CO(2) microdialysis in retrotrapezoid nucleus of the rat increases breathing in wakefulness but not in sleep.

Central chemoreceptors are widespread within the brain stem. We suggest that their function at some sites may vary with the state of arousal. In this study, we tested the hypothesis that the function of chemoreceptors in the retrotrapezoid nucleus (RTN) varies with sleep and wakefulness. In unanesthetized rats, we produced focal acidification of the RTN by means of a microdialysis probe (tip containing the semipermeable membrane = 1-mm length, 240-microm diameter, and 45-nl volume). With the use of a dialysate equilibrated with 25% CO(2), the tissue pH change (measured in anesthetized animals) was 1) limited to within 550 microm of the probe and, 2) at the probe tip, was equivalent to that observed with end-tidal PCO(2) of 63 Torr. This focal acidification of the RTN increased ventilation significantly by 24% above baseline, on average, in 13 trials in seven rats only during wakefulness. The effect was entirely due to an increase in tidal volume. During sleep defined by behavioral criteria, ventilation was unaffected, on average, in 10 trials in seven rats. During sleep, the chemoreceptors in the RTN appear to be inactive, or, if active, the respiratory control system either is not responding or is responding with very low gain. Because ventilation is increased during sleep with all central chemoreceptor sites stimulated via systemic CO(2) application, other central chemoreceptor locations must have enhanced effectiveness.     

CO2 dialysis in the medullary raphe of the rat increases ventilation in sleep.

Central chemoreceptors are widespread within the brain stem. We hypothesize that function at different sites varies with arousal state. In unanesthetized rats, we produced focal acidification at single sites by means of microdialysis using artificial cerebrospinal fluid equilibrated with 25% CO2. Tissue acidosis, measured under anesthesia, is equivalent to that observed with 63 Torr end-tidal PCO2 and is limited to 600 microm. Focal acidification of the retrotrapezoid nucleus increased ventilation by 24% only in wakefulness via an increase in tidal volume (Li A, Randall M, and Nattie E. J Appl Physiol 87: 910-919, 1999). In this study of the medullary raphe, the effect of such focal acidification was in sleep (defined by electroencephalographic and electromyographic criteria): ventilation and frequency increased by 15-20% in non-rapid eye movement sleep, and frequency increased by 15% in rapid eye movement sleep. There was no effect in wakefulness. Chemoreception in the medullary raphe appears to be responsive in sleep. Central chemoreceptors at two different locations appear to vary in effectiveness with arousal state.     

Muscimol dialysis in the retrotrapezoid nucleus region inhibits breathing in the awake rat.

Under anesthesia, inactivation of the retrotrapezoid nucleus (RTN) region markedly inhibits breathing and chemoreception. In conscious rats, we dialyzed muscimol for 30 min to inhibit neurons of the RTN region reversibly. Dialysis of artificial cerebrospinal fluid had no effect. Muscimol (1 or 10 mM) significantly decreased tidal volume (VT) (by 16-17%) within 15 min. VT remained decreased for 50 min or more, with recovery by 90 min. Ventilation (VE) decreased significantly (by 15-20%) within 15 min and then returned to baseline within 40 min as a result of an increase in frequency. This, we suggest, is a compensatory physiological response to the reduced VT. Oxygen consumption was unchanged. In response to 7% CO(2) in the 1 mM group, absolute VE and change in VE were significantly reduced (by 19-22%). In the 10 mM group, the response to dialysis included a time-related increase in frequency and decrease in body temperature, which may reflect greater spread of muscimol. In the awake rat, the RTN region provides a portion of the tonic drive to breathe, as well as a portion of the response to hypercapnia.     

