ABSTRACT
Carotid body (CB) glomus cells depolarize in response to hypoxia, causing a [Ca(2+)](i) increase, at least in part, through activation of voltage-dependent channels. Recently, Turner et al. (2013) showed that mouse glomus cells with knockout of TASK1/3(-/-) channels have near-normal [Ca(2+)](i) response to hypoxia. Thus, we postulated that TRP channels may provide an alternate calcium influx pathway which may be blocked by the TRP channel antagonist, 2-APB (2-aminoethoxydiphenylborane). We confirmed that 2-APB inhibited the afferent nerve response to hypoxia, as previously reported (Lahiri S, Patel G, Baby S, Roy A (2009) 2-APB mediated effects on hypoxic calcium influx in rat carotid body glomus cells. FASEB 2009, Abstract, LB157; Kumar P, Pearson S, Gu Y (2006) A role for TRP channels in carotid body chemotransduction? FASEB J 20:A12-29). To examine the mechanism for this inhibition, we examined dissociated rat CB glomus cells for [Ca(2+)](i) responses to hypoxia, anoxia (with sodium dithionite), 20 mM K(+), NaSH, NaCN, and FCCP in absence/presence of 2-APB (100 µM). Also the effect of 2-APB on hypoxia and/or anoxia were investigated on NADH and mitochondria (MT) membrane potential. Our findings are as follows: (1) 2-APB significantly blocked the [Ca(2+)](i) increase in response to hypoxia and anoxia, but not the responses to 20 mM K(+). (2) The [Ca(2+)](i) responses NaSH, NaCN, and FCCP were significantly blocked by 2-APB. (3) Hypoxia-induced increases in NADH/NAD(+) and MT membrane depolarization were not effected by 2-APB. Thus TRP channels may provide an important pathway for calcium influx in glomus cells in response to hypoxia.
Subject(s)
Carotid Body/physiology , Transient Receptor Potential Channels/physiology , Animals , Boron Compounds/pharmacology , Calcium/metabolism , Carotid Body/cytology , Female , Male , Membrane Potential, Mitochondrial , Mitochondria/physiology , NAD/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
The metabolic hypothesis of carotid body chemoreceptor hypoxia transduction proposes an impairment of ATP production as the signal for activation. We hypothesized that mitochondrial complex IV blockers and hypoxia would act synergistically in exciting afferent nerve activity. Following a pre-treatment with low dosage sodium cyanide (10-20µM), the hypoxia-induced nerve response was significantly reduced along with hypoxia-induced catecholamine release. However, in isolated glomus cells, the intracellular calcium response was enhanced as initially predicted. This suggests a cyanide-mediated impairment in the step between the glomus cell intracellular calcium rise and neurotransmitter release from secretory vesicles. Administration of a PKC blocker largely reversed the inhibitory actions of cyanide on the neural response. We conclude that the expected synergism between cyanide and hypoxia occurs at the level of glomus cell intracellular calcium but not at downstream steps due to a PKC-dependent inhibition of secretion. This suggests that at least one regulatory step beyond the glomus cell calcium response may modulate the magnitude of chemoreceptor responsiveness.
Subject(s)
Carotid Body/cytology , Carotid Body/metabolism , Carotid Body/physiopathology , Electron Transport Complex IV/metabolism , Enzyme Inhibitors/pharmacology , Hypoxia/metabolism , Action Potentials/drug effects , Adenosine Triphosphate/pharmacology , Analysis of Variance , Animals , Animals, Newborn , Calcium/metabolism , Catecholamines/metabolism , Chemoreceptor Cells/drug effects , Chemoreceptor Cells/physiology , Dose-Response Relationship, Drug , Electron Transport Complex IV/antagonists & inhibitors , Patch-Clamp Techniques , Rats , Sodium Cyanide/pharmacologyABSTRACT
Chronic post-natal hyperoxia reduces the hypoxic ventilatory response by reducing the carotid body sensitivity to acute hypoxia as demonstrated by a reduced afferent nerve response, reduced calcium response of carotid body glomus cells and reduced catecholamine secretion in response to acute hypoxia. The present study examined whether hyperoxia alters the electrophysiological characteristics of glomus cells. Rats were treated with hyperoxia for 1 week starting at P1 or P7 and for 2 weeks starting at P1 followed by harvesting and dissociation of their carotid bodies for whole cell, perforated-patch recording. As compared to glomus cells from normoxia animals, hyperoxia treated cells showed a significant reduction in the magnitude of depolarization in response to hypoxia and anoxia, despite little change in the depolarizing response to 20 mM K(+). Resting cell membrane potential in glomus cells from rats exposed to hyperoxia from P1 to P15 and studied at P15 was slightly depolarized compared to other treatment groups and normoxia-treated cells, but conductance normalized to cell size was not different among groups. We conclude that postnatal hyperoxia impairs carotid chemoreceptor hypoxia transduction at a step between hypoxia sensing and membrane depolarization. This occurs without a major change in baseline electrophysiological characteristics, suggesting altered signaling or alterations in the relative abundance of different leak channel isoforms.
