Effect of urocortin 1 infusion in humans with stable ... - Semantic Scholar

Clinical Science (2005) 109, 381–388 (Printed in Great Britain) doi:10.1042/CS20050079

Effect of urocortin 1 infusion in humans with stable congestive cardiac failure Mark E. DAVIS∗ , Christopher J. PEMBERTON∗ , Timothy G. YANDLE∗ , John G. LAINCHBURY∗ , Miriam T. RADEMAKER∗ , M. Gary NICHOLLS†, Christopher M. FRAMPTON∗ and A. Mark RICHARDS∗ ∗

Christchurch Cardioendocrine Research Group, Christchurch School of Medicine and Health Sciences, Christchurch 8001, New Zealand, and †Department of Medicine, University of the United Arab Emirates, Al Ain, United Arab Emirates

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In sheep with HF (heart failure), Ucn1 (urocortin 1) decreases total peripheral resistance and left atrial pressure, and increases cardiac output in association with attenuation of vasopressor hormone systems and enhancement of renal function. In a previous study, we demonstrated in the first human studies that infusion of Ucn1 elevates corticotropin (‘ACTH’), cortisol and ANP (atrial natriuretic peptide), and suppresses the hunger-inducing hormone ghrelin in normal subjects. In the present study, we examined the effects of Ucn1 on pituitary, adrenal and cardiovascular systems in the first Ucn1 infusion study in human HF. In human HF, it is proposed that Ucn1 would augment corticotropin and cortisol release, suppress ghrelin and reproduce the cardiorenal effects seen in animals with HF. On day 3 of a controlled metabolic diet, we studied eight male volunteers with stable HF (ejection fraction < 40 %; New York Heart Association Class II–III) on two occasions, 2 weeks apart, receiving 50 µg of Ucn1 or placebo intravenously over 1 h in a randomized time-matched cross-over design. Neurohormones, haemodynamics and urine indices were recorded. Ucn1 infusion increased plasma Ucn1, corticotropin (baseline, 5.9 + − 0.9 pmol/l; and + + peak, 7.2 + 1.0 pmol/l) and cortisol (baseline, 285 42 pmol/l; and peak, 310 − − − 41 pmol/l) compared with controls (P < 0.001, 0.008 and 0.047 respectively). The plasma Ucn1 half-life was 54 + − 3 min. ANP and ghrelin were unchanged, and no haemodynamic or renal effects were seen. In conclusion, a brief intravenous infusion of 50 µg of Ucn1 stimulates corticotropin and cortisol in male volunteers with stable HF.

INTRODUCTION Ucn1 (urocortin 1) is a 40-amino-acid peptide belonging to the CRF (corticotropin releasing factor) family, which acts via the CRF1 receptor and the three functional mammalian splice variant CRF2 receptor subtypes (CRF2a , CRF2b and CRF2c ) with cAMP as a second messenger [1]. Since its discovery in 1995, Ucn1 immunoreactivity has been found in the central nervous system (pituitary, cerebral cortex and cerebellum), human cardiac

cells (myocytes and non-myocytes), gut, skeletal muscle, adipose tissue, lymphocytes, mast cells, spleen, thymus, ovarian and placental tissue, skin and synovium [2–16]. Reflecting this broad range of tissue expression, a multiplicity of actions have been ascribed to Ucn1, including stress and inflammatory responses, protection of hippocampal neurons from oxidative and excitotoxic cell death [17], influence on gut motility [18], stimulation of pancreatic exocrine function [19], lipolysis [10] and suppression of appetite [20]. Cardiovascular actions include

Key words: corticotropin (‘ACTH’), cortisol, ghrelin, heart failure, urocortin 1. Abbreviations: ANP, atrial natriuretic peptide; AVP, arginine vasopressin; BNP, brain natriuretic peptide; CO, cardiac output; CRF, corticotropin releasing factor; CRF-BP, CRF-binding protein; DBP, diastolic blood pressure; ET-I, endothelin-I; HF, heart failure; HR, heart rate; LV, left ventricular; N-BNP, N-terminal BNP; NYHA, New York Heart Association; PRA, plasma renin activity; SBP, systolic blood pressure; t1/2 , half-life; Ucn1, urocortin 1. Correspondence: Dr Mark E. Davis (email [email protected]).

