Br. J. Pharmacol. (1992), 105, 191-201
,'-.
Macmillan Press Ltd, 1992
Mechanisms contributing to the regional haemodynamic effects of neurotensin in conscious, unrestrained Long Evans rats 1Helene Bachelard, 2Sheila M. Gardiner, Philip A. Kemp & Terence Bennett Department of Physiology & Pharmacology, Medical School, Queen's Medical Centre, Nottingham, NG7 2UH 1 The regional haemodynamic effects of i.v. bolus doses of neurotensin (10-100Ong) were assessed in unrestrained Long Evans rats chronically instrumented with miniaturized, pulsed Doppler probes. 2 Neurotensin caused increases in blood pressure, together with dose-related tachycardias and constrictions in the renal, superior mesenteric and hindquarters vascular beds. The tachycardia elicited by the 1000 ng dose of neurotensin was preceded by a transient bradycardia. 3 In the presence of phentolamine, the pressor effect of neurotensin (1000ng) was converted into a hypotensive effect, accompanied by reduced tachycardic and constrictor responses in the renal, superior mesenteric and hindquarters vascular beds. The tachycardia was not preceded by a bradycardia. 4 In the presence of phentolamine and propranolol, the pressor and bradycardic responses to neurotensin were unaffected, whereas the tachycardia was abolished. The renal vasconstrictor effect was smaller, while the constrictions in the superior mesenteric and hindquarters vasculars beds were not different from those in untreated rats. 5 In rats neonatally treated with capsaicin (50mgkg-1, s.c.), the pressor effects elicited by neurotensin (300 and 1000 ng) were reduced as were the constrictor responses in the renal (at the dose of 300 ng), superior mesenteric (at the dose of 300 ng) and hindquarters (at both doses) vascular beds. The bradycardia elicited by neurotensin (1000 ng) was absent, whereas the tachycardia was potentiated. 6 The results indicate that in conscious, intact rats neurotensin appears to exert cardiovascular influences through activation of sympathoadrenal mechanisms and also through non-adrenergic effects on the heart, renal, superior mesenteric and hindquarters vascular beds. The latter effects appear to involve conscious,
capsaicin-sensitive nerves.
Keywords: Blood pressure; heart rate; neurotensin; regional haemodynamics; capsaicin
Introduction Neurotensin (NT) is a tridecapeptide, first isolated from bovine hypothalami (Carraway & Leeman, 1973), and later found in various areas of the central nervous system, the gastrointestinal tract and in circulating blood in several animal species (Uhl & Snyder, 1976; 1981; Kobayashi et al., 1977; Carraway & Leeman, 1978; Carraway & Bhatnagar, 1980; Hammer et al., 1980; Manberg et al., 1982; Shulkes et al., 1982). NT possesses a wide range of effects on both the central and peripheral nervous systems and on peripheral tissues (Carraway & Leeman, 1973; Bissette et al., 1978; Brown & Miller, 1982). NT was found to produce contraction of some isolated blood vessels, such as the portal vein (Rioux et al., 1980), coronary vasculature (Quirion et al., 1979) and vessels of subcutaneous adipose tissue (Rosell & Rokaeus, 1981), and to have vasoconstrictor properties in the isolated, perfused hindquarters of the rat (Rioux et al., 1982). The peptide also exerts potent positive inotropic and chronotropic effects on rodent isolated, spontaneously beating atria (Quirion et al., 1978; Stene-Larsen & Helle, 1979). Intravenous administration of NT was found to induce hypotension in pentobarbitone anaesthetized rats, possibly through a histamine-mediated process (Quirion et al., 1980), since pretreatment with compound 48/80, a well-known mast cell degranulating agent, was found not only to block the hypotensive effect of NT, but also to unmask hypertensive effects of high doses of NT (Quirion et al., 1980). Using conscious rats, Sumners et al. (1982) reported that i.v. administration of NT elicited a fall in blood pressure which was sometimes preceded 1 Present address: Unite de Recherche en Hypertension, Centre de Recherche du CHUL, Centre Hospitalier de l'Universit6 Laval, 2705 boul. Laurier, St-Foy, P.Q., Canada, G1V 4G2. 2 Author for correspondence.
