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There is evidence that sympathetically evoked vasoconstriction in skeletal muscle is blunted in systemic hypoxia, but the mechanisms underlying this phenomenon are not clear. In Saffan-anaesthetized Wistar rats, we have studied the role of α2-adrenoceptors and neuropeptide Y (NPY) Y1 receptors in mediating vasoconstriction evoked by direct stimulation of the lumbar sympathetic chain by different patterns of impulses in normoxia (N) and systemic hypoxia (H: breathing 8% O2). Patterns comprised 120 impulses delivered in bursts over a 1 min period at 40 or 20 Hz, or continuously at 2 Hz. Hypoxia attenuated the evoked increases in femoral vascular resistance (FVR) by all patterns, the response to 2 Hz being most affected (40 Hz bursts: N = 3.25 ± 0.75 arbitrary resistance units (RU); H = 1.14 ± 0.29 RU). Yohimbine (Yoh, α2-adrenoceptor antagonist) or BIBP 3226 (Y1-receptor antagonist) did not affect baseline FVR. In normoxia, Yoh attenuated the responses evoked by high frequency bursts and 2 Hz, whereas BIBP 3226 only attenuated the response to 40 Hz (40 Hz bursts: N + Yoh = 2.1 ± 0.59 RU; N + BIBP 3226 = 1.9 ± 0.4 RU). In hypoxia, Yoh did not further attenuate the evoked responses, but BIBP 3226 further attenuated the response to 40 Hz bursts. These results indicate that neither α2-adrenoceptors nor Y1 receptors contribute to basal vascular tone in skeletal muscle, but both contribute to constrictor responses evoked by high frequency bursts of sympathetic activity. We propose that in systemic hypoxia, the α2-mediated component represents about 50% of the sympathetically evoked constriction that is blunted, whereas the contribution made by Y1 receptors is resistant. Thus we suggest the importance of NPY in the regulation of FVR and blood pressure increases during challenges such as systemic hypoxia.
The vasodilatation that occurs in skeletal muscle during systemic hypoxia represents a balance between the vasoconstrictor effects of increased muscle sympathetic nerve activity (MSNA) and the dilator influences of local mediators and circulating adrenaline (Marshall, 1994). Thus, in the rat, α-adrenoceptor blockade enhanced the decrease in muscle vascular resistance induced by systemic hypoxia and dilator responses in individual skeletal muscle arterioles, while converting arteriolar constrictor responses to dilator responses (Marshall & Metcalfe, 1988; Mian & Marshall, 1991). Similarly, in human subjects vasodilatation evoked in forearm by systemic hypoxia was augmented by α-adrenoceptor blockade (Weisbrod et al. 2001). Such observations raise the question of whether the vasoconstrictor influence of MSNA is blunted during systemic hypoxia, or there is summation of constrictor and dilator influences and the latter dominates.
There is some evidence that vasoconstriction evoked by increased MSNA is indeed blunted by systemic hypoxia. Reflex vasoconstriction evoked in forearm by lower body negative pressure (LBNP) was reduced by mild and moderate systemic hypoxia (breathing 12 and 10% O2, respectively; Heistad & Wheeler, 1970). Moreover, the increases in MSNA evoked by LBNP were preserved during mild and moderate hypoxia, but the forearm vasoconstrictor responses were reduced (Rowell & Seals, 1990). However, reflex vasoconstriction evoked by LBNP in muscle microcirculation, as assessed by near infra-red spectroscopy, was well maintained during moderate hypoxia (Hansen et al. 2000). Further, forearm vasoconstriction evoked by tyramine, which releases noradrenaline from sympathetic fibres, was preserved during mild and moderate hypoxia (Dinenno et al. 2003).
Such disparities may be attributable to differences in the levels of hypoxia and methods used to activate the sympathetic nerves. In anaesthetized rats, we stimulated the sympathetic chain with three different patterns that delivered the same number of impulses over 1 min (see Coney & Marshall, 2003): continuous stimulation at 2 Hz to represent a low, resting level of MSNA and bursts of impulses at 20 or 40 Hz to represent the high frequency bursts that occur when MSNA is raised, for example by systemic hypoxia (Macefield et al. 1994; Johnson & Gilbey, 1996; Hudson et al. 2002). The increases in femoral vascular resistance (FVR) evoked by bursts at 20 and 40 Hz were not affected by mild or moderate hypoxia, but were considerably blunted by severe hypoxia (8% O2): that evoked by continuous stimulation at 2 Hz was blunted by moderate, as well as severe, hypoxia (Coney & Marshall, 2003).