Subject(s)
Carotid Body/metabolism , Hyperoxia/metabolism , Membrane Potentials/physiology , Animals , Animals, Newborn , Carotid Body/cytology , Cell Hypoxia/physiology , Female , Hyperoxia/complications , Male , Rats , Rats, Sprague-DawleyABSTRACT
Carotid body chemoreceptors increase their action potential (AP) activity in response to a decrease in arterial oxygen tension and this response increases in the post-natal period. The initial transduction site is likely the glomus cell which responds to hypoxia with an increase in intracellular calcium and secretion of multiple neurotransmitters. Translation of this secretion to AP spiking levels is determined by the excitability of the afferent nerve terminals that is largely determined by the voltage-dependence of activation of Na(+) channels. In this review, we examine the biophysical characteristics of Na(+) channels present at the soma of chemoreceptor afferent neurons with the assumption that similar channels are present at nerve terminals. The voltage dependence of this current is consistent with a single Na(+) channel isoform with activation around the resting potential and with about 60-70% of channels in the inactive state around the resting potential. Channel openings, due to transitions from inactive/open or closed/open states, may serve to amplify external depolarizing events or generate, by themselves, APs. Over the first two post-natal weeks, the Na(+) channel activation voltage shifts to more negative potentials, thus enhancing the amplifying action of Na(+) channels on depolarization events and increasing membrane noise generated by channel transitions. This may be a significant contributor to maturation of chemoreceptor activity in the post-natal period.
Subject(s)
Carotid Body/growth & development , Carotid Body/physiology , Chemoreceptor Cells/physiology , Neurons, Afferent/physiology , Voltage-Gated Sodium Channels/physiology , Action Potentials/physiology , Animals , Chemoreceptor Cells/cytology , Humans , Neurons, Afferent/cytologySubject(s)
Carotid Body , Animals , Carotid Body/growth & development , Carotid Body/physiology , HumansABSTRACT
Previous work demonstrated that hyperoxia (30-60% O(2)) exposure in the post-natal period reduces the ventilatory response to acute hypoxia and this impairment may continue considerably beyond the period of hyperoxia exposure. Previous work from our laboratory demonstrated that 1-2 weeks of hyperoxia (60% O(2)) starting between P1 and P14: reduced the single chemoreceptor unit response to hypoxia, reduced the rise in glomus cell calcium caused by acute hypoxia and reduced hypoxia-induced catecholamine release (Donnelly 05, Donnelly 09). The present study asked whether the impairment extended to hypoxia-induced membrane depolarization, an earlier step in the transduction cascade. Perforated patch, whole-cell recordings were obtained from rat glomus cells exposed to hyperoxia from P0-P8 or P8-P15 and age-matched control groups. In both cases, hypoxia-induced membrane depolarization was significantly less in the hyperoxia treated groups compared to controls, while depolarization to 20 mM K(+) was not significantly affected. Resting membrane potential and input resistance were also not different in the hyperoxia treated groups. Whole carotid body quantitative real time PCR showed that TASK-1, TASK-3 and L-type Ca(2+) channel expression was significantly down-regulated at Hyper 8-15 compared to controls. We conclude that 1 week of postnatal hyperoxia during the early and late stage of CB maturation impairs organ function by affecting the coupling between hypoxia and glomus cell depolarization. This may be caused by altered expression of TASK1, TASK3 or L-type Ca(2+) channel gene expression. We speculate that an identification of cellular changes caused by hyperoxia may yield unique insights to the mechanism of oxygen sensing by the carotid bodies.
