Olinical Investigator
Clin Investig (1992) 70: 218-223
Original Article
© Springer-Verlag 1992
The role of vasopressin in the nicotine-induced stimulation of ACTH and cortisol in men* J. Stalke 1, O. Hader ~, V. Bfihr ~, J. Hensen 1, G. Scherer 2, and W. Oelkers ~ 1 Division of Endocrinology, Department of Medicine, Klinikum Steglitz, Freie Universit/it Berlin 2 Analytisch-BiologischesForschungslabor Prof. Adlkofer, Mfinchen
Summary. Experimental evidence indicates that arginine vasopressin (AVP) contributes to the release of A C T H under certain conditions. The present study investigates the role of vasopressin as a secretagogue of A C T H during cigarette smoking or nicotine infusion with additional injection of corticotropin releasing hormone (CRH) and using the specific AVP antagonist d(CH2)sTyr(Me)-AVP. We first tested the effect of the AVP antagonist (10 gg/kg body weight i.v.) on A C T H and cortisol release following cigarette smoking in 15 healthy young male smokers. Smoking led to marked increments in plasma nicotine and to a small rise in plasma A C T H and cortisol. Mean plasma A C T H and cortisol levels were at no time significantly altered by the antagonist. This might be due to a slight agonistic effect of the AVP antagonist, to high interindividual variability of the A C T H and cortisol responses after smoking or to a neglegible role of AVP in smoking-induced A C T H release. In a second study we performed the following tests in six healthy male non-smokers : (1) nicotine infusion (1.0 gg/kg body weight per min); (2) C R H i.v. (100 gg); (3) AVP antagonist i.v. (5 gg/ kg); (4) nicotine infusion plus C R H i.v. ; (5) nicotine infusion plus AVP antagonist i.v. ; (6) nicotine infusion plus C R H and AVP antagonist i.v. ; and (7) sham infusion. Nicotine infusion led to greater increments of AVP, A C T H and cortisol than smoking without causing nausea. Peak nicotine levels after nicotine infusion were lower than after smoking. The AVP antagonist in the reduced dosage given alone had no effect on hormone levels. However, it slightly attenuated the effect of nico-
tine on A C T H and cortisol (P 1.0 mg nicotine per cigarette). Blood samples for plasma nicotine, ACTH and cortisol were drawn from the other arm at 3.45, 3.55, 4.05, 4.10, 4.15, 4.20, 4.30 and 4.40 p.m. Nicotine infusion in combination with intravenous C R H and A VP antagonist Six healthy male non-smoking subjects (age range 25-38 years, weight range 62-80 kg) were each studied seven times on different study days in a single-blind fashion. The subjects were randomly separated into two groups (A and B). Group A was studied in the test order 1 to 7, group B in the test order 7 to 1. The following tests were performed: (1) nicotine infusion; (2) CRH i.v.; (3) AVP antagonist i.v.; (4) nicotine infusion plus CRH i.v. ; (5) nicotine-infusion plus AVP antagonist i.v. ; (6) nicotine infusion plus CRH and AVP antagonist i.v. ; and (7) sham infusion. The subjects assumed the supine position in a test room at 2.30 p.m., and i.v. cannulas were inserted as described above. Nicotine (1.0 gg/kg per rain) was infused over 20 min from 3.50 to 4.10 p.m. According to our own dose-finding study, this dose stimulates the secretion of the hormones measured without causing nausea [23]. CRH (100 gg) was given i.v. 15 min after starting nicotine infusion, and d(CH2)sTyr(Me)-AVP (5 gg/kg) was given i.v. 30 min before infusion of nicotine. Blood was drawn twice at baseline and 5, 10, 15, 20, 25, 30, 40, 55 and 70 min after starting the infusion. Blood for nicotine measurements in plasma was drawn at 10, 20 and 40 min after starting nicotine infusion. The AVP antagonist [d(CHJsTyr(Me)-AVP] was kindly provided by Prof. K.G. Hofbauer (Ciba Geigy Corporation, CH-4002 Basle, Switzerland). Nicotine was purchased from Dr. G. Neurath (2000 Hamburg 56, FRG). CRH was purchased from Bissendorf Peptide GmbH (3002 Wedemark 2, FRG). Laboratory methods Blood for ACTH and cortisol measurements was collected into tubes containing K +-EDTA, put on