Heterogeneous patterns of pH regulation in glial cells in the dorsal and ventral medulla

We examined pH regulation in two chemosensitive areas of the brain, the retrotrapezoid nucleus (RTN) and the nucleus tractus solitarius (NTS), to identify the proton transporters involved in regulation of intracellular pH (pHi) in medullary glia. Transverse brain slices from young rats [postnatal day 8 (P8) to P20] were loaded with the pH-sensitive probe 2',7'-bis (2-carboxyethyl)-5,6-carboxyfluorescein after kainic acid treatment removed neurons. Cells were alkalinized when they were depolarized (extracellular K+ increased from 6.24 to 21.24 mM) in the RTN but not in the NTS. This alkaline shift was inhibited by 0.5 mM DIDS. Removal of CO2/HCO3- or Na+ from the perfusate acidified the glial cells, but the acidification after Na+ removal was greater in the RTN than in the NTS. Treatment of the slice with 5-(N-ethyl-N-isopropyl)amiloride (100 microM) in saline containing CO2/HCO3- acidified the cells in both nuclei, but the acidification was greater in the NTS. Restoration of extracellular Cl- after Cl- depletion during the control condition acidified the cells. Immunohistochemical studies of glial fibrillary acid protein demonstrated much denser staining in the RTN compared with the NTS. We conclude that there is evidence of Na+-HCO3- cotransport and Na+/H+ exchange in glia in the RTN and NTS, but the distribution of glia and the distribution of these pH-regulatory functions are not identical in the NTS and RTN. The differential strength of glial pH regulatory function in the RTN and NTS may also alter CO2 chemosensory neuronal function at these two chemosensitive sites in the brain stem.     

Multiple rostral medullary nuclei can influence breathing in awake goats.

The purpose of this study was to determine the effect on breathing of neuronal dysfunction in the retrotrapezoid (RTN), facial (FN), gigantocellularis reticularis (RGN), or vestibular (VN) nuclei of adult awake goats. Microtubules were chronically implanted to induce neuronal dysfunction by microinjection of an excitatory amino acid (EAA) receptor antagonist or a neurotoxin. The EAA receptor antagonist had minimal effect on eupneic breathing, but 8--10 days after injection of the neurotoxin, 7 of 10 goats hypoventilated (arterial PCO(2) increased 3.2 +/- 0.7 Torr). Overall there were no significant (P > 0.10) effects of the EAA receptor antagonist on CO(2) sensitivity. However, for all nuclei, > or =66% of the antagonist injections altered CO(2) sensitivity by more than the normal 12.7 +/- 1.6% day-to-day variation. These changes were not uniform, inasmuch as the antagonist increased (RTN, n = 2; FN, n = 7; RGN, n = 6; VN, n = 1) or decreased (RTN, n = 2; RGN, n = 3; VN, n = 2) CO(2) sensitivity. Ten days after injection of the neurotoxin into the FN (n = 3) or RGN (n = 5), CO(2) sensitivity was also reduced. Neuronal dysfunction also did not have a uniform effect on the exercise arterial PCO(2) response, and there was no correlation between effects on CO(2) sensitivity and the exercise hyperpnea. We conclude that there is a heterogeneous population of neurons in these rostral medullary nuclei (or adjacent tissue) that can affect breathing in the awake state, possibly through chemoreception or chemoreceptor-related mechanisms.     

Response of membrane potential and intracellular pH to hypercapnia in neurons and astrocytes from rat retrotrapezoid nucleus

We compared the response to hypercapnia (10%) in neurons and astrocytes among a distinct area of the retrotrapezoid nucleus (RTN), the mediocaudal RTN (mcRTN), and more intermediate and rostral RTN areas (irRTN) in medullary brain slices from neonatal rats. Hypercapnic acidosis (HA) caused pH(o) to decline from 7.45 to 7.15 and a maintained intracellular acidification of 0.15 +/- 0.02 pH unit in 90% of neurons from both areas (n = 16). HA excited 44% of mcRTN (7/16) and 38% of irRTN neurons (6/16), increasing firing rate by 167 +/- 75% (chemosensitivity index, CI, 256 +/- 72%) and 310 +/- 93% (CI 292 +/- 50%), respectively. These responses did not vary throughout neonatal development. We compared the responses of mcRTN neurons to HA (decreased pH(i) and pH(o)) and isohydric hypercapnia (IH; decreased pH(i) with constant pH(o)). Neurons excited by HA (firing rate increased 156 +/- 46%; n = 5) were similarly excited by IH (firing rate increased 167 +/- 38%; n = 5). In astrocytes from both RTN areas, HA caused a maintained intracellular acidification of 0.17 +/- 0.02 pH unit (n = 6) and a depolarization of 5 +/- 1 mV (n = 12). In summary, many neurons (42%) from the RTN are highly responsive (CI 248%) to HA; this may reflect both synaptically driven and intrinsic mechanisms of CO(2) sensitivity. Changes of pH(i) are more significant than changes of pH(o) in chemosensory signaling in RTN neurons. Finally, the lack of pH(i) regulation in response to HA suggests that astrocytes do not enhance extracellular acidification during hypercapnia in the RTN.     

