Intrathecal orexin A increases sympathetic outflow and respiratory drive, enhances baroreflex sensitivity and blocks the somato-sympathetic reflex

Abstract

Background: Intrathecal (i.t.) injection of orexin A (OX-A) increases blood pressure and heart rate (HR), but the effects of OX-A on sympathetic and phrenic, nerve activity, and the baroreflex(es), somato-sympathetic and hypoxic chemoreflex(es) are unknown.

Experimental approach: Urethane-anaesthetized, vagotomized and artificially ventilated male Sprague-Dawley rats were examined in this study. The effects of i.t. OX-A (20 nmol 10 µL⁻¹) on cardiorespiratory parameters, and responses to stimulation of the sciatic nerve (electrical), arterial baroreceptors (phenylephrine hydrochloride, 0.01 mg kg⁻¹ i.v.) and peripheral (hypoxia) chemoreceptors were also investigated.

Key results: i.t. OX-A caused a prolonged dose-dependent sympathoexcitation, pressor response and tachycardia. The peak effect was observed at 20 nmol with increases in mean arterial pressure, HR and splanchnic sympathetic nerve activity (sSNA) of 32 mmHg, 52 beats per minute and 100% from baseline respectively. OX-A also dose-dependently increased respiratory drive, as indicated by a rise in phrenic nerve amplitude and a fall in phrenic nerve frequency, an increase in neural minute ventilation, a lengthening of the expiratory period, and a shortening of the inspiratory period. All effects of OX-A (20 nmol) were attenuated by the orexin receptor 1 antagonist SB 334867. OX-A significantly reduced both sympathoexcitatory peaks of somato-sympathetic reflex while increasing baroreflex sensitivity. OX-A increased the amplitude of the pressor response and markedly amplified the effect of hypoxia on sSNA.

Conclusions: Thus, activation of OX receptors in rat spinal cord alters cardiorespiratory function and differentially modulates sympathetic reflexes.

Figure 1

Effect of intrathecal injection of orexin A (OX-A) on mean arterial pressure (MAP), heart rate (HR) and splanchnic sympathetic nerve activity (sSNA). (A) Representative trace of data from a recording of blood pressure (BP), HR and sSNA (arbitrary unit, a.u.) before and after injection of phosphate-buffered saline (PBS) or OX-A (20 nmol). Rectified and integrated sSNA (black) is superimposed over raw sSNA (grey). MAP (black) is superimposed over BP (grey). (B) Comparison of peak cardiovascular effects produced by PBS, OX-A (1, 5, 10 and 20 nmol) or SB334867 (200 nmol) + OX-A (20 nmol). Peak effects are shown as absolute (BP, HR) or percentage (sSNA) change from their respective basal values. (C) Grouped time course effects of PBS (black) or OX-A (20 nmol) (red) on MAP, HR and sSNA. Values are expressed as mean ± standard error. Number of animals are shown in parentheses. bpm, beats per minute; ns, non-significant; ***P < 0.001, **P < 0.01, *P < 0.05 compared with PBS [except SB 334867 (200 nmol) + OX-A (20 nmol) that was compared with OX-A (20 nmol)].

Figure 2

Effect of intrathecal injection of orexin A (OX-A)on phrenic nerve activity (PNA). (A) Representative trace of data from a recordings of rectified PNA (arbitrary unit, a.u.), phrenic nerve frequency (PNf), phrenic nerve amplitude (PNamp), inspiratory period (T_I) and expiratory period (T_E) before and after injection of phosphate-buffered saline (PBS) or OX-A. (B) Grouped time course effects of PBS (black) or OX-A (20 nmol) (red) on PNamp, PNf, neural minute ventilation, T_I and T_E. Following injection of OX-A there is an increase in PNamp associated with a bradypnoea that is due to both an increase in T_E and a decrease in T_I. There is an overall increase in neural minute ventilation over the period of the response. Values are expressed as mean ± standard error. Number of animals is shown in parentheses. bpm, beats per minute; ns, non-significant; ***P < 0.001, **P < 0.01, *P < 0.05 compared with PBS.