Subject(s)
Carotid Body/physiology , Hyperoxia/metabolism , Oxygen/metabolism , Animals , Carotid Body/cytology , Female , Gene Expression Regulation , Male , Membrane Potentials , Rats , Rats, Sprague-DawleyABSTRACT
Carotid body (CB) glomus cells respond to hypoxia by releasing neurotransmitters, such as ATP, which are believed to stimulate excitatory receptors on apposed nerve endings of the carotid sinus nerves as well as bind to autoreceptors on the glomus cell membrane to modulate response magnitude. The CB response to hypoxia is small at birth and increases during postnatal maturation in mammals. As ATP has been shown to inhibit the glomus cell response to hypoxia via an autoreceptor mechanism, we hypothesized that ATP-mediated inhibition may vary with age and play a role in postnatal development of the hypoxia response magnitude. The effects of ATP on CB glomus cell intracellular calcium ([Ca(2+)](i)) responses to hypoxia were studied at two ages, P0-1 and P14-18. The inhibitory effect of ATP or a stable ATP analog on the glomus cell response to hypoxia was greater in newborn rats compared to the more mature age group. Use of selective P2Y receptor agonists and antagonists suggests that the inhibitory effect of ATP on the glomus cell [Ca(2+)](i) response to hypoxia may be mediated by a P2Y12 receptor. Thus, developmental changes in ATP-mediated glomus cell inhibition may play a role in carotid chemoreceptor postnatal maturation.
Subject(s)
Adenosine Triphosphate/pharmacology , Carotid Body/drug effects , Cell Hypoxia/drug effects , Animals , Calcium/metabolism , Carotid Body/cytology , Carotid Body/metabolism , Female , Male , Oxygen/metabolism , Rats , Receptors, Purinergic P2/physiology , Receptors, Purinergic P2Y12ABSTRACT
ATP, acting through P2X(2)/P2X(3) receptor-channel complexes, plays an important role in carotid body chemoexcitation in response to natural stimuli in the rat. Since the channels are permeable to calcium, P2X activation by ATP should induce changes in intracellular calcium ([Ca(2+)](i)). Here, we describe a novel ex vivo approach using fluorescence [Ca(2+)](i) imaging that allows screening of retrogradely labeled chemoafferent neurons in the petrosal ganglion of the rat. ATP-induced [Ca(2+)](i) responses were characterized at postnatal days (P) 5-8 and P19-25. While all labeled cells showed a brisk increase in [Ca(2+)](i) in response to depolarization by high KCl (60 mM), only a subpopulation exhibited [Ca(2+)](i) responses to ATP. ATP (250-1,000 µM) elicited one of three temporal response patterns: fast (R1), slow (R2), and intermediate (R3). At P5-8, R2 predominated and its magnitude was attenuated 44% by the P2X(1) antagonist, NF449 (10 µM), and 95% by the P2X(1)/P2X(3)/P2X(2/3) antagonist, TNP-ATP (10 µM). At P19-25, R1 and R3 predominated and their magnitudes were attenuated 15% by NF449, 66% by TNP-ATP, and 100% by suramin (100 µM), a nonspecific P2 purinergic receptor antagonist. P2X(1) and P2X(2) protein levels in the petrosal ganglion decreased with development, while P2X(3) protein levels did not change significantly. We conclude that the profile of ATP-induced P2X-mediated [Ca(2+)](i) responses changes in the postnatal period, corresponding with changes in receptor isoform expression. We speculate that these changes may participate in the postnatal maturation of chemosensitivity.