CO2-stimulated NaCl absorption in the mouse renal cortical thick ascending limb of Henle. Evidence for synchronous Na +/H+ and Cl-/HCO3- exchange in apical plasma membranes

These experiments evaluated salt transport processes in isolated cortical thick limbs of Henle (cTALH) obtained from mouse kidney. When the external solutions consisted of Krebs-Ringer bicarbonate (KRB), pH 7.4, and a 95% O2-5% CO2 gas phase, the spontaneous transepithelial voltage (Ve, mV, lumen-to-bath) was approximately mV; the net rate of Cl- absorption (JnetCl) was approximately 3,600 pmols s-1 cm-2; the net rate of osmotic solute absorption Jnetosm was twice JnetCl; and the net rate of total CO2 transport (JnetCO2) was indistinguishable from zero. Thus, net Cl- absorption was accompanied by the net absorption of a monovalent cation, presumably Na+, and net HCO3- absorption was negligible. This salt transport process was stimulated by (CO2 + HCO3- ): omission of CO2 from the gas phase and HCO3- from external solutions reduced JnetCl, Jnetosm, and Ve by 50%. Furthermore, 10(-4) M luminal furosemide abolished JnetCl and Ve entirely. The lipophilic carbonic anhydrase inhibitor ethoxzolamide (10(-4) M, either luminal or peritubular) inhibited (CO2 + HCO3-)-stimulated JnetCl, Jnetosm, and Ve by approximately 50%; however, when the combination (CO2 + HCO3-) was absent, ethoxzolamide had no detectable effect on salt transport. Ve was reduced or abolished entirely by omission of either Na+ or Cl- from external solutions, by peritubular K+ removal, by 10(-3) M peritubular ouabain, and by 10(-4) M luminal SITS. However, Ve was unaffected by 10(-3) M peritubular SITS, or by the hydrophilic carbonic anhydrase inhibitor acetazolamide (2.2 x 10(-4) M, lumen plus bath). We interpret these data to indicate that (CO2 + HCO3-)-stimulated NaCl absorption in the cTALH involved two synchronous apical membrane antiport processes: one exchanging luminal Na+ for cellular H+; and the other exchanging luminal Cl- for cellular HCO3- or OH-, operating in parallel with a (CO2+ HCO3-)-independent apical membrane NaCl cotransport mechanism.     

CO2 dialysis in nucleus tractus solitarius region of rat increases ventilation in sleep and wakefulness.

To evaluate the function of widely distributed central chemoreceptors during sleep and wakefulness in the rat, we focally stimulate single chemoreceptor sites during naturally occurring sleep-wake cycles by microdialysis of artificial cerebrospinal fluid equilibrated with 25% CO2. In retrotrapezoid nucleus, this increased ventilation (tidal volume) by 24% only in wakefulness (Li A, Randall M, and Nattie E. J Appl Physiol 87: 910-919, 1999). In caudal medullary raphé, it increased ventilation (frequency) by 15-20% only in sleep (Nattie EE and Li A. J Appl Physiol 90: 1247-1257, 2001). Here, in nucleus tractus solitarius (NTS), focal acidification significantly increased ventilation by 11% in sleep and 7% in wakefulness rostrally (n = 5) and by 16% in sleep and 28% in wakefulness caudally (n = 5). The sleep-wake cycle was unaltered. Dialysis with 5% CO2 had no effect. Dialysis with 50% CO2 caudally did not further stimulate ventilation but did disrupt sleep. Central chemoreceptors in the NTS affect breathing in both sleep and wakefulness. The threshold for arousal in caudal NTS is greater than that for the stimulation of breathing.     