by a slight hypertensive effect. In pentobarbitoneanaesthetized guinea-pigs, NT was found to elicit dosedependent pressor effects, which probably resulted from an influence of the peptide on the sympathetic nervous system, and a tachycardia, which was possibly due to a direct effect of the peptide on the sino-atrial node (Kerouac et al., 1981). Recently, it has been reported that both effects were significantly reduced or abolished in guinea-pigs chronically treated with the neurotoxin, capsaicin (Bachelard et al., 1987). Moreover, in vitro studies have shown that the positive chronotropic and inotropic effects of NT were abolished in atria taken from capsaicin-treated guinea-pigs (Bachelard et al., 1987). Thus, it has been suggested that capsaicin-sensitive sensory neurones participate in the cardiovascular responses to NT in guinea-pigs (Bachelard et al., 1987). The present study was undertaken to investigate the regional haemodynamic effects produced by NT injected i.v. in conscious, unrestrained, rats, chronically instrumented with intravascular catheters and miniaturized pulsed Doppler probes to measure renal, superior mesenteric and hindquarters blood flows. The contribution of adrenoceptors to the cardiovascular responses to NT was examined by peripheral administration of a- and f-adrenoceptor antagonists. Moreover, to assess the involvement of capsaicin-sensitive sensory neurones in the cardiovascular responses to NT, some experiments were carried out in rats neonatally treated with capsaicin or with the vehicle for capsaicin.
Methods The experiments were carried out on male Long Evans rats (350-400 g) bred in the Animal Unit in Nottingham. Details of the techniques used have been published elsewhere (Gardiner
192
H. BACHELARD et al.
Table I Baseline values for heart rate, blood pressure and regional vascular conductance in conscious, untreated (n = 8), vehicle-treated (Veh-treated; n = 9) or capsaicin-treated (Cap-treated; n = 9) Long Evans rats Heart rate (beats min -)
Mean blood pressure (mmHg)
Renal
379 + 12 361 + 11 356 + 11
101 + 2 101 + 3 104 +2
10.1 + 0.9 9.3 + 1.3 10.2 + 0.9
Untreated Veh-treated Cap-treated
Vascular conductance ([kHz mmHg-'] 103) Renal Mesenteric Hindquarters
Doppler shift (kHz) Mesenteric Hindquarters
8.3 + 0.7 9.2 + 1.0 8.0 + 0.3
5.1 +0.6 6.2 + 0.7 5.3 + 0.8
99 + 8 89 + 11 98 + 7
83 + 7 91 + 11 78 + 2
51 + 7 61 + 7 51 + 10
Values are mean + s.e.mean.
et al., 1990a, b). Briefly, animals were anaesthetized (sodium methohexitone, 60 mg kg'- i.p. supplemented as required) and had miniaturized pulsed Doppler probes (Haywood et al., 1981) implanted around the left renal artery, the superior mesenteric artery and the distal abdominal aorta (below the level of the ileocaecal artery). The probe wires were tunnelled s.c. and exteriorized at the back of the neck, where they were sutured in place. Animals were given ampicillin (7mgkg-1, i.m., Penbritin, Beechams) and left to recover for at least 7 days. Thereafter, under brief anaesthesia (sodium methohexitone 40mgkg-1, i.p.), the probes wires were soldered into a microconnector (Microtech Inc., U.S.A.) and one intraarterial (distal abdominal aorta via the caudal artery) and two i.v. (right jugular vein) catheters were implanted. The catheters ran s.c. and emerged with the pulsed Doppler probe leads. The microconnector, soldered to the latter, was clamped in a harness worn by the rat and the catheters passed through a flexible spring (for protection) which was attached to the harness. Animals were returned to their home cages with free access to food and water. Experiments were not begun until the next day. Throughout the experiments, continuous recordings were made of instantaneous heart rate, phasic and mean blood pressures and renal, mesenteric and hindquarters Doppler shift signals (Crystal Biotech VF-1 Doppler flowmeter, Holliston, MA, U.S.A.). Changes in Doppler shift signals were calculated as percentage of the baseline level. The latter have been shown to be a reliable index of volume flow (Haywood et al., 1981; Wright et al., 1987) and are referred to as flows in the text. The Doppler shift and corresponding mean arterial blood pressure signals were used to calculate percentage changes in regional vascular conductances (Gardiner et al., 1990b). At different times, varying between 0.5 and 30min after peptide administration, measurements were made of heart rate, mean blood pressure and mean Doppler shift signals, to represent the full profile of the effects of the peptide, and related to the pre-drug baseline (absolute changes for the former two variables, percentages for the Doppler shifts).
