Davi J. A. Moraes

The carotid body as a putative therapeutic target for the treatment of neurogenic hypertension

In the spontaneously hypertensive (SH) rat, hyperoxic inactivation of the carotid body (CB) produces a rapid and pronounced fall in both arterial pressure and renal sympathetic nerve activity (RSA). Here we show that CB de-afferentation through carotid sinus nerve denervation (CSD) reduces the overactive sympathetic activity in SH rats, providing an effective antihypertensive treatment. We demonstrate that CSD lowers RSA chronically and that this is accompanied by a depressor response in SH but not normotensive rats. The drop in blood pressure is not dependent on renal nerve integrity but mechanistically accompanied by a resetting of the RSA-baroreflex function curve, sensitization of the cardiac baroreflex, changes in renal excretory function and reduced T-lymphocyte infiltration. We further show that combined with renal denervation, CSD remains effective, producing a summative response indicative of an independent mechanism. Our findings indicate that CB de-afferentation is an effective means for robust and sustained sympathoinhibition, which could translate to patients with neurogenic hypertension.

Electrophysiological Properties of Rostral Ventrolateral Medulla Presympathetic Neurons Modulated by the Respiratory Network in Rats

The respiratory pattern generator modulates the sympathetic outflow, the strength of which is enhanced by challenges produced by hypoxia. This coupling is due to the respiratory-modulated presympathetic neurons in the rostral ventrolateral medulla (RVLM), but the underlining electrophysiological mechanisms remain unclear. For a better understanding of the neural substrates responsible for generation of this respiratory-sympathetic coupling, we combined immunofluorescence, single cell qRT-pCR, and electrophysiological recordings of the RVLM presympathetic neurons in in situ preparations from normal rats and rats submitted to a metabolic challenge produced by chronic intermittent hypoxia (CIH). Our results show that the spinally projected cathecholaminergic C1 and non-C1 respiratory-modulated RVLM presympathetic neurons constitute a heterogeneous neuronal population regarding the intrinsic electrophysiological properties, respiratory synaptic inputs, and expression of ionic currents, albeit all neurons presented persistent sodium current-dependent intrinsic pacemaker properties after synaptic blockade. A specific subpopulation of non-C1 respiratory-modulated RVLM presympathetic neurons presented enhanced excitatory synaptic inputs from the respiratory network after CIH. This phenomenon may contribute to the increased sympathetic activity observed in CIH rats. We conclude that the different respiratory-modulated RVLM presympathetic neurons contribute to the central generation of respiratory-sympathetic coupling as part of a complex neuronal network, which in response to the challenges produced by CIH contribute to respiratory-related increase in the sympathetic activity.

Intermittent hypoxia-induced sensitization of central chemoreceptors contributes to sympathetic nerve activity during late expiration in rats.

Hypertension elicited by chronic intermittent hypoxia (CIH) is associated with elevated activity of the thoracic sympathetic nerve (tSN) that exhibits an enhanced respiratory modulation reflecting a strengthened interaction between respiratory and sympathetic networks within the brain stem. Expiration is a passive process except for special metabolic conditions such as hypercapnia, when it becomes active through phasic excitation of abdominal motor nerves (AbN) in late expiration. An increase in CO(2) evokes late-expiratory (late-E) discharges phase-locked to phrenic bursts with the frequency increasing quantally as hypercapnia increases. In rats exposed to CIH, the late-E discharges synchronized in AbN and tSN emerge in normocapnia. To elucidate the possible neural mechanisms underlying these phenomena, we extended our computational model of the brain stem respiratory network by incorporating a population of presympathetic neurons in the rostral ventrolateral medulla that received inputs from the pons, medullary respiratory compartments, and retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG). Our simulations proposed that CIH conditioning increases the CO(2) sensitivity of RTN/pFRG neurons, causing a reduction in both the CO(2) threshold for emerging the late-E activity in AbN and tSN and the hypocapnic threshold for apnea. Using the in situ rat preparation, we have confirmed that CIH-conditioned rats under normal conditions exhibit synchronized late-E discharges in AbN and tSN similar to those observed in control rats during hypercapnia. Moreover, the hypocapnic threshold for apnea was significantly lowered in CIH-conditioned rats relative to that in control rats. We conclude that CIH may sensitize central chemoreception and that this significantly contributes to the neural impetus for generation of sympathetic activity and hypertension.