CFTR inhibition augments NHE3 activity during luminal high CO2 exposure in rat duodenal mucosa

We hypothesized that the function of duodenocyte apical membrane acid-base transporters are essential for H(+) absorption from the lumen. We thus examined the effect of inhibition of Na(+)/H(+) exchanger-3 (NHE3), cystic fibrosis transmembrane regulator (CFTR), or apical anion exchangers on transmucosal CO(2) diffusion and HCO(3)(-) secretion in rat duodenum. Duodena were perfused with a pH 6.4 high CO(2) solution or pH 2.2 low CO(2) solution with the NHE3 inhibitor, S3226, the anion transport inhibitor, DIDS, or pretreatment with the potent CFTR inhibitor, CFTR(inh)-172, with simultaneous measurements of luminal and portal venous (PV) pH and carbon dioxide concentration ([CO(2)]). Luminal high CO(2) solution increased CO(2) absorption and HCO(3)(-) secretion, accompanied by PV acidification and PV Pco(2) increase. During CO(2) challenge, CFTR(inh)-172 induced HCO(3)(-) absorption, while inhibiting PV acidification. S3226 reversed CFTR(inh)-associated HCO(3)(-) absorption. Luminal pH 2.2 challenge increased H(+) and CO(2) absorption and acidified the PV, inhibited by CFTR(inh)-172 and DIDS, but not by S3226. CFTR inhibition and DIDS reversed HCO(3)(-) secretion to absorption and inhibited PV acidification during CO(2) challenge, suggesting that HCO(3)(-) secretion helps facilitate CO(2)/H(+) absorption. Furthermore, CFTR inhibition prevented CO(2)-induced cellular acidification reversed by S3226. Reversal of increased HCO(3)(-) loss by NHE3 inhibition and reduced intracellular acidification during CFTR inhibition is consistent with activation or unmasking of NHE3 activity by CFTR inhibition, increasing cell surface H(+) available to neutralize luminal HCO(3)(-) with consequent CO(2) absorption. NHE3, by secreting H(+) into the luminal microclimate, facilitates net transmucosal HCO(3)(-) absorption with a mechanism similar to proximal tubular HCO(3)(-) absorption.     

Ventilatory effects of impaired glial function in a brain stem chemoreceptor region in the conscious rat.

Glia are thought to regulate ion homeostasis, including extracellular pH; however, their role in modulating central CO2 chemosensitivity is unclear. Using a push-pull cannula in chronically instrumented and conscious rats, we administered a glial toxin, fluorocitrate (FC; 1 mM) into the retrotrapezoid nucleus (RTN), a putative chemosensitive site, during normocapnia and hypercapnia. FC exposure significantly increased expired minute ventilation (VE) to a value 38% above the control level during normocapnia. During hypercapnia, FC also significantly increased both breathing frequency and expired VE. During FC administration, maximal ventilation was achieved at approximately 4% CO2, compared with 8-10% CO2 during control hypercapnic trials. RTN perfusion of control solutions had little effect on any ventilatory measures (VE, tidal volume, or breathing frequency) during normocapnic or hypercapnic conditions. We conclude that unilateral impairment of glial function in the RTN of the conscious rat results in stimulation of respiratory output.     

Neurokinin-1 receptor-expressing neurons in the ventral medulla are essential for normal central and peripheral chemoreception in the conscious rat.

Neurokinin-1 receptor immunoreactive (NK1R-ir) neurons and processes are widely distributed within the medulla, prominently at central chemoreceptor sites. Focal lesions of NK1R-ir neurons in the medullary raphe or the retrotrapezoid nucleus partially reduced the CO(2) response in conscious rats. We ask if NK1R-ir cells and processes over a wide region of the ventral medulla are essential for central and peripheral chemoreception by cisterna magna injection of SSP-SAP, a high-affinity version of substance P-saporin. After 22 days, NK1R-ir cell loss was -79% in the retrotrapezoid nucleus and -65% in the A5 region, which lie close to the ventral surface, and -38% in the medullary raphe and -49% in the pre-B?tzinger complex/rostral ventral respiratory group, which lie deeper. Dorsal chemoreceptor sites, the caudal nucleus tractus solitarius and the A6 region, were unaffected. At 8 and 22 days, these lesions produced 1) hypoventilation during air breathing in wakefulness ( approximately 8%) and in non-rapid eye movement (NREM) ( approximately 9%) and rapid eye movement ( approximately 14%) sleep, as measured over a 4-h period; 2) a substantially reduced ventilatory response to 7% CO(2) by 61% in wakefulness and 46-57% in NREM sleep; and 3) a decreased ventilatory response to 12% O(2) by 40% in wakefulness and 35% in NREM sleep at 8 days, with partial recovery by 22 days. NK1R-ir neurons in the ventral medulla are essential for normal central chemoreception, provide a drive to breathe, and modulate the peripheral chemoreceptor responses. These effects are not state dependent.     