Subject(s)
Adenosine Triphosphate/pharmacology , Calcium/metabolism , Cranial Sinuses/innervation , Ganglia/drug effects , Ganglia/metabolism , Adenosine Triphosphate/analogs & derivatives , Animals , Benzenesulfonates/pharmacology , Calcium Channels/metabolism , Cells, Cultured , Female , Ganglia/cytology , Male , Microscopy, Fluorescence , Models, Animal , Purinergic P2X Receptor Antagonists/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Purinergic P2X/drug effects , Receptors, Purinergic P2X/metabolism , Suramin/pharmacologyABSTRACT
Chronic compression (CCD) of the dorsal root ganglion (DRG) is a model of human radicular pain produced by intraforaminal stenosis and other disorders affecting the DRG, spinal nerve, or root. Previously, we examined electrophysiological changes in small-diameter lumbar level 3 (L3) and L4 DRG neurons treated with CCD; the present study extends these observations to medium-sized DRG neurons, which mediate additional sensory modalities, both nociceptive and non-nociceptive. Whole-cell patch-clamp recordings were obtained from medium-sized somata in the intact DRG in vitro. Compared with neurons from unoperated control animals, CCD neurons exhibited a decrease in the current threshold for action potential generation. In the CCD group, current densities of TTX-resistant and TTX-sensitive Na(+) current were increased, whereas the density of delayed rectifier voltage-dependent K(+) current was decreased. No change was observed in the transient or "A" current after CCD. We conclude that CCD in the mouse produces hyperexcitability in medium-sized DRG neurons, and the hyperexcitability is associated with an increased density of Na(+) current and a decreased density of delayed rectifier voltage-dependent K(+) current.
Subject(s)
Ganglia, Spinal/pathology , Ion Channel Gating/physiology , Neurons/metabolism , Potassium Channels, Voltage-Gated/metabolism , Radiculopathy/pathology , Sodium Channels/metabolism , Analysis of Variance , Animals , Biophysics , Disease Models, Animal , Electric Stimulation , Functional Laterality , In Vitro Techniques , Ion Channel Gating/drug effects , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Neurons/classification , Neurons/drug effects , Patch-Clamp Techniques , Potassium Channel Blockers/pharmacology , Sodium Channel Blockers/pharmacology , Tetraethylammonium/pharmacology , Tetrodotoxin/pharmacologyABSTRACT
Carotid bodies mediate hypoxia sensing for the respiratory system and increase their sensitivity in the post-natal period. The present study examined the characteristics and developmental change of fast Na(+) currents of chemoreceptor afferent neurons. Rat carotid bodies (P2-P19) were harvested intact with the petrosal ganglia and whole-cell recordings obtained from petrosal somas whose axons projected to the carotid body. The magnitude of Na(+) current increased in the post-natal period in parallel with increased conduction velocity and somal size. Voltage-dependence of activation significantly shifted towards negative potentials but no significant change occurred in the voltage dependence of inactivation or the slope factors for activation or inactivation. The leftward shift in activation increased slowly or non-inactivating currents around resting potential which increases afferent neuron excitability, a result confirmed in current clamp recordings. These results suggest that a developmental shift in Na(+) current activation plays a role in chemoreceptor maturation by enhancing excitability of the afferent neuron.
Subject(s)
Carotid Body/cytology , Carotid Body/growth & development , Chemoreceptor Cells/physiology , Sodium Channels/physiology , Age Factors , Analysis of Variance , Animals , Animals, Newborn , Biophysics , Chemoreceptor Cells/drug effects , Electric Stimulation , In Vitro Techniques , Membrane Potentials/drug effects , Membrane Potentials/physiology , Nerve Fibers, Unmyelinated/physiology , Neural Conduction/drug effects , Neural Conduction/physiology , Patch-Clamp Techniques/methods , Rats , Sodium Channel Blockers/pharmacology , Sodium Channels/drug effects , Tetrodotoxin/pharmacologyABSTRACT
A post-natal increase in carotid body (CB) hypoxia responsiveness occurs at the level of carotid sinus nerve activity, intracellular calcium, cell membrane depolarization and hypoxic inhibition of O(2)-sensitive background K(+) conductance. TASK-1, TASK-1/3 and TASK-3 are functionally expressed in CB glomus cells, with TASK-1/3 providing the major part of the O(2)-sensitive TASK-like background K(+) conductance. Here we report the effects of graded hypoxia on TASK-like channel activity in CB glomus cells from rats aged 0 to 1, 6 to 7 and 16 to 18 days; the time frame of postnatal CB functional maturation. TASK was active in nearly all cell-attached patches and TASK activity during normoxia did not differ across ages. Hypoxia produced a progressive decrease in channel opening frequency with graded decreases in O(2) level and also produced glomus cell depolarization, as assessed by the shift in reversal potential of TASK single channel current. Hypoxic inhibition of TASK activity was least at P0-P1 and increased with age mainly between 6-7 and 16-18 days. The O(2)-sensitive TASK activity was significantly greater in glomus cells from P16 to P18 when compared to cells from P0 to P1 day old rats. These results support the hypothesis that postnatal carotid body functional maturation is due, at least in part, to changes in the sensitivity of TASK to the hypoxic signals generated in glomus cells.
