Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R-3 Hand 11S-3 H-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective pro-S hydrogen abstraction from C-11 of the substrate (97-99% pro-S-selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoylphosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S,15S-and 8S,15S-dihydroperoxides, a new product was formed and identified by high-performance liquid chromatography, UV, gas chromatography-mass spectrometry, and NMR as 9R,15S-dihydroperoxyeicosa-5Z,7E,11Z,13E-tetraenoic acid, the R configuration "partner" of the normal 5S,15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site.

Little is known about the role of Ca(2+) in central chemosensitive signaling. We use electrophysiology to examine the chemosensitive responses of tetrodotoxin (TTX)-insensitive oscillations and spikes in neurons of the locus ceruleus (LC), a chemosensitive region involved in respiratory control. We show that both TTX-insensitive spikes and oscillations in LC neurons are sensitive to L-type Ca(2+) channel inhibition and are activated by increased CO(2)/H(+). Spikes appear to arise from L-type Ca(2+) channels on the soma whereas oscillations arise from L-type Ca(2+) channels that are distal to the soma. In HEPES-buffered solution (nominal absence of CO(2)/HCO(3)(-)), acidification does not activate either oscillations or spikes. When CO(2) is increased while extracellular pH is held constant by elevated HCO(3)(-), both oscillation and spike frequency increase. Furthermore, plots of both oscillation and spike frequency vs. intracellular [HCO(3)(-)]show a strong linear correlation. Increased frequency of TTX-insensitive spikes is associated with increases in intracellular Ca(2+) concentrations. Finally, both the appearance and frequency of TTX-insensitive spikes and oscillations increase over postnatal ages day 3-16. Our data suggest that 1) L-type Ca(2+) currents in LC neurons arise from channel populations that reside in different regions of the neuron, 2) these L-type Ca(2+) currents undergo significant postnatal development, and 3) the activity of these L-type Ca(2+) currents is activated by increased CO(2) through a HCO(3)(-)-dependent mechanism. Thus the activity of L-type Ca(2+) channels is likely to play a role in the chemosensitive response of LC neurons and may underlie significant changes in LC neuron chemosensitivity during neonatal development.

This study was designed to investigate brain connections among chemosensitive areas in newborn rats. Rhodamine beads were injected unilaterally into the locus coeruleus (LC) or into the caudal part of the nucleus tractus solitarius (cNTS) in Sprague-Dawley rat pups (P7–P10). Rhodamine-labeled neurons were patched in brainstem slices to study their electrophysiological responses to hypercapnia and to determine if chemosensitive neurons are communicating between LC and cNTS regions. After 7–10 days, retrograde labeling was observed in numerous areas of the brainstem, including many chemosensitive regions, such as the contralateral LC, cNTS and medullary raphe. Whole-cell patch clamp was done in cNTS. In 4 of 5 retrogradely-labeled cNTS neurons that projected to the LC, firing rate increased in response to hypercapnic acidosis (15% CO2), even in synaptic blockade medium (high Mg2+/low Ca2+). In contrast, 2 of 3 retrogradely-labeled LC neurons that projected to cNTS had reduced firing rate in response to hypercapnic acidosis, both in the presence and absence of synaptic blockade medium. Extensive anatomical connections among chemosensitive brainstem regions in newborn rats were found and at least for the LC and cNTS, the connections involve some CO2-sensitive neurons. Such anatomical and functional coupling suggests a complex central respiratory control network, such as seen in adult rats, is already largely present in neonatal rats by at least day P7–P10. Since the NTS and the LC play a major role in memory consolidation, our results may also contribute to the understanding of the development of memory consolidation.

Hypercapnic acidosis activates Ca2+ channels and increases intracellular Ca2+ levels in neurons of the locus coeruleus (LC), a known chemosensitive region involved in respiratory control. We have also shown that large conductance Ca2+-activated K+ channels (BK), in conjunction with this pathway, limits the hypercapnic-induced increase in firing rate in LC neurons. Here, we present evidence that the Ca2+ current is activated by a HCO3−-sensitive pathway. The increase in HCO3− associated with hypercapnia activates HCO3−-sensitive adenylyl cyclase (sAC). This results in an increase in cAMP levels and activation of Ca2+ channels via cAMP-activated protein kinase A (PKA). We also show the presence of sAC in the cytoplasm of LC neurons, and that the cAMP analogue db-cAMP increases Ca2+i. Disrupting this pathway by decreasing HCO3− levels during acidification or inhibiting either sAC or PKA, but not transmembrane adenylyl cyclase (tmAC), can increase the magnitude of the firing rate response to hypercapnia in LC neurons from older neonates to the same extent as inhibition of BK channels.