In view of these findings, the question arises as to whether systemic hypoxia influences the action of the sympathetic cotransmitters. In vitro studies indicate that ATP makes the major contribution to vasoconstriction evoked in arteries by single impulses or short trains at low frequency, while noradrenaline becomes important when the impulse train is longer or the frequency increases (Kennedy et al. 1986; Sjöblom-Widfelt & Nilsson, 1990). On the other hand, in vivo studies on rat hindlimb suggest that ATP and noradrenaline are mutually facilitatory in producing vasoconstriction evoked by sympathetic stimulation with low and high frequencies (Johnson et al. 2001). Further, in vivo studies on skeletal muscle indicate that sympathetic stimulation at constant high frequency, or with irregular activity containing high frequency bursts releases neuropeptide Y (NPY), while in various arterial preparations, high frequency patterns evoke NPY-mediated contstiction (Pernow et al. 1989; Morris & Gibbins, 1992).
Considering these transmitters, the fact that A1 or A2A adenosine receptor blockade had no effect on hypoxia-induced blunting of sympathetically evoked vasoconstriction (see above, Coney & Marshall, 2003) indicates that it is not caused by pre- or postsynaptic actions of adenosine formed from sympathetically released ATP (Ralevic & Burnstock, 1990). Hypoxia may affect the postsynaptic actions of ATP, but this is difficult to test in vivo in the absence of selective P2X receptor antagonists. Severe systemic hypoxia inhibits vasoconstriction evoked by noradrenaline infusion in rats (Coney & Marshall, 2003) and humans (Heistad & Wheeler, 1970). Moreover, graded hypoxia ( 100–40 mmHg) produced graded depression of contraction evoked by stimulation of α1-adrenoceptors in rat iliac and other arteries (Ebeigbe, 1982; Pearce et al. 1992; Franco-Obregon & Lopez-Barneo, 1996; Bartlett & Marshall, 2002). However, sympathetically evoked vasoconstriction in skeletal muscle is partly mediated by α2-adrenoceptors (Ohyanagi et al. 1991; Dinenno et al. 2002) and α2-adrenoceptor-evoked arteriolar constriction is particularly vulnerable to local hypoxia (Tateishi & Faber, 1995). Whether the α2-adrenoceptor component of sympathetic vasoconstriction is blunted by systemic hypoxia has not been examined. Further, there is little pharmacological evidence on the contribution of NPY to sympathetically evoked vasoconstriction in skeletal muscle in vivo (see Jackson et al. 2004).
Thus, we tested the hypotheses that severe systemic hypoxia blunts muscle vasoconstriction evoked by sympathetic chain stimulation with high and low frequency patterns chosen to mimic the range of MSNA that occur at rest and during reflex activation, by limiting the components that are mediated by α2-adrenoceptors and by NPY acting on Y1 receptors, the NPY receptors that are mainly responsible for inducing vasoconstriction (Modin et al. 1991).
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Experiments were performed on two groups of male Wistar rats. All experiments were approved by UK legislation under the Home Office Animals (Scientific Procedures) Act 1986. Anaesthesia was induced with halothane (3.5% in O2) and judged to be of a surgical level when pedal withdrawal reflexes were absent. A jugular vein was then cannulated for continuous infusion of the anaesthetic Saffan (Schering-Plough Animal Health, Welwyn Garden City, UK) at 7–12 mg kg−1 h−1. Thereafter, the level of anaesthesia was judged to be adequate by the absence of withdrawal reflexes and stability of arterial blood pressure (ABP), as we have previously described (Coney & Marshall, 2003). At the end of the experiments all animals were killed by anaesthetic overdose.
The surgical preparation of the animal and the recording techniques were essentially as we have described before (Coney & Marshall, 2003). Briefly, the trachea was cannulated and the side-arm of the cannula was connected to a system of rotameters in a gas proportioner frame (CP Instruments, Hanwell, London, UK), allowing the rats to breathe either a normoxic (21% O2 in N2) or a hypoxic (8% O2 in N2) gas mixture, as described below. Both brachial arteries were cannulated, one being used to monitor arterial blood pressure (ABP), the other allowing 150 μl samples to be taken anaerobically for analysis by a blood gas analyser (IL1640; Instrumentation Laboratories, Warrington, Cheshire, UK): samples were taken in normoxia and during hypoxia following the period of sympathetic stimulation (see below). The caudal ventral tail artery was retrogradely cannulated to allow administration of drugs predominantly to the right hindlimb from which blood flow was recorded (see below). The left femoral vein was cannulated to allow administration of pharmacological antagonists.