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protection against cardiac ischaemic/reperfusion insult, coronary and peripheral vasodilatation and stimulation of cardiac natriuretic peptide secretion [21–25]. In normal sheep, Ucn1 infusion increased cardiac inotropy and mean arterial pressure, whereas, in experimental ovine HF (heart failure) at the same doses, Ucn1 increased CO (cardiac output), decreased calculated total peripheral resistance and left atrial pressure, all in association with marked attenuation of vasopressor hormone systems and enhancement of renal function [26]. These actions in HF parallel many of the goals of HF treatment. Subsequently, two further members of the family have been identified, Ucn2 [27,28] and Ucn3 [29], which act specifically through CRF2 receptors and have similar cardioprotective activity [30]. We have demonstrated previously [31] in healthy humans infused with 50 µg of Ucn1 over 1 h the sequential stimulation of corticotropin (‘ACTH’) and cortisol, a small rise in plasma ANP (atrial natriuretic peptide) and suppression of the hunger-inducing hormone ghrelin, but no haemodynamic response. We hypothesized that Ucn1 administered to patients with stable congestive HF [NYHA (New York Heart Association) class II–III; LV (left ventricular) ejection fraction 40 %; and creatinine < 0.15 mmol/l) would (i) augment corticotropin and cortisol release, (ii) suppress ghrelin, and (iii) replicate in humans some of the enhanced neurohormonal, haemodynamic and renal actions (including augmented CO and renal function) seen in sheep with HF compared with normal sheep. Therefore, in the present study, we report the first controlled study of Ucn1 infusion in humans with stable HF, examining effects on ghrelin, stress and cardiovascular hormones as well as haemodynamics and renal function.

MATERIALS AND METHODS Subjects We studied eight males with stable congestive HF (LV ejection fraction 40 %; NYHA class II–III; and creatinine < 0.15 mmol/l). Their age was 68.1 + − 5.8 years (values are means + S.D.; range 58–74 years), weight was − 78.1 + 14.4 kg (range 53.2–95.1 kg), plasma creatinine − was 0.11 + − 0.03 mmol/l, LV ejection fraction was 30.1 + − 10.0 % and N-BNP [N-terminal BNP (brain natriuretic peptide)] was 275 + − 249 pmol/l. All subjects were taking either an ACE-I (angiotensin-converting-enzyme inhibitor; n = 7) or AIIRB (angiotensin II receptor blocker; n = 1), seven were receiving loop diuretics, six were receiving a β-blocker and three were receiving spironolactone.

Study protocol Participants gave written informed consent, and the protocol was approved by the Ethics Committee of the C


