Differential modulation of inspiratory motoneurons and respiratory rhythm generating circuits by ATP

Abstract

Adenosine triphosphate (ATP) gates a diverse family of P2 (P2X1-7 and P2Y1-11) receptors that are expressed throughout the CNS, including respiratory regions of the brainstem [1]. Activation of ATP receptors potentiates activity of inspiratory motoneurons (MN) [2], modulates respiratory behaviour in vivo when applied to medullary respiratory nuclei and, via activation of pH-sensitive P2X2 receptors, may contribute to central respiratory chemosensitivity [3]. Aims of this study were to: i) determine the effects of ATP receptor activation within the pre-Botzinger complex (preBotC, proposed site of rhythm generation) on respiratory behaviour; ii) test the hypothesis that respiratory networks are endogenously modulated by ATP; and iii) compare the sensitivity of inspiratory MN pools and rhythm generating networks to purinergic modulation. To determine the effects of ATP on respiratory rhythm and inspiratory motor output, ATP was pressure-injected into the pre-BotC and XII nuclei of rhythmically-active medullary slice preparations from neonatal rats, while monitoring XII nerve and MN output. Effects on phrenic MNs were determined by locally applying drugs over the phrenic MN pool of brainstem spinal cord preparations. ATP (10 s, 0.01–1 mM) caused up to a 4-fold, suramin-sensitive (0.05–1.0 mM), increase in frequency, that was followed by a brief (22 ± 5%) reduction. To test whether this post-ATP inhibition was due to hydrolysis of ATP to adenosine and activation of adenosine receptors, we applied ATPγs, a non-hydrolyzable ATP analogue. Peak potentiation of frequency by 0.1 mM ATPγs (3.20 ± 0.3 fold increase, n = 7) was similar to that evoked by ATP (3.30 ± 0.3, n = 7), but the effects of ATPγs were longer lasting (102.9 s ± 10.64 vs 29.3 s ± 2.06 for ATP). The secondary reduction in frequency was also absent following ATPγs. Since P2 receptor antagonists also antagonize glutamate receptors which are essential for rhythm generation, the role of endogenous ATP in modulating respiratory rhythm was investigated via bath application of ectoATPase inhibitors (DEPC, pCMPS) and an allosteric modulator of P2X2 recptors (Cu2+). DEPC (100 μM, n = 4) and pCMPS (30 μM, n = 3) increased respiratory frequency 1.30–1.40 fold, while Cu2+ (10–50 μM, n = 6) increased frequency ~1.5-fold. Local application of ATP (1–10 mM) over XII and phrenic nuclei produced a biphasic response comprising an initial potentiation of burst amplitude (1.40 ± 0.20 of control and 1.22 ± 0.7 respectively) followed by a decrease in burst amplitude (to 0.82 ± 0.05 and 0.90 ± 0.05 of control respectively) that was theophylline-sensitive and absent following application of ATP-γ-s. The doses of ATP required to potentiate burst amplitude by 1.20–1.40 of control [2] were ~100 times higher than required to increase rhythm 2.5- to 4-fold. Results show that exogenous ATP potently increases respiratory frequency and that rhythm generating networks are significantly more sensitive to ATP than respiratory motoneurons. In addition, effects of ectoATPase inhibitors and allosteric modulators of ATP receptors suggest that respiratory networks are endogenously modulated by ATP. The differential sensitivity of rhythm generating elements and motoneurons provides an opportunity to explore the physiological significance of P2 receptor diversity to respiratory control.