A bipolar, silver-wire stimulating electrode was attached to the right lumbar sympathetic chain between L3 and L4 via a laparotomy, the great vessels being temporarily retracted to expose the sympathetic chain. The electrode tips were embedded in dental impression material (President, Light Body, Colténe, Switzerland) to both mechanically fix and electrically isolate them. The electrodes were used to deliver three different patterns of nerve stimulation at constant current via an isolated stimulator (DS2A; Digitimer, UK). The patterns delivered the same number of 1 ms pulses at a constant current of 1 mA in a 1 min period (see Coney & Marshall, 2003): (1) continuous stimulation at 2 Hz, (2) a 20 Hz burst for 1 s repeated every 10 s (bursts at 20 Hz), and (3) a 40 Hz burst for 0.5 s repeated every 10 s (bursts at 40 Hz). Each pattern resulted in 120 impulses being delivered over the 1 min stimulation period: they were delivered in random order.
Blood flow was recorded from the right femoral artery (FBF) via a perivascular flowprobe (0.7V; Transonic Systems, Ithaca, NY, USA) connected to a flowmeter (T106; Transonic Systems). ABP and FBF were acquired into Chart software (ADInstruments) via a MacLab/8 s (ADInstruments) at a sampling frequency of 100 Hz. Mean arterial pressure (MAP) and heart rate (HR) were derived on-line from the ABP signal, and femoral vascular resistance (FVR) was calculated on-line, by the division of ABP by FBF.
Group 1: sympathetic stimulation before and after α2-adrenoceptor antagonism The responses evoked by sympathetic stimulation with bursts at 40 and 20 Hz and by continuous stimulation at 2 Hz were tested in seven rats (body mass 218 ± 8 g: mean ±s.d.) during normoxia. The inspirate was then switched to hypoxia (8% O2) and at least 1 min after a new steady baseline had been achieved, the responses evoked by sympathetic stimulation were re-tested. The inspirate was then returned to normoxia and the animal allowed to recover for at least 10–15 min. The α2-adrenoceptor antagonist yohimbine (Sigma, UK) was administered intravenously at a dose of 0.5 mg kg−1 and following an equilibration period of 10–15 min, the responses to stimulation were re-tested under both normoxic and hypoxic conditions. In previous studies, this dose of yohimbine was shown to selectively and maximally inhibit α2-adrenoceptor-mediated sympathoinhibition (Szabo et al. 1993). In pilot studies this dose abolished the increase in ABP evoked by infusion of the α2-adrenoceptor agonist UK14304 (A. M. Coney & J. M. Marshall, unpublished observations).
Group 2: sympathetic stimulation before and during infusion of an NPY Y1 receptor antagonist This protocol was performed in seven rats (mean ±s.d., body mass 213 ± 11 g). The protocol was essentially the same as for Group 1, except that when the responses to all three patterns of sympathetic stimulation had been tested in normoxia and during hypoxia, the animal was allowed to recover in normoxia for at least 10–15 min and then the NPY Y1 receptor antagonist BIBP 3226 (Sigma, UK) was infused through the caudal ventral artery at a rate of 10 μg kg−1 min−1. Responses evoked in normoxia, and in acute hypoxia were then re-tested as described above. This regime for administration of BIBP 3226 was shown in a previous study on the rat to selectively reduce the facilitation produced by NPY of peripheral vasoconstrictor responses evoked by α1-adrenoceptor stimulation (Bischoff et al. 1997). Further, in a pilot study on four rats, we found that administration of Leu-Pro NPY (a Y1 agonist) at 128 μg kg−1 increased ABP by 32 ± 3 mmHg. This was completely abolished by the above regime of BIBP 3226 administration.
All data are expressed as mean ±s.e.m. Data were analysed as described in Coney & Marshall (2003). Thus, FVR (mmHg ml−1 min) was computed on-line by the division of ABP by FBF and the size of the constrictor response to sympathetic stimulation was calculated by subtracting the integral of baseline FVR calculated from the 1 min preceding the stimulus, from the integral of FVR measured during the 1 min stimulus. The integrated constrictor response was expressed in arbitrary resistance units (RU). This allowed comparison between responses evoked by different patterns of stimulation as well as between responses evoked from different baseline values of FVR, i.e. during normoxia and hypoxia. Differences in baseline and in integrated FVR before and after each antagonist were determined by repeated measures ANOVA followed by Student–Newman–Keuls post hoc test if P < 0.05.