma. Neuropeptides 3:193–212; 1983. Spampinato, S.; Paradisi, R.; Canossa, M.; et al. Immunoreactive dynorphin A-like material in extracted human hypothalamic–hypophysial plasma. Life Sci. 52:223–230; 1993. Specchia, G.; Falcone, C.; Traversi, E.; et al. Mental stress as a provocative test in patients with various clinical syndromes of coronary heart disease. Circulation 83(Suppl II):II-108–114; 1991. Ushiyama, K.; Ogawa, T.; Ishii, M.; Ajisaka, R.; Sugishita, Y.; Ito, I. Physiologic neuroendocrine arousal by mental arithmetic stress test in healthy subjects. Am. J. Cardiol. 67:101–103; 1991. Venn, R. F. Combined high-performance liquid chromatographicradioimmunoassay method for the analysis of endorphins, enkephalins and other neurotransmitter peptides. J. Liq. Chromatogr. 423:93–104; 1987. Viveros, O.; Diliberto, E., Jr.; Hazum, E.; Chang, K. J. Opiate-like materials in the adrenal medulla: Evidence for storage and secretion with catecholamines. Mol. Pharmacol. 16:1101–1108; 1979. Yanagisawa, M.; Kurihara, H.; Kimura, S.; et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332:411–415; 1988. Yang, H. Y. T.; Hong, J. S.; Costa, E. Regional distribution of Leu- and Met-enkephalin in rat brain. Neuropharmacology 16:303–307; 1977. Zamir, N.; Simantov, R.; Segal, M. Pain sensitivity and opioid activity in genetically and experimetally hypertensive rats. Brain Res. 184:299–310; 1980.
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