Before every experiment a 30 min baseline recording period was made. Animals had free access to water and food for the duration of the experiment. All i.v. injections were given in 100lu isotonic saline (the dead space of the catheter). NT was dissolved in isotonic saline containing 1% bovine serum albumin (BSA). The following experiments were performed:
Cardiovascular responses to neurotensin Animals were used on 3 consecutive days during which they received 6 randomized i.v. injections of vehicle (0.1 ml isotonic saline containing 1% bovine serum albumin (BSA)) and NT at doses varying between 10 and 1000ng; randomized between days, but the lower doses were always given before the higher doses on any particular day. Each animal received no more than 2 i.v. injections on the same day, separated by at least 120min. Measurements were made before, during and for 30min following i.v. injections of vehicle or NT. Injections were given over a period of 5 s.
Effect of pretreatment with phentolamine, or phentolamine and propranolol on responses to neurotensin The a-adrenoceptor antagonist, phentolamine, was administered i.v. as a bolus (1 mgkg- 1, 0.1 ml) followed by continuous infusion of I mg kg- h -t (0.3 ml h -1) (Winn et al., 1985) to a group of 11 rats. Fifteen min after the start of the phentolamine infusion the animals received an i.v. injection of NT (1000 ng). The next day, in the same animals, a mixture of phentolamine (lmgkg'-, i.v. bolus, lmgkg'-h-1, infusion) and the fJ-adrenoceptor antagonist, propranolol, (1 mgkg- ', i.v. bolus, 0.5mgkg-1h-1, infusion) (Gardiner & Bennett, 1988) was administered and 15min after onset of the coinfusion, NT (1000ng) was injected i.v. Measurements were made before, during, and for 30 min following i.v. injection of NT.
Table 2 Peak cardiovascular changes following intravenous administration of saline-bovine serum albumin (saline-BSA, 1%) or increasing doses of neurotensin (NT) in conscious, untreated Long Evans rats
Saline-BSA 1% NT 10 ng NT 30 ng NT 100 ng NT 300 ng NT 1000 ng
AHeart rate
AMean blood pressure
(beats min 1)
(mmHg)
+19+6t +16 + 6 +16 + 8 +25 + 10 +43 + 20*
+3 + 2 +8 + 2 +11 + 1*
-57 + 24* +99 + 12*
0+ 1
+ 14 2* + 15 + 2*
Renal -3 + 2 0+3 -3 + 2 0+2 -6 + 2 -24 + 4*
ADoppler shift (%) Mesenteric Hindquarters +3 ± 4 -3 + 4 +8 + 7 - 14 + 3* -41 + 5* -52 + 6*
+5
+ 5
+5 + 4 -8 -11 -28 -59
+ + + +
2 3 7* 5*
A Vascular conductance (%)
Renal -3+ -2 + -9 + -8 + -15 + -25 +
1 3 2 3 2* 4*
Mesenteric
Hindquarters
-2 + 3 -2 + 5 -4+4 -17 + 4* -45 + 4* -55 + 4*
-2 + 4 0+6 -11 + 3 -11 +4 -35 + 6* -62 + 4*
Values are mean + s.e.mean; n = 8, at least (some injections were given in the same animals). *P < 0.05 versus control injection, analysis of variance followed by Fisher's test. t P < 0.05 versus baseline, paired Student's t test for control injection of saline-BSA (1%). With NT 1000 ng a biphasic effect was observed on heart rate; the first number indicates the initial change and below is the later change.
HAEMODYNAMIC EFFECTS OF NEUROTENSIN
193
Cardiovascular responses to neurotensin in rats neonatally treated with capsaicin At least 48 h after birth, Long Evans rats were anaesthetized with 1.5-2% halothane (in oxygen) and injected s.c. with either vehicle (10% ethanol, 10% Tween 80 in isotonic saline) or capsaicin (50mgkg-1) in a volume of 100pl. After weaning (day 21-30 after birth) only male rats were selected for the experiments and housed in groups of 4 or 5 with free access to food and tap water. About 20 weeks later, two groups of rats (vehicle-treated, n = 9 and capsaicin-treated, n = 9), previously instrumented with pulsed Doppler probes and intravascular catheters (as earlier described), were used on 3 consecutive days during which they received i.v. injections of vehicle (0.1 ml isotonic saline containing 1% BSA) and NT (300 and 100ng), in random order. Measurements were made before, during and for 30 min following i.v. injections of vehicle or NT.