Subject(s)
Carotid Body/cytology , Carotid Body/growth & development , Cell Hypoxia/physiology , Chemoreceptor Cells/physiology , Oxygen/metabolism , Potassium Channels, Tandem Pore Domain/metabolism , Age Factors , Analysis of Variance , Animals , Animals, Newborn , Biophysics , Cell Hypoxia/drug effects , Cells, Cultured , Chemoreceptor Cells/drug effects , Electric Stimulation , Gene Expression Regulation, Developmental/physiology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Nerve Tissue Proteins , Oxygen/pharmacology , Patch-Clamp Techniques , RatsABSTRACT
Carotid body glomus (CB) cells express different types of K(+) channels such as TASK, BK, and Kv channels, and hypoxia has been shown to inhibit these channels. Here we report the presence of a â¼72-pS channel that has not been described previously in CB cells. In cell-attached patches with 150 mM K(+) in the pipette and bath solutions, TASK-like channels were present (â¼15 and â¼36-pS). After formation of inside-out patches, a 72-pS channel became transiently active in â¼18% of patches. The 72-pS channel was K(+)-selective, inhibited by 2-4 mM ATP and 10-100 µM glybenclamide. The 72-pS channel was observed in CB cells isolated from newborn, 2-3 week and 10-12 week-old rats. Reverse transcriptase-PCR and immunocytochemistry showed that Kir6.1, Kir6.2, SUR1 and SUR2 were expressed in CB glomus cells as well as in non-glomus cells. Acute hypoxia (â¼15 mmHg O(2)) inhibited TASK-like channels but failed to activate the 72-pS channel in cell-attached CB cells. K(+) channel openers (diazoxide, pinacidil, levcromakalim), sodium cyanide and removal of extracellular glucose also did not activate the 72-pS channel in the cell-attached state. The hypoxia-induced elevation of intracellular [Ca(2+)] was unchanged by glybenclamide or diazoxide. NaCN-induced increase in [Ca(2+)] was not affected by 10 µM glybenclamide but inhibited by 100 µM glybenclamide. Acute glucose deprivation did not elevate [Ca(2+)] in the presence or absence of glybenclamide. These results show that an ATP-sensitive K(+) channel is expressed in the plasma membrane of CB cells, but is not activated by short-term metabolic inhibition. The functional relevance of the 72-pS channel remains to be determined.