Figure 3

Effect of intrathecal injection of orexin A (OX-A)on phrenic nerve activity (PNA). (A) Comparison of peak effects produced by phosphate-buffered saline (PBS), OX-A (1, 5, 10 and 20 nmol) or SB 334867 (200 nmol) + OX-A (20 nmol) on PNamp, PNf and neural minute ventilation. Peak effects are shown as absolute or percentage change from respective basal values. (B) Grouped data illustrating the effects of PBS and OX-A (20 nmol) on T_I and T_E. Values are expressed as mean ± standard error. Number of animals is shown in parentheses. bpm, beats per minute; ns, non-significant; ***P < 0.001, **P < 0.01, *P < 0.05 compared with PBS [except SB 334867 (200 nmol) + OX-A (20 nmol) that was compared with OX-A (20 nmol)].

Figure 4

Effect of orexin A (OX-A) on phrenic nerve discharge-related rhythmicity of sSNA. (A) Phrenic-triggered average of sSNA before (black) and after (red) intrathecal injection of OX-A (20 nmol). (B) Time course effect of OX-A (20 nmol) on inspiratory (I) and post-inspiratory (PI) related activity of sSNA. (C) Grouped data illustrating the effects of OX-A (20 nmol, n= 5) on I and PI peaks of sSNA. Note that there is a marked increase in PI following OX-A injection. Values are expressed as mean ± standard error. ns, non-significant; *P < 0.05 compared with phosphate-buffered saline (PBS). Both PBS and OX-A values were normalized to the control period prior to injections.

Figure 5

Effect of intrathecal orexin A (OX-A; 20 nmol) on mean arterial pressure (MAP), heart rate (HR), splanchnic sympathetic nerve activity (sSNA) and phrenic nerve activity (PNA) in an intact rat (A) and a C8 anaesthetized rat (B). (A) Representative trace of data from a recording of PNA, blood pressure (BP), HR and sSNA (arbitrary unit, a.u.) in intact rat. Integrated sSNA (black) is superimposed over raw sSNA (grey). MAP (black) is superimposed over blood pressure (BP) (grey). (B) Representative trace of data from a recording of PNA, BP, HR and sSNA in a C8 anaesthetized rat. (C) Comparison of peak cardio-respiratory effects produced by OX-A (20 nmol) in intact (n= 6) and C8 anaesthetized rat (n= 3). Peak effects are shown as absolute or percentage change from respective basal values. Values are expressed as mean ± standard error. Note that OX-A causes a greater increase in MAP, HR and sSNA for a shorter period when compared with the response prior to C8 anaesthesia. The effect on PNA was abolished. Number of animals is shown in parentheses. bpm, beats·per minute (HR) or bursts·per minute (PNf); ns, non-significant; *P < 0.05 compared with OX-response in intact animal.

Figure 6

Effect of intrathecal injection of orexin A (OX-A) on somato-sympathetic, baroreceptor and peripheral chemoreceptor reflex. (A) Effect on somato-sympathetic reflex evoked by sciatic nerve (SN) stimulation. Left 3 traces represent grouped effect of SN-evoked stimulation of sSNA at control period and after injection of phosphate-buffered saline and OX-A. Data are mean (black) ± standard error (grey). Arrows indicate the time of stimulation. Trace at right represents group data illustrating the heights of 1st and 2nd sympathoexcitatory peaks. (B) Effect on baroreceptor reflex evoked by intravenous injection of SNP and PE. Left trace represents experimental recording of the effect of changes in BP on sSNA due to SNP or PE after phosphate-buffered saline (PBS) and OX-A injection. Middle trace shows average sympathetic baroreflex functional curves generated for data after PBS (black) or OX-A (red) injection. Trace at right represents baroreflex gain for sSNA (error bars are omitted for clarity). (C) Effect of peripheral chemoreceptor reflex activated by brief hypoxia with 100% N2 for 12–14 s. Left trace shows experimental recording of hypoxic episodes at control period and after PBS or OX-A injection. 3 traces of right represent comparison of peak effects on MAP, HR and sSNA after intrathecal injection of PBS and OX-A in response to brief hypoxia. Values are expressed as mean ± standard error. Number of animals is shown in parentheses. bpm, beats per minute; ns, non-significant; **P < 0.01, *P < 0.05 compared with PBS.