Br. J. Pharmacol. (1991), 103, 2009-2015
,'-.
Macmillan Press Ltd, 1991
The effects of phosphoramidon on the regional haemodynamic responses to human proendothelin [1-38] in conscious rats 'Sheila M. Gardiner, Alix M. Compton, Philip A. Kemp & Terence Bennett Department of Physiology and Pharmacology, Medical School, Queen's Medical Centre, Nottingham, NG7 2UH 1 Cardiovascular responses to human proendothelin [1-38], in the absence and presence of phosphoramidon, were studied in conscious Long Evans rats, chronically instrumented for the continuous recording of heart rate, systemic arterial blood pressure and renal, mesenteric and hindquarters blood flows. 2 A dose of 0.1nmolkg-' human proendothelin [1-38] caused a slight pressor effect (maximum 5 + 2mmHg), but a clear bradycardia (maximum -29 + 7beatsmin-1). Renal haemodynamics were unchanged but there was mesenteric vasoconstriction and a vasodilatation followed by a vasoconstriction in the hindquarters. 3 A dose of 1.0nmolkg-' human proendothelin [1-38] caused a gradual hypertension (maximum 42 + 4 mmHg at 10 min) and a profound bradycardia (- 149 + 10 beats min'- at 30 min). There were gradual but marked, renal and hindquarters vasoconstrictions, whereas there was a substantial mesenteric vasoconstriction that was relatively rapid in onset. 4 In 2 animals, administration of human proendothelin [1-38] at a dose of 10nmolkg-1 caused an initial hypotension followed by a rapidly-developing pressor effect; there were renal and mesenteric vasoconstrictions and vasodilatation followed by vasoconstriction in the hindquarters. These changes were very similar to those seen following injection of endothelin-1 (0.1 nmol kg- '). 5 Phosphoramidon (2pumol kg 1) had no cardiovascular effects itself and it did not affect significantly the pressor or mesenteric vasoconstrictor effects of human proendothelin [1-38], but it reduced the bradycardia and renal and hindquarters vasoconstrictor responses. A higher dose of phosphoramidon (lOpmolkg-') caused significant attenuation of all the responses to human proendothelin [1-38], but a substantial mesenteric vasoconstrictor response still occurred under these conditions. 6 The results are consistent with the involvement of phosphoramidon-sensitive enzyme systems in the conversion of human proendothelin [1-38] to endothelin-1 in vivo. In addition, considering the different patterns of responses to human proendothelin [1-38] in the effector tissues studied (heart, and renal, mesenteric and hindquarters vascular beds), and the differential degrees of inhibition of them by phosphoramidon, it is likely that the effects of human proendothelin [1-38] were due to its local (rather than systemic) conversion to endothelin-1 by processes with differing degrees of susceptibility to phosphoramidon. Keywords: Phosphoramidon; human proendothelin [1-38]; regional haemodynamics
Introduction In their original paper, Yanagisawa et al. (1988) proposed that porcine proendothelin [1-39] was converted into endothelin (i.e. endothelin-1) by an endothelin-converting enzyme. Considering the unusual proteolytic processing involved, Yanagisawa et al. (1988) suggested the enzyme was likely to be an endopeptidase with a chymotrypsin-like specificity. Recently, both Matsumura et al. (1990) and Fukuroda et al. (1990) found that phosphoramidon caused substantial inhibition of the pressor effects of porcine proendothelin [1-39] and human proendothelin [1-38], respectively, in anaesthetized rats, indicating that a neutral endopeptidase-like, endothelinconverting enzyme system might be involved in the production of endothelin-1 in vivo. A similar conclusion was reached by McMahon et al. (1991). We considered it feasible that the in vivo conversion of human proendothelin [1-38] to endothelin-1 might vary in different regional vascular beds and, in addition, that such processes might be differently sensitive to phosphoramidon. Therefore, in the present work we compared the regional haemodynamic effects of human proendothelin [1-38] in the same conscious rats in the absence and presence of phos-
phoramidon.
Methods Male, Long Evans rats (350-450g) were used in all experiments since our earlier studies on endothelin-1 (Gardiner et '
Author for correspondence.