Ministry of Health (Canterbury, New Zealand) and has been carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association. Human Ucn1 for infusion was purchased from Clinalfa. Subjects were studied on two occasions, 2 weeks apart, and received Ucn1 and placebo in a balanced randomized single-blind cross-over design. On the morning of day 3 of a controlled metabolic diet (80 mmol/day sodium and 100 mmol/day potassium), participants ate breakfast by 07.30 hours, presented to the study room and completed a 24 h urine collection at 08.00 hours. The subjects fasted until provision of lunch at 12.00 hours. Participants were weighed and drank 10 ml of water/kg of body weight, followed by 200 ml of water/h between 09.00 and 14.00 hours. Subjects remained seated throughout the day, except when standing to collect urine. At 08.15 hours, venous cannulae were placed in each forearm, one for infusion of Ucn1 and placebo, and the other for blood sampling. All subjects received 50 µg (10 622 pmol) of Ucn1 [dissolved in 1 ml of normal saline, ultrasonicated and made up to 1 µg/ml with Hemaccel (Hoechst Marion Roussel), i.e. total infusate volume was 50 ml] or matching placebo vehicle infusion (50 ml of Hemaccel) over 1 h commencing at 09.00 hours. The Ucn1 dose was based on that which achieved haemodynamic, neurohormonal and renal effects in sheep and neurohormonal response in normal humans. Our own previous unpublished work (M. E. Davis, unpublished work) has shown a 20 % loss to adsorption to infusion apparatus, and this has been accounted for in pharmacokinetic calculations. Venous samples were drawn from the opposite arm at 08.30, 09.00, 09.30, 10.00, 10.30, 11.00, 12.00 and 14.00 hours. Blood was collected into chilled tubes containing EDTA, centrifuged at 4 ◦ C and the plasma stored at − 80 ◦ C prior to assay for Ucn1 [31], cAMP (Biotrak; Amersham Pharamacia Biotech), cGMP [32], ghrelin [33], corticotropin (Nichols Institute Diagnostics), cortisol [34], AVP (arginine vasopressin) [35], BNP [36], ANP [37], N-BNP [38], PRA (plasma renin activity) [39], aldosterone [40], adrenaline [41], noradrenaline [41], ET-I (endothelin-I) [42] and adrenomedullin [43]. Insulin was measured on an Elecsys 2010 automated analyser (Roche Diagnostics). At the conclusion of infusions, further plasma Ucn1 sampling was conducted at 10.05, 10.10, 10.15 and 10.20 hours for kinetic calculations. For each hormone, all samples from an individual were analysed in a single assay. Intra-assay and interassay coefficients of variation, measured at concentrations similar to those extant during these experiments, were all < 17.8 %, except the inter-assay coefficients of variation for BNP (23.6 %) and ET-I (23.5 %). Plasma sodium, potassium, creatinine and glucose and haematocrit were also measured at 09.00, 10.00, 11.00 and 14.00 hours. After blood sampling, the subjects stood to pass urine at 09.00, 10.00, 11.00 and 14.00 hours for measurement

Urocortin 1 infusion in human heart failure

of volume, urinary Ucn1, cAMP, sodium, potassium and creatinine. SBP (systolic blood pressure), DBP (diastolic blood pressure) and HR (heart rate) were recorded with an automatic sphygmomanometer (PRO 300 monitor; Dinamap, Critikon) in duplicate at 30 min intervals until 4 h post-infusion, and then hourly. CO was measured by the thoracic impedance method at the same time intervals (Minnesota impedance cardiograph, model 304B; Surcom). Echocardiography was undertaken immediately preand post-infusion while sitting semi-recumbant (using standard techniques with a Vivid 3 echocardiogram; General Electric). LV volume was measured in the fourchamber view using the modified Simpson’s rule. Data was stored digitally for subsequent analysis of LV volumes (diastolic and systolic), ejection fraction, E (early diastolic flow velocity), A (mitral valve diastolic flow velocity with atrial contraction), the ratio of E/A and E (early diastolic mitral valve anulus tissue velocity). Transmitral flow was measured by pulse Doppler at the mitral valve leaflet tips in the four-chamber view. Tissue Doppler velocities at the medial and lateral mitral valve annulus were measured using the machine presets. Acquisition of echocardiographic data was non-blinded to the study protocol.

Statistics The effects of Ucn1 infusion were analysed using repeated-measures ANOVA (version 10.0 statistical package; SPSS). Associations between measured and derived variables were analysed using Pearson’s correlation coefficient. Areas under the curve were calculated using the standard trapezoidal rule. Ucn1 plasma t1/2 (halflife), metabolic clearance rate and volume of distribution were calculated using a one-compartment model during and after the infusion period (WinNonLin Professional 3.1; Pharsight Corporation) and corrected for a 20 % dose loss from adsorption to the infusion apparatus (M. E. Davis, unpublished work) and subtracting each individual subject’s baseline endogenous Ucn1 from achieved plasma concentrations. A value of P < 0.05 was taken to indicate statistical significance. Results are means + − S.E.M.