E 0)a) .0
20 0
I -0
20 CD
0 2-1-I
-r I
-
-
1
0
2 E -20[gL**
'**
Q.
-40
20 r
ROnol *enai
-20
Drugs
-40 40
Neurotensin (Bachem), phentolamine mesylate (Ciba), propranolol hydrochloride (Imperial Chemical Industries) and 8 methyl-N-vanillyl-6-nonenamide (capsaicin; Sigma) were used.
20
7-
Mesenteric
0
Statistical analysis
~-20
Values are expressed as the mean + s.e.mean; n is the number of animals. The paired Student's t test was used to analyse the effects of control injection of saline-BSA, while comparisons between groups were made by an analysis of variance followed by Fisher's test. A P value < 0.05 was taken to indicate a significant difference.
C -40 a)
Q-60 o 60
0
t
40
20
&II
,1
*
*
0
Hindquarters
-20
Results The baseline values for cardiovascular variables are given in Table 1.
-40
-60 I
,
,
10
Haemodynamic responses to neurotensin Control injections caused a slight tachycardia (significant at 1 min), but had no significant effect on the other measured or calculated variables (Table 2). Table 2 shows that the lowest doses of NT tested (10 and 30ng) did not have effects significantly different from those of control injections. However, with the 100ng dose of NT there was a slight increase in blood pressure (significant at 0.5 min) accompanied by falls in superior mesenteric flow (significant at 1-1.5min) and vascular conductance (significant at 1-2 min); these effects were significantly different from those following control injections. There were no changes in heart rate or renal or hindquarters flows or vascular conductances. NT at a dose of 300 ng produced a slight increase in blood pressure (significant at 0.5 and 1.5-5 min) and a tachycardia (significant at 4-10 min). There were long-lasting falls in superior mesenteric (significant at 1-30 min) and hindquarters flows (significant at 3-30min), but no significant change in renal flow. These responses were associated with constrictions in renal (significant at 2-5 min), superior mesenteric (significant at 1-30min) and hindquarters (significant at 230 min) vascular beds (Table 2). The highest dose of NT (1000 ng) caused a slight increase in blood pressure (significant at 2-5 min) accompanied by a transient bradycardia (significant at 1 min) followed by a tachycardia (significant at 3-30 min), and reductions in renal (significant at 4-30min), superior mesenteric (significant at 1-
15
30 min
t
Neurotensin 1000 ng Phentolamine Figure 1 Cardiovascular responses to i.v. bolus injections of neurotensin (1000ng) in conscious, unrestrained, Long Evans rats, in the absence, (0) or in the presence (Q) of i.v. treatment with phentolamine (1 mg kg- 1, i.v. bolus, 1 mg kg- I h- 1, infusion). Values are mean with s.e.mean shown by vertical lines. *P < 0.05 for changes in untreated group (n = 15) versus changes in treated group (n = 11) (analysis of variance followed by Fisher's test). t P < 0.05 versus predrug baseline (Student's t test for paired data). HR = heart rate; BP = blood pressure.
30 min) and hindquarters flows (significant at 0.5-30 min) (Figure 1 and Table 2). Hence, there were constrictions in renal (significant at 2-30 min), superior mesenteric (significant at 1-30 min) and hindquarters (significant at 0.5-30 min) vascular beds (Figure 2 and Table 2).
Haemodynamic responses to neurotensin (1000 ng) in the presence of phentolamine Fifteen min after the onset of pretreatment with phentolamine there was no change in blood pressure, but there were persistent increases in heart rate, and hindquarters flow and reductions in renal and superior mesenteric flows. There were
194
H. BACHELARD et al.
20
Renal
0 -20
-40 60 40 20 _
0
Mesenteric
-
a) C.)
Co
-20
0
C cJ 'ua 0 C
CD
-40 I
--
-
-
--/
-60 60
Hindquarters _~~
40 20 0
Hindquarters -20 -40 -60 -80 I0
1
2
3
4
10
5
15
30 min
1i~~~~~~~01
Neurotensin 1000 Phentolamine
ng
Figure 2 Changes in regional vascular conductances elicited by i.v. bolus injections of neurotensin (1000ng) in conscious, unrestrained, Long Evans rats, in the absence, (0) or in the presence (Q) of i.v. treatment with phentolamine (1 mgkg-', i.v. bolus, 1 mg kg'- h -1, infusion). These data were derived from those shown in Figure 1. Values are mean with s.e.mean shown by vertical lines. *P < 0.05 for changes in untreated group (n = 15) versus changes in treated group (n = 11) (analysis of variance followed by Fisher's test). t P 0.05 versus pre-drug baseline (Student's t test for paired data).