Medullary Respiratory Network Drives Sympathetic Overactivity and Hypertension in Rats Submitted to Chronic Intermittent Hypoxia

Hypertension is a pathological condition affecting up to one third of adult population worldwide.1 Over the last few decades, the remarkable progress of the pharmacological antihypertensive therapies, such as β-adrenergic blockers, angiotensin-converting enzyme inhibitors, angiotensin II receptor type 1 antagonists, and diuretics, combined with physical activities and dietary orientations, contributed significantly to improve life quality of millions of hypertensive patients around the world by lowering their arterial pressure. However, in ≈30% of hypertensive patients the arterial blood pressure remains elevated in spite of the use of different pharmacological strategies,1–4 indicating not only that these therapies are not effective for all patients but also that the mechanisms underpinning the chronic increase of arterial pressure are not completely understood. In this context, a relevant example is the hypertension observed in patients suffering of the syndrome of obstructive sleep apnea (OSA). Over the last 2 decades, it has been demonstrated that the OSA, a condition that affects ≈20% of adult population in the United States,5 is an important risk factor for the development of arterial hypertension,6 and it is one of the major causes of secondary hypertension in patients presenting hypertension resistant to pharmacological treatment.7 Regardless of the marked correlation between OSA and arterial hypertension,8 the mechanisms contributing to the development of hypertension in OSA patients are not fully elucidated. There is evidence that hypertension in OSA patients is markedly associated with the long-term exposure to episodic hypoxemia (or intermittent hypoxia) as a consequence of recurrent obstructions of upper airways during sleep.9 The critical role of chronic intermittent hypoxia (CIH) exposure in the development of cardiovascular changes induced by OSA is supported not only by clinical studies reporting the beneficial cardiovascular effects of the treatment of OSA patients with continuous positive airway pressure10 …

Purinergic receptors in the carotid body as a new drug target for controlling hypertension

In view of the high proportion of individuals with resistance to antihypertensive medication and/or poor compliance or tolerance of this medication, new drugs to treat hypertension are urgently needed. Here we show that peripheral chemoreceptors generate aberrant signaling that contributes to high blood pressure in hypertension. We discovered that purinergic receptor P2X3 (P2rx3, also known as P2x3) mRNA expression is upregulated substantially in chemoreceptive petrosal sensory neurons in rats with hypertension. These neurons generate both tonic drive and hyperreflexia in hypertensive (but not normotensive) rats, and both phenomena are normalized by the blockade of P2X3 receptors. Antagonism of P2X3 receptors also reduces arterial pressure and basal sympathetic activity and normalizes carotid body hyperreflexia in conscious rats with hypertension; no effect was observed in rats without hypertension. We verified P2X3 receptor expression in human carotid bodies and observed hyperactivity of carotid bodies in individuals with hypertension. These data support the identification of the P2X3 receptor as a potential new target for the control of human hypertension.

Specific Respiratory Neuron Types Have Increased Excitability That Drive Presympathetic Neurones in Neurogenic Hypertension