RESULTS Baseline pre-infusion hormone, biochemical and haemodynamic variables did not differ between the two experimental days (placebo compared with Ucn1). No hypotensive episodes or other adverse effects occurred. Baseline Ucn1 plasma concentrations were 11.1 + − 0.6 and 10.9 + − 1.1 pmol/l prior to Ucn1 and placebo infusions respectively. Ucn1 infusion significantly increased (P < 0.001) plasma Ucn1 compared with placebo, with peak

Figure 1 Plasma Ucn1, corticotropin and cortisol concentrations with 50 µg of Ucn1 (䊉) or placebo (䊊) infusions over 1 h in eight men with stable congestive HF

Values are displayed as means + − S.E.M. Sequential elevation in corticotropin (ACTH) and cortisol was seen (P = 0.008 and 0.047 respectively, compared with placebo). plasma immunoreactive Ucn1 achieved at the end of 1 h of infusion (178 + − 35 pmol/l compared with 15.5 + − 1.6 pmol/l in time-matched control; P = 0.002; Figure 1). Ucn1 kinetics were consistent with a one-compartment model. The t1/2 for immunoreactive Ucn1 was 54 + − 3 min. Calculated metabolic clearance rate was 35.9 + − 7.1 litres/h and volume of distribution was 47.1 + − 10.0 litres. Compared with placebo, Ucn1 infusion induced significant increases in plasma corticotropin and cortisol (P = 0.008 and 0.047 respectively). Plasma corticotropin and cortisol were elevated sequentially, the peak increment in corticotropin occurring at the end of the infusion period (5.9 + − 0.9 pmol/l at baseline rising to 7.2 + − 1.0 pmol/l), whereas peak post-infusion cortisol levels C


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M. E. Davis and others were achieved 30 min later (285 + − 42 pmol/l at baseline rising to 310 + 41 pmol/l; Figure 1). Ucn1 did not alter − the plasma levels of ghrelin, insulin, glucose, ANP, BNP, N-BNP, cAMP, adrenaline, noradrenaline, adrenomedullin, AVP, PRA, aldosterone, ET-I, sodium, potassium, creatinine and haematocrit (Table 1). Haemodynamics (HR, CO, SBP and DBP; Table 2), echocardiographic parameters [LV volumes (diastolic and systolic), ejection fraction and mitral valve Doppler indices (E, A, E/A ratio and E ), measures of LV diastolic function and filling pressures (results not shown)], urine volume and urinary excretion of Ucn1, sodium, potassium, creatinine and cAMP were also unchanged (Table 3).

DISCUSSION Having previously provided the first report of the biological effects of Ucn1 infused in normal humans [31], in the present study we report, for the first time using a randomized balanced single-blind controlled design, the biological effects of Ucn1 infused in humans with stable HF. Ucn1 increased plasma levels of corticotropin and cortisol, but in contrast with previous studies in normal humans at the same dose [31], we observed no elevation in ANP and no significant suppression of plasma ghrelin. In contrast with earlier studies in sheep with HF [26], but in keeping with results seen in our normal human study (also performed with eight subjects using the same regimen of Ucn1 administration and hormonal, haemodynamic and renal assessments) [31], there were no haemodynamic or renal effects observed at the dose used.

Pituitary–adrenal response The sequential rise in corticotropin and cortisol was of lesser magnitude in the patients with stable congestive HF than in normal volunteers [31]. A dose of 50 µg of Ucn1 induced increments above time-matched controls in plasma corticotropin of 34 % and 127 % in HF and normal subjects respectively, and in plasma cortisol of 26 % and 77 % in HF and normal subjects respectively. The peak plasma Ucn1 concentrations achieved were similar in the present study (178 + − 35 pmol/l) compared with normal humans (200 + − 29 pmol/l) using the same dosing regimen [31], approx. 16 and 21 times baseline plasma levels respectively. Whether this relatively small difference in relative increment achieved underlies the blunted response of corticotropin and cortisol and the lack of response of ANP and ghrelin is unclear. It is possible that receptor down-regulation contributed to the attenuated response. Ucn1 has been shown previously [44] to be elevated in human HF, and we observed a similar trend when comparing our normal subjects studied previously [31] with the patients in the present study. Studies on human endomyocardial biopsy C