Recent progresses on the genetic basis of the regulation of CO2 acquisition systems in response to CO2 concentration

Marine diatoms, the major primary producer in ocean environment, are known to take up both CO(2) and HCO(3)(-) in seawater and efficiently concentrate them intracellularly, which enable diatom cells to perform high-affinity photosynthesis under limiting CO(2). However, mechanisms so far proposed for the inorganic carbon acquisition in marine diatoms are significantly diverse despite that physiological studies on this aspect have been done with only limited number of species. There are two major hypotheses about this; that is, they take up and concentrate both CO(2) and HCO(3)(-) as inorganic forms, and efficiently supply CO(2) to Rubisco by an aid of carbonic anhydrases (biophysical CO(2)-concentrating mechanism: CCM); and as the other hypothesis, biochemical conversion of HCO(3)(-) into C(4) compounds may play a major role to supply concentrated CO(2) to Rubisco. At moment however, physiological evidence for these hypotheses were not related well to molecular level evidence. In this study, recent progresses in molecular studies on diatom-carbon-metabolism genes were related to the physiological aspects of carbon acquisition. Furthermore, we discussed the mechanisms regulating CO(2) acquisition systems in response to changes in pCO(2). Recent findings about the participation of cAMP in the signaling pathway of CO(2) concentration strongly suggested the occurrences of mammalian-type-signaling pathways in diatoms to respond to changes in pCO(2). In fact, there were considerable numbers of putative adenylyl cyclases, which may take part in the processes of CO(2) signal capturing.     

Role of gap junctions in CO(2) chemoreception and respiratory control

Gap junctions are composed of connexins, which are organized into intercellular channels that form transmembrane pathways between neurons (cell-cell coupling), and in some cases, neurons and glia, for exchange of ions and small molecules (metabolic coupling) and ionic current (electrical coupling). Cell-cell coupling via gap junctions has been identified in brain stem neurons that function in CO(2)/H(+) chemoreception and respiratory rhythmogenesis; however, the exact roles of gap junctions in respiratory control are undetermined. Here we review the methods commonly used to study gap junctions in the mammalian brain stem under in vitro and in vivo conditions and briefly summarize the anatomical, pharmacological, and electrophysiological evidence to date supporting roles for cell-cell coupling in respiratory rhythmogenesis and central chemoreception. Specific research questions related to the role of gap junctions in respiratory control are suggested for future research.     

Lesions in retrotrapezoid nucleus decrease ventilatory output in anesthetized or decerebrate cats.

Kainic acid (KA) injections into the retrotrapezoid nucleus (RTN) of anesthetized deafferented cats profoundly decreased phrenic activity (PA) and CO2 sensitivity (J. Appl. Physiol. 68: 1157-1166, 1990). In this study small electrolytic lesions of the RTN produced the same results, indicating that the KA destroyed cells. We then asked whether anesthetic depression or the absence of peripheral chemoreceptors could explain the degree of respiratory depression observed. In decerebrate cats electrolytic lesions of the RTN resulted in a decrease in PA similar to that seen under anesthesia. CO2 sensitivity was decreased by RTN lesions that extended into the caudal RTN but less so than under anesthesia. KA injections resulted in an initial increase in PA followed by a continuous decrease, a pattern similar to that seen under anesthesia but with a slower time course. CO2 sensitivity was essentially absent. Peripheral chemodenervation produced a small further decrease in PA and a downward shift of the CO2 response without change in slope. Blood pressure was unaffected by RTN lesions but was decreased by more-caudal lesions without respiratory effects. The RTN appears to be necessary for the maintenance of eupneic phrenic activity and CO2 sensitivity even in decerebrate cats with intact peripheral chemoreceptors.     