A major aspect of hypertension is excessive sympathetic activity but the reasons for this remain elusive. Sympathetic tone is increased in the spontaneously hypertensive (SH) rat reflecting, in part, enhanced respiratory–sympathetic coupling. We aimed to identify which respiratory cells might have altered properties. Using the working heart–brain stem preparation, we monitored simultaneously sympathetic and respiratory nerve activity in combination with intracellular recordings of physiologically characterized medullary presympathetic or respiratory neurons. In SH rats, respiratory modulation of both inspiratory and postinspiratory phases of sympathetic activity was larger relative to Wistar rats. An additional burst of sympathetic activity in the preinspiratory phase was also present in SH rats. After synaptic isolation of rostral medullary presympathetic neurons, there was no difference in their excitability compared with neurons in Wistar rats. Rather, both pre-Bötzinger preinspiratory and Bötzinger postinspiratory neurons had increased neuronal excitability in SH rats relative to Wistar rats; this was attributed to higher input resistance/reduced leak current in preinspiratory neurons and reduced calcium activated potassium conductance in postinspiratory neurons. Thus, the respiratory network of the SH rat is reconfigured to a pattern dominated by heightened excitability of preinspiratory and postinspiratory neurons. These neurons both provide augmented excitatory synaptic drive to rostral medullary presympathetic neurons contributing to excessive sympathetic nerve activity associated with hypertension in the in situ SH rat. Our data indicate selective modulation of potassium conductances in 2 subsets of respiratory neurons contributing to neurogenic hypertension.

Sympathoexcitation during chemoreflex active expiration is mediated by l-glutamate in the RVLM/Bötzinger complex of rats

The involvement of glutamatergic neurotransmission in the rostral ventrolateral medulla/Bötzinger/pre-Bötzinger complexes (RVLM/BötC/pre-BötC) on the respiratory modulation of sympathoexcitatory response to peripheral chemoreflex activation (chemoreflex) was evaluated in the working heart-brain stem preparation of juvenile rats. We identified different types of baro- and chemosensitive presympathetic and respiratory neurons intermingled within the RVLM/BötC/pre-BötC. Bilateral microinjections of kynurenic acid (KYN) into the rostral aspect of RVLM (RVLM/BötC) produced an additional increase in frequency of the phrenic nerve (PN: 0.38 ± 0.02 vs. 1 ± 0.08 Hz; P < 0.05; n = 18) and hypoglossal (HN) inspiratory response (41 ± 2 vs. 82 ± 2%; P < 0.05; n = 8), but decreased postinspiratory (35 ± 3 vs. 12 ± 2%; P < 0.05) and late-expiratory (24 ± 4 vs. 2 ±1%; P < 0.05; n = 5) abdominal (AbN) responses to chemoreflex. Likewise, expiratory vagal (cVN; 67 ± 6 vs. 40 ± 2%; P < 0.05; n = 5) and expiratory component of sympathoexcitatory (77 ± 8 vs. 26 ± 5%; P < 0.05; n = 18) responses to chemoreflex were reduced after KYN microinjections into RVLM/BötC. KYN microinjected into the caudal aspect of the RVLM (RVLM/pre-BötC; n = 16) abolished inspiratory responses [PN (n = 16) and HN (n = 6)], and no changes in magnitude of sympathoexcitatory (n = 16) and expiratory (AbN and cVN; n = 10) responses to chemoreflex, producing similar and phase-locked vagal, abdominal, and sympathetic responses. We conclude that in relation to chemoreflex activation 1) ionotropic glutamate receptors in RVLM/BötC and RVLM/pre-BötC are pivotal to expiratory and inspiratory responses, respectively; and 2) activation of ionotropic glutamate receptors in RVLM/BötC is essential to the coupling of active expiration and sympathoexcitatory response.

Contribution of the retrotrapezoid nucleus/parafacial respiratory region to the expiratory-sympathetic coupling in response to peripheral chemoreflex in rats