crushed ice and immediately centrifuged at 4° C. Plasma was stored at - 7 0 ° C. ACTH was measured in unextracted plasma using a commercial kit purchased from Nichols Institute (San Juan Capistrano, Calif., USA). The minimum plasma ACTH concentration detectable with this assay was 3 rig/1. The intra-assay variability was 3%, and the inter-assay variability 9.8%. Plasma cortisol was measured using a commercial kitt (Sorin Biomedica, Saluggia, Italy) with antibody-coated tubes. The sensitivity of the assay was 15 nmol/1. The intra-assay variabilities at the cortisol levels (100, 350 and 1000 nmol/1) were 5.4%, 3.9% and 4.5%, respectively. The inter-assay variabilities at the same concentrations were 9.6%, 5.7% and 5.5%, respectively. AVP in plasma was measured radioimmunologically by the method of Morton et al. [16] after extraction of the plasma using SepPak C~s cartridges (Waters Ass., Milford, Mass., USA). As AVP binds to platelets, 1 ml of plasma above the red cell layer after centrifugation was not removed for AVP measurements [3]. The detection limit of AVP in plasma was 0.4 rig/1. The intra-assay variability was 4.9%, and the inter-assay variability 8.8%. The antibody was kindly provided by Dr. J.J. Morton, MRC Blood Pressure Unit, Glasgow, UK. Nicotine in plasma was measured by gas chromatography after extraction [10]. Statistical analysis was performed using twoway analysis of variance (ANOVA) with paired t-test using.the Stats Plus statistics program (Starsoft, Tulsa, Okla., USA). All data are expressed as means +_SEM. Results
Cigarette smoking In response to smoking, mean plasma nicotine levels rose significantly (P < 0.01) from 7.74_ 1.85 ng/ ml to peak levels of 27.66 + 2.83 ng/ml after 15 min without the AVP antagonist and from 7.51+ 1.67 ng/ml to 28.05 ± 1.85 ng/ml with the AVP antagonist. The ACTH and cortisol responses to cigarette smoking with and without application of the AVP inhibitor are shown in Fig. 1. ACTH levels increased slightly from 14.8 -4-1.0 ng/1 to peak levels of 19.5___1.5 ng/1 after 20 min without application of the antagonist (P
Clin Investig (1992) 70: 218-223
Original Article
© Springer-Verlag 1992
The role of vasopressin in the nicotine-induced stimulation of ACTH and cortisol in men* J. Stalke 1, O. Hader ~, V. Bfihr ~, J. Hensen 1, G. Scherer 2, and W. Oelkers ~ 1 Division of Endocrinology, Department of Medicine, Klinikum Steglitz, Freie Universit/it Berlin 2 Analytisch-BiologischesForschungslabor Prof. Adlkofer, Mfinchen
Summary. Experimental evidence indicates that arginine vasopressin (AVP) contributes to the release of A C T H under certain conditions. The present study investigates the role of vasopressin as a secretagogue of A C T H during cigarette smoking or nicotine infusion with additional injection of corticotropin releasing hormone (CRH) and using the specific AVP antagonist d(CH2)sTyr(Me)-AVP. We first tested the effect of the AVP antagonist (10 gg/kg body weight i.v.) on A C T H and cortisol release following cigarette smoking in 15 healthy young male smokers. Smoking led to marked increments in plasma nicotine and to a small rise in plasma A C T H and cortisol. Mean plasma A C T H and cortisol levels were at no time significantly altered by the antagonist. This might be due to a slight agonistic effect of the AVP antagonist, to high interindividual variability of the A C T H and cortisol responses after smoking or to a neglegible role of AVP in smoking-induced A C T H release. In a second study we performed the following tests in six healthy male non-smokers : (1) nicotine infusion (1.0 gg/kg body weight per min); (2) C R H i.v. (100 gg); (3) AVP antagonist i.v. (5 gg/ kg); (4) nicotine infusion plus C R H i.v. ; (5) nicotine infusion plus AVP antagonist i.v. ; (6) nicotine infusion plus C R H and AVP antagonist i.v. ; and (7) sham infusion. Nicotine infusion led to greater increments of AVP, A C T H and cortisol than smoking without causing nausea. Peak nicotine levels after nicotine infusion were lower than after smoking. The AVP antagonist in the reduced dosage given alone had no effect on hormone levels. However, it slightly attenuated the effect of nico-