Castration of piglets under CO2-gas anaesthesia

It has become common practice in pig fattening production systems to castrate young boar piglets without the use of anaesthesia. In this study, we examined whether or not CO2 gas is capable of inducing an acceptable anaesthetic state during which castration can be performed. The first step was to identify the most promising CO2/O2 mixture. Based on the results from this first experiment, a mixture of 70% CO2 + 30% O2 was chosen for further investigation as a potential anaesthetic during the castration of young piglets. Thereby, it was established whether the duration and depth of anaesthesia were acceptable for castration where the animal has to be insensible and unconscious. Physiological effects were assessed based on electroencephalogram (EEG) and electrocardiogram (ECG) measurements, blood gas values and behavioural responses. During the induction phase, the only typical behaviour the piglets exhibited when exposed to the 70/30 gas mixture was heavy breathing. All piglets (n = 25) lost consciousness after approximately 30 s according to the EEG. Heart rate decreased slowly during the induction phase, a serious drop occurred when piglets lost their posture. Immediately after this drop, the heart rate neared zero or showed a very irregular pattern. Shortly after loss of posture, most animals showed a few convulsions. None of the animals showed any reaction to castration in behaviour and/or on the EEG and ECG. On average, the piglets recovered within 59 s, i.e. EEG returned to its pre-induction pattern and piglets were able to regain a standing position. After 120 s, heart rate returned to pre-induction levels. In order to explore the usage range of CO2 concentration, 24 piglets were exposed to 60% CO2 + 20% O2 + 20% N2 for up to 30 s after loss of consciousness (as registered on EEG), and castrated after removal from the chamber. Sixteen of the 24 animals showed a reaction to the castration on the EEG. To establish the maximum time piglets survive in 70% CO2 + 30% O2, five piglets were placed in this mixture for 3 min. Two of them died. After that, four piglets were placed in this mixture for 2 min after unconsciousness, one died after 2 min. It was concluded from this study that it is possible to anaesthetise piglets with a mixture of 70% CO2 + 30% O2, but that there are limits to its safety in terms of CO2 concentration and duration of exposure. Before implementation for practical use, further research is essential to assess the limits of gas concentration and exposure times.     

Diethyl pyrocarbonate (an imidazole binding substance) inhibits rostral VLM CO2 sensitivity

Diethyl pyrocarbonate (DEPC) has been useful in vitro as an agent relatively specific for binding to imidazole of histidine. Administered via the cisterna magna DEPC inhibits central chemosensitivity in conscious rabbits, supporting the alphastat hypothesis for central chemoreceptor function. In this study I have applied DEPC via 1 X 3 mm cottonoid pledgets to each of the three ventrolateral medulla (VLM) chemosensitive areas in glomectomized, vagotomized, paralyzed, and servo-ventilated alpha-chloralose-urethan-anesthetized cats. CO2 responses measured by integrated phrenic nerve output were evaluated before and after DEPC application. A dose of 40 mmol/l applied to the rostral chemosensitive area increased the CO2 threshold (5.3%) and significantly decreased (P less than 0.03; Wilcoxon sign rank test) the initial slope (-43%) and the maximum (-41%) of the CO2 response. No significant effects were observed with DEPC application in the intermediate or caudal areas. Treatment with 40 mmol/l hydroxylamine immediately after DEPC in the rostral area prevented the effects supporting the interpretation that imidazole was the reactant with DEPC. The results are consistent with the hypothesis that imidazole-histidine is involved in the mechanism of central chemoreception and indicate that only the rostral area utilizes a DEPC inhibitable mechanism.     