Subject(s)
Carotid Body/cytology , Chemoreceptor Cells/metabolism , Potassium Channels, Inwardly Rectifying/physiology , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphate/pharmacology , Animals , Animals, Newborn , Calcium/metabolism , Cell Hypoxia/drug effects , Cell Hypoxia/physiology , Cells, Cultured , Chemoreceptor Cells/drug effects , Cyanides/pharmacology , Diazoxide/pharmacology , Glucose/pharmacology , Glyburide/pharmacology , Hypoglycemic Agents/pharmacology , Ion Channel Gating/drug effects , Ion Channel Gating/physiology , KATP Channels/genetics , KATP Channels/physiology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Patch-Clamp Techniques , Potassium Channels, Inwardly Rectifying/genetics , Potassium Channels, Inwardly Rectifying/metabolism , RNA, Messenger/metabolism , Rats , Receptors, Drug/genetics , Receptors, Drug/metabolism , Sulfonylurea Receptors , Sweetening Agents/pharmacology , Vasodilator Agents/pharmacologyABSTRACT
We investigated the effects of chronic compression (CCD) of the L3 and L4 dorsal root ganglion (DRG) on pain behavior in the mouse and on the electrophysiological properties of the small-diameter neuronal cell bodies in the intact ganglion. CCD is a model of human radicular pain produced by intraforaminal stenosis and other disorders affecting the DRG, spinal nerve, or root. On days 1, 3, 5, and 7 after the onset of compression, there was a significant decrease from preoperative values in the threshold mechanical force required to elicit a withdrawal of the foot ipsilateral to the CCD (tactile allodynia). Whole cell patch-clamp recordings were obtained, in vitro, from small-sized somata and, for the first time, in the intact DRG. Under current clamp, CCD neurons exhibited a significantly lower rheobase compared with controls. A few CCD but no control neurons exhibited spontaneous action potentials. CCD neurons showed an increase in the density of TTX-resistant and TTX-sensitive Na(+) current. CCD neurons also exhibited an enhanced density of voltage-dependent K(+) current, due to an increase in delayed rectifier K(+) current, without a change in the transient or "A" current. We conclude that CCD in the mouse produces a model of radicular pain, as we have previously demonstrated in the rat. While the role of enhanced K(+) current remains to be elucidated, we speculate that it represents a compensatory neuronal response to reduce ectopic or aberrant levels of neuronal activity produced by the injury.
Subject(s)
Ganglia, Spinal/physiopathology , Radiculopathy/physiopathology , Action Potentials/drug effects , Action Potentials/physiology , Animals , Ganglia, Spinal/drug effects , Hyperalgesia/drug therapy , Hyperalgesia/physiopathology , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Pain/drug therapy , Pain/physiopathology , Patch-Clamp Techniques , Potassium Channels, Voltage-Gated/drug effects , Potassium Channels, Voltage-Gated/physiology , Radiculopathy/drug therapy , Sodium Channels/drug effects , Sodium Channels/physiology , Tetrodotoxin/pharmacologyABSTRACT
Carotid body chemoreceptors transduce a decrease in arterial oxygen tension into increased sinus nerve action potential (AP) activity which undergoes a maturational increase in the post-natal period. MaxiK-channels channels are proposed to play a major role in organ function based on their maturation-dependent expression in glomus cells and inhibition by acute hypoxia. To better resolve the role of this channel, single-unit AP activity of rat chemoreceptor neurons was recorded, in vitro, during a progressive decrease in oxygen from normoxia (â¼150 Torr) to moderate hypoxia (â¼60 Torr). Blockade of MaxiK channels with charybdotoxin (100 nM) in both older (P16-P18) and younger (P2-P3) animals resulted in no significant change in AP activity, but increased nerve conduction speed in the older animals. In dissociated glomus cells, charybdotoxin slightly enhanced the intracellular calcium response to acute hypoxia at both ages. We conclude that MaxiK channels play little or no role in mediating the response to acute, moderate hypoxia, either in the newborn or older animal.
Subject(s)
Carotid Body/metabolism , Chemoreceptor Cells/metabolism , Hypoxia/metabolism , Large-Conductance Calcium-Activated Potassium Channels/metabolism , Mechanotransduction, Cellular/physiology , Animals , Carotid Body/physiopathology , Electrophysiology , Hypoxia/physiopathology , RatsABSTRACT
Chronic postnatal hyperoxia blunts the hypoxic ventilatory response (HVR) in rats, an effect that persists for months after return to normoxia. To determine whether decreased carotid body O(2) sensitivity contributes to this lasting impairment, single-unit chemoafferent nerve and glomus cell calcium responses to hypoxia were recorded from rats reared in 60% O(2) through 7d of age (P7) and then returned to normoxia. Single-unit nerve responses were attenuated by P4 and remained low through P7. After return to normoxia, hypoxic responses were partially recovered within 3d and fully recovered within 7-8d (i.e., at P14-15). Glomus cell calcium responses recovered with a similar time course. Hyperoxia altered carotid body mRNA expression for O(2)-sensitive K(+) channels TASK-1, TASK-3, and BK(Ca), but only TASK-1 mRNA paralleled changes in chemosensitivity (i.e., downregulation by P7, partial recovery by P14). Collectively, these data do not support a role for reduced O(2) sensitivity of individual chemoreceptor cells in long-lasting reduction of the HVR after developmental hyperoxia.