,'-.
Macmillan Press Ltd, 1992
Mechanisms contributing to the regional haemodynamic effects of neurotensin in conscious, unrestrained Long Evans rats 1Helene Bachelard, 2Sheila M. Gardiner, Philip A. Kemp & Terence Bennett Department of Physiology & Pharmacology, Medical School, Queen's Medical Centre, Nottingham, NG7 2UH 1 The regional haemodynamic effects of i.v. bolus doses of neurotensin (10-100Ong) were assessed in unrestrained Long Evans rats chronically instrumented with miniaturized, pulsed Doppler probes. 2 Neurotensin caused increases in blood pressure, together with dose-related tachycardias and constrictions in the renal, superior mesenteric and hindquarters vascular beds. The tachycardia elicited by the 1000 ng dose of neurotensin was preceded by a transient bradycardia. 3 In the presence of phentolamine, the pressor effect of neurotensin (1000ng) was converted into a hypotensive effect, accompanied by reduced tachycardic and constrictor responses in the renal, superior mesenteric and hindquarters vascular beds. The tachycardia was not preceded by a bradycardia. 4 In the presence of phentolamine and propranolol, the pressor and bradycardic responses to neurotensin were unaffected, whereas the tachycardia was abolished. The renal vasconstrictor effect was smaller, while the constrictions in the superior mesenteric and hindquarters vasculars beds were not different from those in untreated rats. 5 In rats neonatally treated with capsaicin (50mgkg-1, s.c.), the pressor effects elicited by neurotensin (300 and 1000 ng) were reduced as were the constrictor responses in the renal (at the dose of 300 ng), superior mesenteric (at the dose of 300 ng) and hindquarters (at both doses) vascular beds. The bradycardia elicited by neurotensin (1000 ng) was absent, whereas the tachycardia was potentiated. 6 The results indicate that in conscious, intact rats neurotensin appears to exert cardiovascular influences through activation of sympathoadrenal mechanisms and also through non-adrenergic effects on the heart, renal, superior mesenteric and hindquarters vascular beds. The latter effects appear to involve conscious,
capsaicin-sensitive nerves.
Keywords: Blood pressure; heart rate; neurotensin; regional haemodynamics; capsaicin
Introduction Neurotensin (NT) is a tridecapeptide, first isolated from bovine hypothalami (Carraway & Leeman, 1973), and later found in various areas of the central nervous system, the gastrointestinal tract and in circulating blood in several animal species (Uhl & Snyder, 1976; 1981; Kobayashi et al., 1977; Carraway & Leeman, 1978; Carraway & Bhatnagar, 1980; Hammer et al., 1980; Manberg et al., 1982; Shulkes et al., 1982). NT possesses a wide range of effects on both the central and peripheral nervous systems and on peripheral tissues (Carraway & Leeman, 1973; Bissette et al., 1978; Brown & Miller, 1982). NT was found to produce contraction of some isolated blood vessels, such as the portal vein (Rioux et al., 1980), coronary vasculature (Quirion et al., 1979) and vessels of subcutaneous adipose tissue (Rosell & Rokaeus, 1981), and to have vasoconstrictor properties in the isolated, perfused hindquarters of the rat (Rioux et al., 1982). The peptide also exerts potent positive inotropic and chronotropic effects on rodent isolated, spontaneously beating atria (Quirion et al., 1978; Stene-Larsen & Helle, 1979). Intravenous administration of NT was found to induce hypotension in pentobarbitone anaesthetized rats, possibly through a histamine-mediated process (Quirion et al., 1980), since pretreatment with compound 48/80, a well-known mast cell degranulating agent, was found not only to block the hypotensive effect of NT, but also to unmask hypertensive effects of high doses of NT (Quirion et al., 1980). Using conscious rats, Sumners et al. (1982) reported that i.v. administration of NT elicited a fall in blood pressure which was sometimes preceded 1 Present address: Unite de Recherche en Hypertension, Centre de Recherche du CHUL, Centre Hospitalier de l'Universit6 Laval, 2705 boul. Laurier, St-Foy, P.Q., Canada, G1V 4G2. 2 Author for correspondence.
