LY317615 This exercise was chosen as the stimulus since it evokes activation of the sympathetic nervous system and an acute marked increase in afterload, which affects myocardial contractility (Siegel et al., 1972). Material and Methods Subjects The study was performed in 24 older (mean age 66.3 ��2.4 years) male volunteers. They were recruited from the general population by an advertisement and found to be in good health. All were normotensive, non-obese, non-smokers and were not taking any medication. A comprehensive clinical evaluation was performed in all subjects by physician, with testing including exercise electrocardiography, echocardiography, hematological and multipanel serum biochemistry screening. All the subjects gave their informed consent to participate in the study.

The investigation conformed with the principles outlined in the Declaration of Helsinki and was approved by the Local Ethics Committee. General characteristics of the subjects is presented in Table 1. Table 1 Characteristics of the subjects (the values are means �� SEM, n=24) Procedure All the tests were carried out under similar environmental conditions (24��C and 40�C50% relative humidity) between 4:00 and 5:00 P.M. Each subject had the maximal voluntary contraction (MVC) of the right and left hand determined using hand dynamometers (Medipan, Poland). Then, they had a catheter inserted into the antecubital vein in one arm and were allowed to rest in the supine position for 30 min. After the rest period, blood samples were taken for determinations of baseline plasma adrenomedullin, noradrenaline, adrenaline and endothelin-1 concentrations.

Next, the subjects performed 3-min handgrip at 30% MVC with right hand and then 3-min handgrip at the same percentage of MVC with left hand, with no resting interval between the bouts, and more blood samples were taken at the end of each 3-min exercise bout, and 5-min after termination of the exercise. To avoid Valsalva manoeuvre, the subjects were instructed not to hold their breath during the handgrip bouts. The subjects respiratory pattern was monitored continuously during the experiment. The protocol with two exercise bouts was used with the intention to prolong the duration of the stimulus, since the static handgrip at 30% MVC performed by one hand cannot usually be maintained longer than 3�C4 min, which was thought to be too short time period for marked activation of the endocrine system.

Measurements Biochemical analysis All plasma hormone determinations were performed in duplicate. The plasma ADM was determined using a specific and sensitive radioimmunoassay kit for ADM (1�C52) produced by Phoenix Brefeldin_A Pharmaceuticals Inc., Belmont 94002 CA, USA. The limit of detection for this assay was 0.5 pg ADM per tube, and the half-maximal inhibition dose of radiodinated ligand binding was 10 pg ADM per tube. The intra-assay coefficient of variance was 5.8%.