specimens have also shown elevated immunoreactive Ucn1 in samples from patients with dilated and hypertrophic cardiomyopathy compared with normals [25]. Systemic administration of Ucn1 in rats (5 µg/kg of body weight), at approx. seven times the dose administered (by body weight) in the present study, decreases CRF1 receptor mRNA in the anterior pituitary and CRF2 receptor mRNA in the heart [45,46]. Whether the much smaller rise in Ucn1 seen in HF patients in the present study compared with our previous study in normal humans (15 % higher; [31]) are enough to cause a similar down-regulation of CRF2 receptors is unknown. However, Ucn1 is most probably a predominantly paracrine hormone, given the proximity of its synthesis to receptor sites (particularly notable in the heart), and even slightly raised systemic plasma concentrations may reflect a considerably greater increase in local tissue levels over a sustained period. If elevated levels of Ucn1 in humans with HF do cause down-regulation of CRF1 and CRF2 receptors it might partially explain the blunted corticotropin and cortisol response, thought to be CRF1 receptor mediated, the lack of ANP stimulation, which is thought to be CRF2 -receptor mediated [24] and possibly also the lack of ghrelin suppression. The effect of Ucn1 on ghrelin may be mediated via pancreatic β-cell CRF2 receptor stimulation of insulin synthesis with subsequent ghrelin suppression [47,48]. Our own data in 87 normal people (age range, 16.2– 81.3 years; A. M. Richards and T. G. Yandle, unpublished work) suggests no significant age trend in Ucn1, thus making the older age of the HF group (68.1 + − 5.8 years) compared with the previously studied normal group (36.3 + − 2.2 years) less likely to be responsible for the blunted response. However, we cannot exclude other factors, such as the inverse relationship between plasma ghrelin and age [49], from having an influence on our results. The literature is sparse regarding the effect of drugs on Ucn1 activity. There is no information relating effects of any of the β-blockers used by our subjects on Ucn1. However, esmolol (a short-acting β 1 -selective adrenergic blocking agent) does not affect Ucn2 (CRF2 -receptorspecific ligand) activity in mice [50]. There is no information on the effects of ACE-I/AIIRB or loop diuretics on Ucn1.

Pharmacokinetics The measured Ucn1 plasma t1/2 is very similar to that we reported in normal humans [31] (54 + − 3 min in HF compared to 52 + − 3 min in normal humans), suggesting that metabolism is not altered in stable HF. As we have noted previously [26], this is markedly different to both normal sheep and sheep with HF, where pharmacokinetics best fit a two-compartment model with t1/2 α of approx. 90 min and t1/2 β of 18–20 h. A potentially relevant regulator is CRF-BP (CRF-binding protein)

Urocortin 1 infusion in human heart failure Table 1 Effect of infusion of 50 µg of human Ucn1 over 1 h on plasma hormones, second messengers, biochemical parameters and haematocrit

Values are means + − S.E.M. Time 0 relates to the start of infusion. Time (min) − 30 Ghrelin (pmol/l) Ucn1 Placebo ANP (pmol/l) Ucn1 Placebo BNP (pmol/l) Ucn1 Placebo N-BNP (pmol/l) Ucn1 Placebo cAMP (nmol/l) Ucn1 Placebo cGMP (nmol/l) Ucn1 Placebo Adrenaline (pmol/l) Ucn1 Placebo Noradrenaline (pmol/l) Ucn1 Placebo PRA (nmol · l−1 · h−1 ) Ucn1 Placebo Aldosterone (pmol/l) Ucn1 Placebo ET-I (pmol/l) Ucn1 Placebo Insulin (pmol/l) Ucn1 Placebo Adrenomedullin (pmol/l) Ucn1 Placebo Sodium (mmol/l) Ucn1 Placebo Potassium (mmol/l) Ucn1 Placebo Glucose (mg/dl) Ucn1 Placebo Creatinine (mmol/l) Ucn1 Placebo Haematocrit Ucn1 Placebo