Central mechanisms of coupling between respiratory and sympathetic systems are essential for the entrainment between the enhanced respiratory drive and sympathoexcitation in response to hypoxia. However, the brainstem nuclei and neuronal network involved in these respiratory-sympathetic interactions remain unclear. Here, we evaluated whether the increase in expiratory activity and expiratory-modulated sympathoexcitation produced by the peripheral chemoreflex activation involves the retrotrapezoid nucleus/parafacial respiratory region (RTN/pFRG). Using decerebrated arterially perfused in situ rat preparations (60-80 g), we recorded the activities of thoracic sympathetic (tSN), phrenic (PN), and abdominal nerves (AbN) as well as the extracellular activity of RTN/pFRG expiratory neurons, and reflex responses to chemoreflex activation were evaluated before and after inactivation of the RTN/pFRG region with muscimol (1 mM). In the RTN/pFRG, we identified late-expiratory (late-E) neurons (n = 5) that were silent at resting but fired coincidently with the emergence of late-E bursts in AbN after peripheral chemoreceptor activation. Bilateral muscimol microinjections into the RTN/pFRG region (n = 6) significantly reduced basal PN frequency, mean AbN activity, and the amplitude of respiratory modulation of tSN (P < 0.05). With respect to peripheral chemoreflex responses, muscimol microinjections in the RTN/pFRG enhanced the PN inspiratory response, abolished the evoked late-E activity of AbN, but did not alter either the magnitude or pattern of the tSN reflex response. These findings indicate that the RTN/pFRG region is critically involved in the processing of the active expiratory response but not of the expiratory-modulated sympathetic response to peripheral chemoreflex activation of rat in situ preparations.

Modulation of respiratory responses to chemoreflex activation by l-glutamate and ATP in the rostral ventrolateral medulla of awake rats

Presympathetic neurons in the different anteroposterior aspects of rostral ventrolateral medulla (RVLM) are colocalized with expiratory [Bötzinger complex (BötC)] and inspiratory [pre-Bötzinger complex (pre-BötC)] neurons of ventral respiratory column (VRC), suggesting that this region integrates the cardiovascular and respiratory chemoreflex responses. In the present study, we evaluated in different anteroposterior aspects of RVLM of awake rats the role of ionotropic glutamate and purinergic receptors on cardiorespiratory responses to chemoreflex activation. The bilateral ionotropic glutamate receptors antagonism with kynurenic acid (KYN) (8 nmol/50 nl) in the rostral aspect of RVLM (RVLM/BötC) enhanced the tachypneic (120 ± 9 vs. 180 ± 9 cpm; P < 0.01) and attenuated the pressor response (55 ± 2 vs. 15 ± 1 mmHg; P < 0.001) to chemoreflex activation (n = 7). On the other hand, bilateral microinjection of KYN into the caudal aspect of RVLM (RVLM/pre-BötC) caused a respiratory arrest in four awake rats used in the present study. Bilateral P2X receptors antagonism with PPADS (0.25 nmol/50 nl) in the RVLM/BötC reduced chemoreflex tachypneic response (127 ± 6 vs. 70 ± 5 cpm; P < 0.001; n = 6), but did not change the chemoreflex pressor response. In addition, PPADS into the RVLM/BötC attenuated the enhancement of the tachypneic response to chemoreflex activation elicited by previous microinjections of KYN into the same subregion (188 ± 2 vs. 157 ± 3 cpm; P < 0.05; n = 5). Our findings indicate that: 1) L-glutamate, but not ATP, in the RVLM/BötC is required for pressor response to peripheral chemoreflex and 2) both transmitters in the RVLM/BötC are required for the processing of the ventilatory response to peripheral chemoreflex activation in awake rats.

Short-term sustained hypoxia induces changes in the coupling of sympathetic and respiratory activities in rats

Hypoxia activates peripheral chemoreceptors producing an increase in breathing and arterial pressure. In conditions of sustained hypoxia, an increase in ventilation and arterial blood pressure is observed that persists after the return to normoxia. We show in rats that sustained hypoxia for 24 h produces glutamate‐dependent changes in the activity of expiratory and sympathetic neurones of the rostral ventrolateral medulla, which are essential for the control of respiratory and sympathetic activities. These neuronal changes induced by sustained hypoxia are critical for the emergence of coupled active expiration and augmented sympathetic activity. These findings contribute to a better understanding of cardiorespiratory adjustments associated with sustained hypoxia in individuals experiencing high altitudes.