al., 1990a,b) had used this strain. Under sodium methohexitone anaesthesia (60mgkg-1 i.p. supplemented as required) miniaturized, pulsed Doppler probes (Haywood et al., 1981) were implanted around the left renal and superior mesenteric arteries and the distal abdominal aorta, below the level of the ileocaecal artery. Following surgery, animals were given ampicillin (7mgkg1' i.m., Penbritin, Beechams) and returned to their home cages with free access to food and water for 7-14 days. After this time intravascular catheters (right jugular vein and distal abdominal aorta) were implanted under brief anaesthesia (sodium methohexitone, 40mgkg-' i.p.), and animals were then left to recover for at least 24h before experiments were begun (Gardiner et al., 1990c). The protocols were completed over 3 days. In preliminary experiments, human proendothelin [1-38] was given in i.v. bolus doses of 0.1, 1.0 and 10nmolkg-'. However, the pressor effects and bradycardia produced by the latter dose were very marked and the 2 animals which received it showed progressive cardiovascular deterioration. Therefore, only the 0.1 and 1.0 nmol kg1- doses of human proendothelin [1-38] were used in the full experiment. On the first experimental day i.v. bolus doses (0.1 and 1.0 nmol kg- 1) of human proendothelin [1-38] and of endothelin-1 (0.01 and O.lnmolkg-1) were given at least 2h apart, with the lower doses of both peptides being given first. Since the lower dose of human proendothelin [1-38] had only slight effects (see Results), on the second and third experimen-
tal days the higher dose (1nmolkg-1 i.v. bolus) only of the peptide was given 5min after injection of phosphoramidon in an i.v. bolus dose of either 2 or 10umolkg- . Animals
2010
S.M. GARDINER et al.
received the 2 doses of phosphoramidon in random order on days 2 and 3. Throughout an experiment, continuous recordings were made of phasic and mean systemic arterial blood pressures, instantaneous heart rate, and phasic and mean Doppler shift signals. Percentage changes in the latter were taken as indices of flow changes (Haywood et al., 1981) and from the mean arterial pressure and mean Doppler shift signals, % changes in regional vascular conductances were calculated (Gardiner et al., 1990a, b, c). The mean values quoted were derived from averaging (by eye) over 20s around the times given, except in the case of values at zero time, in which case they refer to the 20 s period immediately prior to the intervention.
Drugs and peptides Phosphoramidon (Sigma UK) was dissolved in saline
(157mmoll-1 NaCl) and human proendothelin [1-38] and endothelin-1 (Bachem UK) were dissolved in distilled water and then diluted in saline containing 1% bovine serum albumin (Sigma). Injections were given in 100pl and flushed in with 100,ul saline. Concentrated aliquots of peptide solutions were stored at -800C before being thawed and diluted for use.
Results Absolute values for average resting cardiovascular variables under control conditions and after phosphoramidon are given in Table 1.
Data analysis Changes relative to baseline were assessed by Friedman's test (Theodorsson-Norheim, 1987), while comparisons of responses to human proendothelin [1-38] in the absence and presence of phosphoramidon were compared by the Kruskal-Wallis test applied to areas under or over curves (AUC or AOC) obtained by computer analysis (Gardiner et al., 1990c). A P value
,'-.
Macmillan Press Ltd, 1991
The effects of phosphoramidon on the regional haemodynamic responses to human proendothelin [1-38] in conscious rats 'Sheila M. Gardiner, Alix M. Compton, Philip A. Kemp & Terence Bennett Department of Physiology and Pharmacology, Medical School, Queen's Medical Centre, Nottingham, NG7 2UH 1 Cardiovascular responses to human proendothelin [1-38], in the absence and presence of phosphoramidon, were studied in conscious Long Evans rats, chronically instrumented for the continuous recording of heart rate, systemic arterial blood pressure and renal, mesenteric and hindquarters blood flows. 2 A dose of 0.1nmolkg-' human proendothelin [1-38] caused a slight pressor effect (maximum 5 + 2mmHg), but a clear bradycardia (maximum -29 + 7beatsmin-1). Renal haemodynamics were unchanged but there was mesenteric vasoconstriction and a vasodilatation followed by a vasoconstriction in the hindquarters. 3 A dose of 1.0nmolkg-' human proendothelin [1-38] caused a gradual hypertension (maximum 42 + 4 mmHg at 10 min) and a profound bradycardia (- 149 + 10 beats min'- at 30 min). There were gradual but marked, renal and hindquarters vasoconstrictions, whereas there was a substantial mesenteric vasoconstriction that was relatively rapid in onset. 4 In 2 animals, administration of human proendothelin [1-38] at a dose of 10nmolkg-1 caused an initial hypotension followed by a rapidly-developing pressor effect; there were renal and mesenteric vasoconstrictions and vasodilatation followed by vasoconstriction in the hindquarters. These changes were very similar to those seen following injection of endothelin-1 (0.1 nmol kg- '). 5 Phosphoramidon (2pumol kg 1) had no cardiovascular effects itself and it did not affect significantly the pressor or mesenteric vasoconstrictor effects of human proendothelin [1-38], but it reduced the bradycardia and renal and hindquarters vasoconstrictor responses. A higher dose of phosphoramidon (lOpmolkg-') caused significant attenuation of all the responses to human proendothelin [1-38], but a substantial mesenteric vasoconstrictor response still occurred under these conditions. 6 The results are consistent with the involvement of phosphoramidon-sensitive enzyme systems in the conversion of human proendothelin [1-38] to endothelin-1 in vivo. In addition, considering the different patterns of responses to human proendothelin [1-38] in the effector tissues studied (heart, and renal, mesenteric and hindquarters vascular beds), and the differential degrees of inhibition of them by phosphoramidon, it is likely that the effects of human proendothelin [1-38] were due to its local (rather than systemic) conversion to endothelin-1 by processes with differing degrees of susceptibility to phosphoramidon. Keywords: Phosphoramidon; human proendothelin [1-38]; regional haemodynamics