0

30

60

90

120

180

300

183 + − 27 168 + − 24

200 + − 31 215 + − 37

233 + − 34 212 + − 37

231 + − 33 221 + − 37

232 + − 33 222 + − 37

229 + − 33 225 + − 41

240 + − 37 250 + − 43

177 + − 25 192 + − 31

49 + −9 51 + − 10

47 + −9 58 + − 14

46 + −9 54 + − 12

51 + − 11 52 + − 10

52 + − 12 52 + − 10

47 + − 11 47 + −8

45 + −9 44 + −8

47 + − 10 47 + −9

32 + −7 32 + −8

33 + −7 34 + −7

33 + −7 36 + −8

34 + −7 37 + −8

35 + −8 37 + −8

34 + −7 36 + −8

35 + −8 37 + −8

34 + −8 39 + −8

165 + − 34 171 + − 35

162 + − 33 176 + − 37

165 + − 34 188 + − 38

176 + − 34 189 + − 38

181 + − 37 195 + − 40

180 + − 36 195 + − 39

180 + − 36 200 + − 40

185 + − 40 210 + − 46

19.7 + − 1.3 19.6 + − 1.5

20.1 + − 1.5 19.1 + − 0.7

19.8 + − 0.9 18.5 + − 1.7

19.6 + − 2.0 18.1 + − 1.1

19.9 + − 1.3 20.7 + − 0.9

18.0 + − 0.9 18.7 + − 0.6

18.5 + − 1.4 18.2 + − 1.7

16.5 + − 1.0 15.6 + − 0.6

12.2 + − 1.8 13.4 + − 1.8

11.4 + − 1.9 12.4 + − 12.1

10.4 + − 1.8 11.3 + − 1.9

11.3 + − 1.6 11.0 + − 1.5

11.3 + − 1.7 10.3 + − 1.5

10.7 + − 1.6 7.3 + − 3.2

9.2 + − 1.1 10.3 + − 1.6

9.4 + − 1.3 8.2 + − 1.1

140 + − 32 148 + − 27

127 + − 31 148 + − 28

112 + − 30 138 + − 24

124 + − 32 133 + − 23

109 + − 28 135 + − 24

123 + − 34 123 + − 18

126 + − 23 136 + − 23

107 + − 21 115 + − 28

3914 + − 708 3675 + − 434

3300 + − 630 3068 + − 321

3358 + − 676 2856 + − 298

3178 + − 558 2695 + − 325

3150 + − 537 2580 + − 343

3203 + − 473 2584 + − 407

3124 + − 474 2775 + − 429.7

3150 + − 467 3156 + − 641

5.7 + − 2.0 5.1 + − 1.2

5.0 + − 1.9 4.3 + − 1.0

4.6 + − 1.5 3.8 + − 0.8

4.5 + − 1.2 3.9 + − 0.8

4.8 + − 1.4 3.7 + − 0.8

4.6 + − 1.4 3.8 + − 1.0

4.1 + − 1.1 3.7 + − 1.0

5.6 + − 1.4 6.0 + − 1.4

305 + − 55 303 + − 46

271 + − 40 242 + − 27

244 + − 35 241 + − 32

232 + − 41 204 + − 25

252 + − 40 204 + − 28

220 + − 35 194 + − 27

201 + − 35 185 + − 19

217 + − 33 207 + − 32

1.4 + − 0.1 1.3 + − 0.1

1.4 + − 0.1 1.4 + − 0.1

1.4 + − 0.1 1.3 + − 0.1

1.4 + − 0.1 1.4 + − 0.1

.4 + − 0.1 11.3 + − 0.1

1.4 + − 0.1 1.3 + − 0.1

1.5 + − 0.1 1.4 + − 0.1

1.6 + − 0.2 1.7 + − 0.1

433 + − 176 350 + − 113

170 + − 91 121 + − 46

119 + − 47 71 + − 20

80 + − 31 67 + − 17

71 + − 18 56 + − 13

53 + − 13 69 + − 27

56 + − 10 52 + − 10

534 + − 121 624 + − 145

7.7 + − 0.5 7.4 + − 0.3

7.6 + − 0.6 8.2 + − 0.5

8.6 + − 0.7 9.0 + − 0.6

8.7 + − 0.5 8.5 + − 0.6

8.7 + − 0.5 8.5 + − 0.6