by a slight hypertensive effect. In pentobarbitoneanaesthetized guinea-pigs, NT was found to elicit dosedependent pressor effects, which probably resulted from an influence of the peptide on the sympathetic nervous system, and a tachycardia, which was possibly due to a direct effect of the peptide on the sino-atrial node (Kerouac et al., 1981). Recently, it has been reported that both effects were significantly reduced or abolished in guinea-pigs chronically treated with the neurotoxin, capsaicin (Bachelard et al., 1987). Moreover, in vitro studies have shown that the positive chronotropic and inotropic effects of NT were abolished in atria taken from capsaicin-treated guinea-pigs (Bachelard et al., 1987). Thus, it has been suggested that capsaicin-sensitive sensory neurones participate in the cardiovascular responses to NT in guinea-pigs (Bachelard et al., 1987). The present study was undertaken to investigate the regional haemodynamic effects produced by NT injected i.v. in conscious, unrestrained, rats, chronically instrumented with intravascular catheters and miniaturized pulsed Doppler probes to measure renal, superior mesenteric and hindquarters blood flows. The contribution of adrenoceptors to the cardiovascular responses to NT was examined by peripheral administration of a- and f-adrenoceptor antagonists. Moreover, to assess the involvement of capsaicin-sensitive sensory neurones in the cardiovascular responses to NT, some experiments were carried out in rats neonatally treated with capsaicin or with the vehicle for capsaicin.
Methods The experiments were carried out on male Long Evans rats (350-400 g) bred in the Animal Unit in Nottingham. Details of the techniques used have been published elsewhere (Gardiner
192
H. BACHELARD et al.
Table I Baseline values for heart rate, blood pressure and regional vascular conductance in conscious, untreated (n = 8), vehicle-treated (Veh-treated; n = 9) or capsaicin-treated (Cap-treated; n = 9) Long Evans rats Heart rate (beats min -)
Mean blood pressure (mmHg)
Renal
379 + 12 361 + 11 356 + 11
101 + 2 101 + 3 104 +2
10.1 + 0.9 9.3 + 1.3 10.2 + 0.9
Untreated Veh-treated Cap-treated
Vascular conductance ([kHz mmHg-'] 103) Renal Mesenteric Hindquarters
Doppler shift (kHz) Mesenteric Hindquarters
8.3 + 0.7 9.2 + 1.0 8.0 + 0.3
5.1 +0.6 6.2 + 0.7 5.3 + 0.8
99 + 8 89 + 11 98 + 7
83 + 7 91 + 11 78 + 2
51 + 7 61 + 7 51 + 10
Values are mean + s.e.mean.
et al., 1990a, b). Briefly, animals were anaesthetized (sodium methohexitone, 60 mg kg'- i.p. supplemented as required) and had miniaturized pulsed Doppler probes (Haywood et al., 1981) implanted around the left renal artery, the superior mesenteric artery and the distal abdominal aorta (below the level of the ileocaecal artery). The probe wires were tunnelled s.c. and exteriorized at the back of the neck, where they were sutured in place. Animals were given ampicillin (7mgkg-1, i.m., Penbritin, Beechams) and left to recover for at least 7 days. Thereafter, under brief anaesthesia (sodium methohexitone 40mgkg-1, i.p.), the probes wires were soldered into a microconnector (Microtech Inc., U.S.A.) and one intraarterial (distal abdominal aorta via the caudal artery) and two i.v. (right jugular vein) catheters were implanted. The catheters ran s.c. and emerged with the pulsed Doppler probe leads. The microconnector, soldered to the latter, was clamped in a harness worn by the rat and the catheters passed through a flexible spring (for protection) which was attached to the harness. Animals were returned to their home cages with free access to food and water. Experiments were not begun until the next day. Throughout the experiments, continuous recordings were made of instantaneous heart rate, phasic and mean blood pressures and renal, mesenteric and hindquarters Doppler shift signals (Crystal Biotech VF-1 Doppler flowmeter, Holliston, MA, U.S.A.). Changes in Doppler shift signals were calculated as percentage of the baseline level. The latter have been shown to be a reliable index of volume flow (Haywood et al., 1981; Wright et al., 1987) and are referred to as flows in the text. The Doppler shift and corresponding mean arterial blood pressure signals were used to calculate percentage changes in regional vascular conductances (Gardiner et al., 1990b). At different times, varying between 0.5 and 30min after peptide administration, measurements were made of heart rate, mean blood pressure and mean Doppler shift signals, to represent the full profile of the effects of the peptide, and related to the pre-drug baseline (absolute changes for the former two variables, percentages for the Doppler shifts).