Subject(s)
Carotid Body/metabolism , Chemoreceptor Cells/metabolism , Hyperoxia/metabolism , Animals , Animals, Newborn , Calcium Signaling/physiology , Carotid Body/physiopathology , Hyperoxia/physiopathology , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits , Nerve Tissue Proteins/biosynthesis , Patch-Clamp Techniques , Potassium Channels/biosynthesis , Potassium Channels, Tandem Pore Domain/biosynthesis , RNA, Messenger/analysis , Rats , Rats, Sprague-DawleyABSTRACT
Adenosine triphosphate, acting through purinergic P2X receptors, has been shown to stimulate ventilation and increase carotid body chemoreceptor activity in adult rats. However, its role during postnatal development of the ventilatory response to hypoxia is yet unknown. Using whole body plethysmography, we measured ventilation in normoxia and in moderate hypoxia (12% fraction of inspired O2, 20 min) before and after intraperitoneal injection of suramin (P2X2 and P2X3 receptor antagonist, 40 mg/kg) in 4-, 7-, 12-, and 21-day-old rats. Suramin reduced baseline breathing (â¼20%) and the response to hypoxia (â¼30%) in all rats, with a relatively constant effect across ages. We then tested the effect of the specific P2X3 antagonist, A-317491 (150 mg/kg), in rats aged 4, 7, and 21 days. As with suramin, A-317491 reduced baseline ventilation (â¼55%) and the hypoxic response (â¼40%) at all ages studied. Single-unit carotid body chemoreceptor activity was recorded in vitro in 4-, 7-, and 21-day-old rats. Suramin (100 µM) and A-317491 (10 µM) significantly depressed the sinus nerve chemosensory discharge rate (â¼80%) in normoxia (Po2 â¼150 Torr) and hypoxia (Po2 â¼60 Torr), and this decrease was constant across ages. We conclude that, in newborn rats, P2X purinergic receptors are involved in the regulation of breathing under basal and hypoxic condition, and P2X3-containing receptors play a major role in carotid body function. However, these effects are not age dependent within the age range studied.
Subject(s)
Aging/physiology , Carotid Body/physiology , Chemoreceptor Cells/physiology , Pulmonary Ventilation/physiology , Purinergic P2X Receptor Antagonists/pharmacology , Receptors, Purinergic P2X/metabolism , Aging/drug effects , Animals , Animals, Newborn , Carotid Body/drug effects , Chemoreceptor Cells/drug effects , Female , Male , Pulmonary Ventilation/drug effects , Rats , Rats, Sprague-DawleyABSTRACT
In vivo electrophysiological recordings from cell bodies of primary sensory neurons are used to determine sensory function but are commonly performed blindly and without access to voltage- (patch-clamp) electrophysiology or optical imaging. We present a procedure to visualize and patch-clamp the neuronal cell body in the dorsal root ganglion, in vivo, manipulate its chemical environment, determine its receptive field properties, and remove it either to obtain subsequent molecular analyses or to gain access to deeper lying cells. This method allows the association of the peripheral transduction capacities of a sensory neuron with the biophysical and chemical characteristics of its cell body.