Ventilatory effects of gap junction blockade in the RTN in awake rats

We tested the hypothesis that carbenoxolone, a pharmacological inhibitor of gap junctions, would reduce the ventilatory response to CO(2) when focally perfused within the retrotrapezoid nucleus (RTN). We tested this hypothesis by measuring minute ventilation (V(E)), tidal volume (V(T)), and respiratory frequency (F(R)) responses to increasing concentrations of inspired CO(2) (Fi(CO(2)) = 0-8%) in rats during wakefulness. We confirmed that the RTN was chemosensitive by perfusing the RTN unilaterally with either acetazolamide (AZ; 10 microM) or hypercapnic artificial cerebrospinal fluid equilibrated with 50% CO(2) (pH approximately 6.5). Focal perfusion of AZ or hypercapnic aCSF increased V(E), V(T), and F(R) during exposure to room air. Carbenoxolone (300 microM) focally perfused into the RTN decreased V(E) and V(T) in animals <11 wk of age, but V(E) and V(T) were increased in animals >12 wk of age. Glyzyrrhizic acid, a congener of carbenoxolone, did not change V(E), V(T), or F(R) when focally perfused into the RTN. Carbenoxolone binds to the mineralocorticoid receptor, but spironolactone (10 microM) did not block the disinhibition of V(E) or V(T) in older animals when combined with carbenoxolone. Thus the RTN is a CO(2) chemosensory site in all ages tested, but the function of gap junctions in the chemosensory process varies substantially among animals of different ages: gap junctions amplify the ventilatory response to CO(2) in younger animals, but appear to inhibit the ventilatory response to CO(2) in older animals.     

Partitioning of the Leaf CO2 Exchange into Components Using CO2 Exchange and Fluorescence Measurements

Photorespiration was calculated from chlorophyll fluorescence and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) kinetics and compared with CO2 evolution rate in the light, measured by three gas-exchange methods in mature sunflower (Helianthus annuus L.) leaves. The gas-exchange methods were (a) postillumination CO2 burst at unchanged CO2 concentration, (b) postillumination CO2 burst with simultaneous transfer into CO2-free air, and (c) extrapolation of the CO2 uptake to zero CO2 concentration at Rubisco active sites. The steady-state CO2 compensation point was proportional to O2 concentration, revealing the Rubisco specificity coefficient (Ksp) of 86. Electron transport rate (ETR) was calculated from fluorescence, and photorespiration rate was calculated from ETR using CO2 and O2 concentrations, Ksp, and diffusion resistances. The values of the best-fit mesophyll diffusion resistance for CO2 ranged between 0.3 and 0.8 s cm-1. Comparison of the gas-exchange and fluorescence data showed that only ribulose-1,5-bisphosphate (RuBP) carboxylation and photorespiratory CO2 evolution were present at limiting CO2 concentrations. Carboxylation of a substrate other than RuBP, in addition to RuBP carboxylation, was detected at high CO2 concentrations. A simultaneous decarboxylation process not related to RuBP oxygenation was also detected at high CO2 concentrations in the light. We propose that these processes reflect carboxylation of phosphoenolpyruvate, formed from phosphoglyceric acid and the subsequent decarboxylation of malate.     

Modulation of the heteromeric Kir4.1-Kir5.1 channels by P(CO(2)) at physiological levels

Several inward rectifier K(+) (Kir) channels are pH-sensitive, making them potential candidates for CO(2) chemoreception in cells. However, there is no evidence showing that Kir channels change their activity at near physiological level of P(CO(2)), as most previous studies were done using high concentrations of CO(2). It is known that the heteromeric Kir4.1-Kir5.1 channels are highly sensitive to intracellular protons with pKa value right at the physiological pH level. Such a pKa value may allow these channels to regulate membrane potentials with modest changes in P(CO(2)). To test this hypothesis, we studied the Kir4.1-Kir5.1 currents expressed in Xenopus oocytes and membrane potentials in the presence and absence of bicarbonate. Evident inhibition of these currents (by approximately 5%) was seen with P(CO(2)) as low as 8 torr. Higher P(CO(2)) levels (23-60 torr) produced stronger inhibitions (by 30-40%). The inhibitions led to graded depolarizations (5-45 mV with P(CO(2)) 8-60 torr). Similar effects were observed in the presence of 24 mM bicarbonate and 5% CO(2). Indeed, the Kir4.1-Kir5.1 currents were enhanced with 3% CO(2) and suppressed with 8% CO(2) in voltage clamp, resulting in hyper- (-9 mV) and depolarization (16 mV) in current clamp, respectively. With physiological concentration of extracellular K(+), the Kir4.1-Kir5.1 channels conduct substantial outward currents that were similarly inhibited by CO(2) as their inward rectifying currents. These results therefore indicate that the heteromeric Kir4.1-Kir5.1 channels are modulated by a modest change in P(CO(2)) levels. Such a modulation alters cellular excitability, and enables the cell to detect hypercapnia and hypocapnia in the presence of bicarbonate.