Before every experiment a 30 min baseline recording period was made. Animals had free access to water and food for the duration of the experiment. All i.v. injections were given in 100lu isotonic saline (the dead space of the catheter). NT was dissolved in isotonic saline containing 1% bovine serum albumin (BSA). The following experiments were performed:
Cardiovascular responses to neurotensin Animals were used on 3 consecutive days during which they received 6 randomized i.v. injections of vehicle (0.1 ml isotonic saline containing 1% bovine serum albumin (BSA)) and NT at doses varying between 10 and 1000ng; randomized between days, but the lower doses were always given before the higher doses on any particular day. Each animal received no more than 2 i.v. injections on the same day, separated by at least 120min. Measurements were made before, during and for 30min following i.v. injections of vehicle or NT. Injections were given over a period of 5 s.
Effect of pretreatment with phentolamine, or phentolamine and propranolol on responses to neurotensin The a-adrenoceptor antagonist, phentolamine, was administered i.v. as a bolus (1 mgkg- 1, 0.1 ml) followed by continuous infusion of I mg kg- h -t (0.3 ml h -1) (Winn et al., 1985) to a group of 11 rats. Fifteen min after the start of the phentolamine infusion the animals received an i.v. injection of NT (1000 ng). The next day, in the same animals, a mixture of phentolamine (lmgkg'-, i.v. bolus, lmgkg'-h-1, infusion) and the fJ-adrenoceptor antagonist, propranolol, (1 mgkg- ', i.v. bolus, 0.5mgkg-1h-1, infusion) (Gardiner & Bennett, 1988) was administered and 15min after onset of the coinfusion, NT (1000ng) was injected i.v. Measurements were made before, during, and for 30 min following i.v. injection of NT.
Table 2 Peak cardiovascular changes following intravenous administration of saline-bovine serum albumin (saline-BSA, 1%) or increasing doses of neurotensin (NT) in conscious, untreated Long Evans rats
Saline-BSA 1% NT 10 ng NT 30 ng NT 100 ng NT 300 ng NT 1000 ng
AHeart rate
AMean blood pressure
(beats min 1)
(mmHg)
+19+6t +16 + 6 +16 + 8 +25 + 10 +43 + 20*
+3 + 2 +8 + 2 +11 + 1*
-57 + 24* +99 + 12*
0+ 1
+ 14 2* + 15 + 2*
Renal -3 + 2 0+3 -3 + 2 0+2 -6 + 2 -24 + 4*
ADoppler shift (%) Mesenteric Hindquarters +3 ± 4 -3 + 4 +8 + 7 - 14 + 3* -41 + 5* -52 + 6*
+5
+ 5
+5 + 4 -8 -11 -28 -59
+ + + +
2 3 7* 5*
A Vascular conductance (%)
Renal -3+ -2 + -9 + -8 + -15 + -25 +
1 3 2 3 2* 4*
Mesenteric
Hindquarters
-2 + 3 -2 + 5 -4+4 -17 + 4* -45 + 4* -55 + 4*
-2 + 4 0+6 -11 + 3 -11 +4 -35 + 6* -62 + 4*
Values are mean + s.e.mean; n = 8, at least (some injections were given in the same animals). *P < 0.05 versus control injection, analysis of variance followed by Fisher's test. t P < 0.05 versus baseline, paired Student's t test for control injection of saline-BSA (1%). With NT 1000 ng a biphasic effect was observed on heart rate; the first number indicates the initial change and below is the later change.
HAEMODYNAMIC EFFECTS OF NEUROTENSIN
193
Cardiovascular responses to neurotensin in rats neonatally treated with capsaicin At least 48 h after birth, Long Evans rats were anaesthetized with 1.5-2% halothane (in oxygen) and injected s.c. with either vehicle (10% ethanol, 10% Tween 80 in isotonic saline) or capsaicin (50mgkg-1) in a volume of 100pl. After weaning (day 21-30 after birth) only male rats were selected for the experiments and housed in groups of 4 or 5 with free access to food and tap water. About 20 weeks later, two groups of rats (vehicle-treated, n = 9 and capsaicin-treated, n = 9), previously instrumented with pulsed Doppler probes and intravascular catheters (as earlier described), were used on 3 consecutive days during which they received i.v. injections of vehicle (0.1 ml isotonic saline containing 1% BSA) and NT (300 and 100ng), in random order. Measurements were made before, during and for 30 min following i.v. injections of vehicle or NT.