Subject(s)
Electrophysiology/methods , Ganglia, Spinal/cytology , Ganglia, Spinal/physiology , Neurophysiology/methods , Patch-Clamp Techniques/methods , Sensory Receptor Cells/cytology , Sensory Receptor Cells/physiology , Animals , Electrophysiology/instrumentation , Female , Ganglia, Spinal/blood supply , Male , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence/instrumentation , Microscopy, Fluorescence/methods , Microscopy, Video/instrumentation , Microscopy, Video/methods , Neurophysiology/instrumentation , Patch-Clamp Techniques/instrumentation , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Sensory Receptor Cells/drug effectsABSTRACT
Carotid body chemoreceptors transduce a decrease in arterial oxygen tension into increased action potential (AP) activity on the sinus nerve, which increases the drive to breathe. The mechanism by which AP activity increases is unresolved, but acetylcholine (ACh), acting through nicotinic receptors, is postulated to be a major contributor to nerve excitation based partly on the demonstration that pharmacological antagonism of nicotinic receptors reduces the afferent nerve response in some studies. However, most previous studies relied on indirect measures of chemoreceptor activity or utilized a recording configuration that is sensitive to AP morphology in addition to AP frequency. In the present study, single-unit AP activity was recorded from the soma of rat chemoreceptor neurons in vitro. The nicotinic blocker mecamylamine (50 microM) ablated the excitatory actions of exogenous ACh and increased, rather than decreased, AP activity during moderate hypoxia. At higher dosage (500 microM) AP height was reduced, conduction velocity slowed, and conduction failure occurred, especially during hypoxia, producing the appearance of a decreased response to hypoxia. Recovery from mecamylamine block was slow (>10 min). In contrast to mecamylamine, suramin, a P2X receptor blocker, reversibly inhibited the response to hypoxia, suggesting relatively free diffusion of drugs to the glomus cell/nerve synaptic site. These results strongly suggest that ACh acting through nicotinic receptors does not mediate excitatory transmission in rat carotid body and that previous results demonstrating such a role may have been partially influenced by changes in AP morphology or conduction failure.
Subject(s)
Action Potentials/physiology , Carotid Body/physiology , Receptors, Nicotinic/physiology , Synaptic Transmission/physiology , Acetylcholine/metabolism , Acetylcholine/pharmacology , Action Potentials/drug effects , Analysis of Variance , Animals , Carotid Body/drug effects , Carotid Body/metabolism , Chemoreceptor Cells , Dose-Response Relationship, Drug , Female , Hypoxia , Male , Mecamylamine/pharmacology , Neural Conduction/drug effects , Neural Conduction/physiology , Neurons/drug effects , Neurons/physiology , Nicotinic Antagonists/pharmacology , Patch-Clamp Techniques , Purinergic P2 Receptor Antagonists , Rats , Rats, Sprague-Dawley , Receptors, Purinergic P2/physiology , Suramin/pharmacology , Synaptic Transmission/drug effectsABSTRACT
Peripheral arterial chemoreceptors, particularly the carotid body chemoreceptors, are the primary sites for the detection of hypoxia and reflexly increase ventilatory drive and behavioral arousal during hypoxic or asphyxial events. Newborn infants are at risk for hypoxic and asphyxial events during sleep, yet, the strength of the chemoreceptor responses is low or absent at birth and then progressively increases with early postnatal development. This review summarizes the available data showing that even though the "oxygen sensor" in the glomus cells has not been unequivocally identified, it is clear that development affects many of the other properties of the chemoreceptor unit (glomus cell, afferent nerve fibers and neurotransmitter profile at the synapse) that are necessary and essential for the propagation of the "sensing" response, and exposure to hypoxia, hyperoxia and nicotine can modify normal development of each of the components leading to altered peripheral chemoreceptor responses.
Subject(s)
Arteries/metabolism , Chemoreceptor Cells/metabolism , Hypoxia/metabolism , Animals , Arteries/pathology , Carotid Body/metabolism , Humans , Neurotransmitter Agents/metabolismABSTRACT
Postnatal hyperoxia exposure reduces the carotid body response to acute hypoxia and produces a long-lasting impairment of the ventilatory response to hypoxia. The present work investigated the time course of pre- and post-synaptic alterations following exposure to hyperoxia (Fl(O2) = 0.6) for 1, 3, 5, 8 and 14 days (d) starting at postnatal day 7 (P7) as compared to age-matched controls. Hyperoxia exposure for 1d enhanced the nerve response and glomus cell calcium response to acute hypoxia, but exposure for 3-5d caused a significant reduction in both. Hypoxia-induced catecholamine release and nerve conduction velocity were significantly decreased by 5d hyperoxia. We conclude that hyperoxia exerts pre-synaptic (glomus cell calcium and secretory responses) and post-synaptic (afferent nerve excitability) actions to initially enhance and then reduce the chemoreceptor response to acute hypoxia. The parallel changes in glomus cell calcium response and nerve response suggest causality between the two and that environmental hyperoxia can affect the coupling between acute hypoxia and glomus cell calcium regulation.