Introduction In their original paper, Yanagisawa et al. (1988) proposed that porcine proendothelin [1-39] was converted into endothelin (i.e. endothelin-1) by an endothelin-converting enzyme. Considering the unusual proteolytic processing involved, Yanagisawa et al. (1988) suggested the enzyme was likely to be an endopeptidase with a chymotrypsin-like specificity. Recently, both Matsumura et al. (1990) and Fukuroda et al. (1990) found that phosphoramidon caused substantial inhibition of the pressor effects of porcine proendothelin [1-39] and human proendothelin [1-38], respectively, in anaesthetized rats, indicating that a neutral endopeptidase-like, endothelinconverting enzyme system might be involved in the production of endothelin-1 in vivo. A similar conclusion was reached by McMahon et al. (1991). We considered it feasible that the in vivo conversion of human proendothelin [1-38] to endothelin-1 might vary in different regional vascular beds and, in addition, that such processes might be differently sensitive to phosphoramidon. Therefore, in the present work we compared the regional haemodynamic effects of human proendothelin [1-38] in the same conscious rats in the absence and presence of phos-
phoramidon.
Methods Male, Long Evans rats (350-450g) were used in all experiments since our earlier studies on endothelin-1 (Gardiner et '
Author for correspondence.
al., 1990a,b) had used this strain. Under sodium methohexitone anaesthesia (60mgkg-1 i.p. supplemented as required) miniaturized, pulsed Doppler probes (Haywood et al., 1981) were implanted around the left renal and superior mesenteric arteries and the distal abdominal aorta, below the level of the ileocaecal artery. Following surgery, animals were given ampicillin (7mgkg1' i.m., Penbritin, Beechams) and returned to their home cages with free access to food and water for 7-14 days. After this time intravascular catheters (right jugular vein and distal abdominal aorta) were implanted under brief anaesthesia (sodium methohexitone, 40mgkg-' i.p.), and animals were then left to recover for at least 24h before experiments were begun (Gardiner et al., 1990c). The protocols were completed over 3 days. In preliminary experiments, human proendothelin [1-38] was given in i.v. bolus doses of 0.1, 1.0 and 10nmolkg-'. However, the pressor effects and bradycardia produced by the latter dose were very marked and the 2 animals which received it showed progressive cardiovascular deterioration. Therefore, only the 0.1 and 1.0 nmol kg1- doses of human proendothelin [1-38] were used in the full experiment. On the first experimental day i.v. bolus doses (0.1 and 1.0 nmol kg- 1) of human proendothelin [1-38] and of endothelin-1 (0.01 and O.lnmolkg-1) were given at least 2h apart, with the lower doses of both peptides being given first. Since the lower dose of human proendothelin [1-38] had only slight effects (see Results), on the second and third experimen-
tal days the higher dose (1nmolkg-1 i.v. bolus) only of the peptide was given 5min after injection of phosphoramidon in an i.v. bolus dose of either 2 or 10umolkg- . Animals
2010
S.M. GARDINER et al.
received the 2 doses of phosphoramidon in random order on days 2 and 3. Throughout an experiment, continuous recordings were made of phasic and mean systemic arterial blood pressures, instantaneous heart rate, and phasic and mean Doppler shift signals. Percentage changes in the latter were taken as indices of flow changes (Haywood et al., 1981) and from the mean arterial pressure and mean Doppler shift signals, % changes in regional vascular conductances were calculated (Gardiner et al., 1990a, b, c). The mean values quoted were derived from averaging (by eye) over 20s around the times given, except in the case of values at zero time, in which case they refer to the 20 s period immediately prior to the intervention.
Drugs and peptides Phosphoramidon (Sigma UK) was dissolved in saline
(157mmoll-1 NaCl) and human proendothelin [1-38] and endothelin-1 (Bachem UK) were dissolved in distilled water and then diluted in saline containing 1% bovine serum albumin (Sigma). Injections were given in 100pl and flushed in with 100,ul saline. Concentrated aliquots of peptide solutions were stored at -800C before being thawed and diluted for use.
Results Absolute values for average resting cardiovascular variables under control conditions and after phosphoramidon are given in Table 1.
Data analysis Changes relative to baseline were assessed by Friedman's test (Theodorsson-Norheim, 1987), while comparisons of responses to human proendothelin [1-38] in the absence and presence of phosphoramidon were compared by the Kruskal-Wallis test applied to areas under or over curves (AUC or AOC) obtained by computer analysis (Gardiner et al., 1990c). A P value