E 0)a) .0
20 0
I -0
20 CD
0 2-1-I
-r I
-
-
1
0
2 E -20[gL**
'**
Q.
-40
20 r
ROnol *enai
-20
Drugs
-40 40
Neurotensin (Bachem), phentolamine mesylate (Ciba), propranolol hydrochloride (Imperial Chemical Industries) and 8 methyl-N-vanillyl-6-nonenamide (capsaicin; Sigma) were used.
20
7-
Mesenteric
0
Statistical analysis
~-20
Values are expressed as the mean + s.e.mean; n is the number of animals. The paired Student's t test was used to analyse the effects of control injection of saline-BSA, while comparisons between groups were made by an analysis of variance followed by Fisher's test. A P value < 0.05 was taken to indicate a significant difference.
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Results The baseline values for cardiovascular variables are given in Table 1.
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Haemodynamic responses to neurotensin Control injections caused a slight tachycardia (significant at 1 min), but had no significant effect on the other measured or calculated variables (Table 2). Table 2 shows that the lowest doses of NT tested (10 and 30ng) did not have effects significantly different from those of control injections. However, with the 100ng dose of NT there was a slight increase in blood pressure (significant at 0.5 min) accompanied by falls in superior mesenteric flow (significant at 1-1.5min) and vascular conductance (significant at 1-2 min); these effects were significantly different from those following control injections. There were no changes in heart rate or renal or hindquarters flows or vascular conductances. NT at a dose of 300 ng produced a slight increase in blood pressure (significant at 0.5 and 1.5-5 min) and a tachycardia (significant at 4-10 min). There were long-lasting falls in superior mesenteric (significant at 1-30 min) and hindquarters flows (significant at 3-30min), but no significant change in renal flow. These responses were associated with constrictions in renal (significant at 2-5 min), superior mesenteric (significant at 1-30min) and hindquarters (significant at 230 min) vascular beds (Table 2). The highest dose of NT (1000 ng) caused a slight increase in blood pressure (significant at 2-5 min) accompanied by a transient bradycardia (significant at 1 min) followed by a tachycardia (significant at 3-30 min), and reductions in renal (significant at 4-30min), superior mesenteric (significant at 1-
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Neurotensin 1000 ng Phentolamine Figure 1 Cardiovascular responses to i.v. bolus injections of neurotensin (1000ng) in conscious, unrestrained, Long Evans rats, in the absence, (0) or in the presence (Q) of i.v. treatment with phentolamine (1 mg kg- 1, i.v. bolus, 1 mg kg- I h- 1, infusion). Values are mean with s.e.mean shown by vertical lines. *P < 0.05 for changes in untreated group (n = 15) versus changes in treated group (n = 11) (analysis of variance followed by Fisher's test). t P < 0.05 versus predrug baseline (Student's t test for paired data). HR = heart rate; BP = blood pressure.
30 min) and hindquarters flows (significant at 0.5-30 min) (Figure 1 and Table 2). Hence, there were constrictions in renal (significant at 2-30 min), superior mesenteric (significant at 1-30 min) and hindquarters (significant at 0.5-30 min) vascular beds (Figure 2 and Table 2).
Haemodynamic responses to neurotensin (1000 ng) in the presence of phentolamine Fifteen min after the onset of pretreatment with phentolamine there was no change in blood pressure, but there were persistent increases in heart rate, and hindquarters flow and reductions in renal and superior mesenteric flows. There were
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Neurotensin 1000 Phentolamine
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Figure 2 Changes in regional vascular conductances elicited by i.v. bolus injections of neurotensin (1000ng) in conscious, unrestrained, Long Evans rats, in the absence, (0) or in the presence (Q) of i.v. treatment with phentolamine (1 mgkg-', i.v. bolus, 1 mg kg'- h -1, infusion). These data were derived from those shown in Figure 1. Values are mean with s.e.mean shown by vertical lines. *P < 0.05 for changes in untreated group (n = 15) versus changes in treated group (n = 11) (analysis of variance followed by Fisher's test). t P 0.05 versus pre-drug baseline (Student's t test for paired data).