7.3 + − 0.4 7.4 + − 0.5

134.7 + − 1.0 135.2 + − 1.1

135.7 + − 0.7 135.4 + − 1.0

135.5 + − 0.8 135.0 + − 1.1

134.8 + − 1.1 135.5 + − 1.2

4.4 + − 0.1 4.3 + − 0.1

4.5 + − 0.1 4.5 + − 0.1

4.3 + − 0.1 4.3 + − 0.1

4.0 + − 0.1 3.9 + − 0.1

5.8 + − 0.4 5.6 + − 0.4

4.9 + − 0.2 4.6 + −4

5.2 + − 0.2 4.8 + − 0.2

5.1 + − 0.2 4.9 + − 0.2

5.1 + − 0.2 5.0 + − 0.2

5.2 + − 0.2 5.2 + − 0.2

5.4 + − 0.2 5.3 + − 0.1

.5 + − 1.1 99.5 + − 0.9

0.1 + − 0.01 0.1 + − 0.01

0.1 + − 0.01 0.1 + − 0.01

0.1 + − 0.01 0.1 + − 0.01

0.1 + − 0.01 0.1 + − 0.01

0.4 + − 0.01 0.4 + − 0.01

0.4 + − 0.01 0.4 + − 0.01

0.4 + − 0.01 0.4 + − 0.01

0.4 + − 0.01 0.4 + − 0.01

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Table 2 Haemodynamic measurements during Ucn1 infusion

Values are mean + − S.E.M. Time 0 relates to the start of infusion. Time (min) − 30 HR (beats/min) Ucn1 Placebo SBP (mmHg) Ucn1 Placebo DBP (mmHg) Ucn1 Placebo CO (litres/min) Ucn1 Placebo

0

30

60

90

70 + −3 75 + −4

66 + −3 71 + −4

66 + −3 68 + −3

69 + −3 70 + −4

68 + −3 69 + −4

108 + −5 114 + −8

103 + −4 110 + −7

100 + −5 112 + −8

101 + −8 108 + −8

100 + −5 106 + −7

60 + −1 64 + −1

58 + −2 64 + −2

56 + −4 62 + −2

55 + −2 62 + −2

62 + −2 65 + − 11

120

150

66 + −3 70 + −4

180

210

240

270

300

66 + −3 69 + −4

67 + −3 70 + −4

72 + −3 71 + −4

72 + −3 76 + −4

72 + −2 73 + −3

71 + −2 75 + −3

102 + − 4 106 + −5 + 109 + 10 108 − −7

104 + −5 108 + −8

110 + −5 115 + −7

104 + −6 106 + −7

97 + −4 106 + −8

96 + −4 108 + −8

60 + −1 64 + −3

63 + −1 66 + −2

58 + −3 60 + −2

55 + −3 61 + −3

54 + −3 61 + −2

57 + −2 63 + −2

59 + −1 64 + −3

4.1 + − 1.0 − 1.0 5.7 + − 1.0 6.1 + − 1.2 6.3 + − 1.4 6.6 + − 1.2 6.2 + − 1.1 5.5 + − 1.0 4.9 + − 0.9 4.4 + − 0.7 5.6 + − 0.7 6.5 + − 1.3 5.4 + + + + + + + + + + + + 4.9 + 0.8 4.4 0.8 4.3 0.7 5.0 0.6 5.0 0.7 5.0 0.9 4.9 0.7 5.0 0.7 5.1 0.8 5.5 0.9 6.0 1.1 5.4 − − − − − − − − − − − − 1.0

Table 3 Urine analytes upon infusion of 50 µg of human Ucn1 over 1 h

Values are means + − S.E.M. uUcn1, urinary Ucn1. Pre-infusion, 08.00–09.00 hours; Infusion, 09.00–10.00 hours; 0–1 h post-infusion, 10.00–11.00 hours; 1–4 h post-infusion, 11.00–14.00 hours. Time post-infusion (h) Analytes Volume (ml/h) Ucn1 Placebo Sodium (mmol/h) Ucn1 Placebo Potassium (mmol/h) Ucn1 Placebo Creatinine (mmol/h) Ucn1 Placebo uUcn1 (pmol/h) Ucn1 Placebo cAMP (nmol/h) Ucn1 Placebo