tine on A C T H and cortisol (P 1.0 mg nicotine per cigarette). Blood samples for plasma nicotine, ACTH and cortisol were drawn from the other arm at 3.45, 3.55, 4.05, 4.10, 4.15, 4.20, 4.30 and 4.40 p.m. Nicotine infusion in combination with intravenous C R H and A VP antagonist Six healthy male non-smoking subjects (age range 25-38 years, weight range 62-80 kg) were each studied seven times on different study days in a single-blind fashion. The subjects were randomly separated into two groups (A and B). Group A was studied in the test order 1 to 7, group B in the test order 7 to 1. The following tests were performed: (1) nicotine infusion; (2) CRH i.v.; (3) AVP antagonist i.v.; (4) nicotine infusion plus CRH i.v. ; (5) nicotine-infusion plus AVP antagonist i.v. ; (6) nicotine infusion plus CRH and AVP antagonist i.v. ; and (7) sham infusion. The subjects assumed the supine position in a test room at 2.30 p.m., and i.v. cannulas were inserted as described above. Nicotine (1.0 gg/kg per rain) was infused over 20 min from 3.50 to 4.10 p.m. According to our own dose-finding study, this dose stimulates the secretion of the hormones measured without causing nausea [23]. CRH (100 gg) was given i.v. 15 min after starting nicotine infusion, and d(CH2)sTyr(Me)-AVP (5 gg/kg) was given i.v. 30 min before infusion of nicotine. Blood was drawn twice at baseline and 5, 10, 15, 20, 25, 30, 40, 55 and 70 min after starting the infusion. Blood for nicotine measurements in plasma was drawn at 10, 20 and 40 min after starting nicotine infusion. The AVP antagonist [d(CHJsTyr(Me)-AVP] was kindly provided by Prof. K.G. Hofbauer (Ciba Geigy Corporation, CH-4002 Basle, Switzerland). Nicotine was purchased from Dr. G. Neurath (2000 Hamburg 56, FRG). CRH was purchased from Bissendorf Peptide GmbH (3002 Wedemark 2, FRG). Laboratory methods Blood for ACTH and cortisol measurements was collected into tubes containing K +-EDTA, put on
crushed ice and immediately centrifuged at 4° C. Plasma was stored at - 7 0 ° C. ACTH was measured in unextracted plasma using a commercial kit purchased from Nichols Institute (San Juan Capistrano, Calif., USA). The minimum plasma ACTH concentration detectable with this assay was 3 rig/1. The intra-assay variability was 3%, and the inter-assay variability 9.8%. Plasma cortisol was measured using a commercial kitt (Sorin Biomedica, Saluggia, Italy) with antibody-coated tubes. The sensitivity of the assay was 15 nmol/1. The intra-assay variabilities at the cortisol levels (100, 350 and 1000 nmol/1) were 5.4%, 3.9% and 4.5%, respectively. The inter-assay variabilities at the same concentrations were 9.6%, 5.7% and 5.5%, respectively. AVP in plasma was measured radioimmunologically by the method of Morton et al. [16] after extraction of the plasma using SepPak C~s cartridges (Waters Ass., Milford, Mass., USA). As AVP binds to platelets, 1 ml of plasma above the red cell layer after centrifugation was not removed for AVP measurements [3]. The detection limit of AVP in plasma was 0.4 rig/1. The intra-assay variability was 4.9%, and the inter-assay variability 8.8%. The antibody was kindly provided by Dr. J.J. Morton, MRC Blood Pressure Unit, Glasgow, UK. Nicotine in plasma was measured by gas chromatography after extraction [10]. Statistical analysis was performed using twoway analysis of variance (ANOVA) with paired t-test using.the Stats Plus statistics program (Starsoft, Tulsa, Okla., USA). All data are expressed as means +_SEM. Results
Cigarette smoking In response to smoking, mean plasma nicotine levels rose significantly (P < 0.01) from 7.74_ 1.85 ng/ ml to peak levels of 27.66 + 2.83 ng/ml after 15 min without the AVP antagonist and from 7.51+ 1.67 ng/ml to 28.05 ± 1.85 ng/ml with the AVP antagonist. The ACTH and cortisol responses to cigarette smoking with and without application of the AVP inhibitor are shown in Fig. 1. ACTH levels increased slightly from 14.8 -4-1.0 ng/1 to peak levels of 19.5___1.5 ng/1 after 20 min without application of the antagonist (P