24 h urine collection pre-study

Pre-infusion


0–1

1–4

58 + − 12 65 + − 10

95 + − 53 90 + − 45

204 + − 93 167 + − 74

134 + − 75 141 + − 60

97 + − 30 102 + − 34

4.2 + − 0.6 3.4 + − 0.5

2.4 + − 1.3 1.7 + − 0.9

4.1 + − 2.2 3.3 + − 1.8

3.7 + − 2.7 3.4 + − 1.9

2.6 + − 1.3 2.4 + − 1.2

2.0 + − 0.3 2.1 + − 0.3

1.4 + − 0.7 1.6 + − 0.7

2.7 + − 1.0 1.8 + − 0.7

1.8 + − 0.8 1.8 + − 0.7

1.5 + − 0.5 1.3 + − 0.5

0.7 + − 0.3 0.4 + − 0.1

0.2 + − 0.1 0.3 + − 0.1

0.4 + − 0.2 0.3 + − 0.1

0.3 + − 0.2 0.3 + − 0.1

0.3 + − 0.1 0.2 + − 0.1

1.5 + − 0.2 1.6 + − 0.3

4.1 + − 0.9 3.0 + − 0.4

3.8 + − 0.5 3.3 + − 0.3

2.2 + − 0.4 2.6 + − 0.3

3.2 + − 0.9 3.2 + − 1.0

117 + − 17 114 + − 21

240 + − 34 237 + − 51

281 + − 80 238 + − 71

129 + − 37 191 + − 45

141 + − 28 145 + − 40

which is present in plasma in humans and some primates, but not in sheep [51]. It binds both CRF and Ucn1 and is thought to have a role in modulating free active peptide and possibly in peptide clearance [52,53]. There is no reported work comparing CRF-BP activity in normal humans with HF patients. Our Ucn1 assay measures free and bound Ucn1, but we have no information on the activity of the CRF-BP–Ucn1 complex or the relative concentrations of the free compared with bound moieties. If the CRF-BP does contribute to clearance of Ucn1, it C

Infusion

could underlie the shorter t1/2 and lower peak achieved plasma Ucn1 concentrations in humans compared with sheep. There was no increase in urinary Ucn1 over the course of the active study days, suggesting either Ucn1 is not metabolized significantly in the kidney or that our assay does not recognize the metabolite. In contrast with studies in sheep with HF and normal sheep [26], we observed no haemodynamic, renal or neurohormonal activity, other than for corticotropin and

Urocortin 1 infusion in human heart failure

cortisol, and no increase in those responses in humans with HF compared with normal humans [31]. It is possible that the markedly lower plasma levels of Ucn1 achieved in humans with HF (178 + − 35 pmol/l) compared with those seen in sheep at the same Ucn1 dose (1900 + − 143 pmol/l) might explain the lack of haemodynamic, renal and other hormonal responses (decrements in AVP, PRA, aldosterone, ET-1, ANP and BNP) seen in the present study (a finding that may be related to the presence of CRF-BP in human, but not ovine, plasma) [51]. This suggests there is a lower threshold for neurohormonal pituitary activation, which was seen in the present study, than for the effects noted above. In summary we present the first Ucn1 infusion study in humans with stable HF. Ucn1 significantly elevated circulating levels of corticotropin and cortisol, but to a lesser degree than seen in our previous work on normal humans using the same dosing regimen [31]. Unlike that group, there was no rise in ANP or suppression of ghrelin. Ucn1 at the dose used had no demonstrable acute haemodynamic effects, nor did it alter circulating levels of second messenger cAMP, PRA, aldosterone or AVP. The pharmacokinetics of infused Ucn1 were similar in patients with HF to normal humans, but differ markedly from sheep. We found no evidence for renal clearance of Ucn1. Future directions should include extending our Ucn1 work to use higher dose studies to determine if there is a haemodynamic and hormonal dose response and further infusion studies to examine bioactivity and metabolism of Ucn2 and Ucn3, which are not bound to the CRF-BP, and are specific for the CRF2 receptor, through which the majority of the cardiovascular actions of Ucn1 are mediated [54,55].

ACKNOWLEDGMENTS We gratefully acknowledge the provision of Ucn1 antiserum by Professor Wylie Vale and Dr Joan Vaughan (Salk Institute for Biological Studies, San Diego, CA, U.S.A.). We thank the technical staff at Endolab, in particular Steve Fisher and Greg Hammond, and the nurses from the Endocrine Special Test Centre. Barbara Griffin provided secretarial assistance. Funding for this project was provided by the Health Research Council and the National Heart Foundation of New Zealand.

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Received 25 February 2005/22 April 2005; accepted 9 May 2005 Published as Immediate Publication 9 May 2005, doi:10.1042/CS20050079

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