The cellular mechanisms by which LC neurons respond to hypercapnia are usually attributed to an "accelerator" whereby hypercapnic acidosis causes an inhibition of K channels or activation of Na and Ca channels to depolarize CO-sensitive neurons. Nevertheless, it is still unknown if this "accelerator" mechanism could be controlled by a brake phenomenon. Whole-cell patch clamping, fluorescence imaging microscopy and plethysmography were used to study the chemosensitive response of the LC neurons. Hypercapnic acidosis activates L-type Ca channels and large conductance Ca-activated K (BK) channels, which function as a "brake" on the chemosensitive response of LC neurons. Our findings indicate that both Ca and BK currents develop over the first 2 weeks of postnatal life in rat LC slices and that this brake pathway may cause the developmental decrease in the chemosensitive firing rate response of LC neurons to hypercapnic acidosis. Inhibition of this brake by paxilline (BK channel inhibitor) returns the magnitude of the chemosensitive firing rate response from LC neurons in rats older than P10 to high values similar to those in LC neurons from younger rats. Inhibition of BK channels in LC neurons by bilateral injections of paxilline into the LC results in a significant increase in the hypercapnic ventilatory response of adult rats. Our findings indicate that a BK channel-based braking system helps to determine the chemosensitive respiratory drive of LC neurons and contributes to the hypercapnic ventilatory response. Perhaps, abnormalities of this braking system could result in hypercapnia-induced respiratory disorders and panic responses.

Chemosensitive LC neurons increase their firing rate in response to CO2 via inhibition of K+ channels. This increase gets smaller during neonatal development (from P3‐P16). Changes of this K+ channel‐based “accelerating” pathway may account for the developmental changes. We used whole cell patch clamp and fluorescence imaging microscopy to study a different basis for these developmental changes in chemosensitive gain, a “braking” pathway. We found that an L‐type Ca2+ current increased in LC neurons during development. This current was activated by CO2, resulting in increases in [Ca2+]i and the activation of hyperpolarizing Ca2+‐activated K+ currents (KCa), which should reduce the firing rate response of LC neurons to CO2. We used the BK (a large conductance KCa) channel inhibitor paxilline and voltage clamp to study this KCa current. Inhibition of the BK current removed the “brake” and increased the firing rate response to CO2 in LC neurons. Notably, the “braking” pathway increased during neonatal development in a fashion that paralleled the decrease in the chemosensitive firing rate response of LC neurons to CO2. These findings suggest a unique role for Ca2+ in controlling central chemoreceptor gain. Abnormalities in this pathway could be associated with disorders involving elevated central gain, such as sleep apnea and panic disorders.[NIH R01 HL56683, AHA Great Rivers Affiliate Predoctoral Fellowship]

To study the communication among chemosensitive brainstem areas, we injected rhodamine beads into the LC in rat pups (P7–P10). After 8–10 days, retrograde labeling was seen in the brainstem, including neurons from many chemosensitive regions, such as contralateral LC, caudal NTS (cNTS), retrotrapezoid nucleus, medullary raphé and periaqueductal gray. Similar connections were seen with cNTS injection, although less labeling was seen in each area. We patched cNTS neurons that project to the LC and found that 4 of 5 retrogradely‐labeled cNTS neurons had increased firing rate (FR) in response to hypercapnic acidosis (HA‐15% CO2), even in synaptic blockade medium (SNB—high Mg2+/low Ca2+). In contrast, 2 of 3 retrogradely‐labeled LC neurons that project to the cNTS had reduced FR in response to HA both in the presence and absence of SNB. We loaded the patched neuron with Lucifer Yellow (LY) from the pipette and verified that the neuron was labeled for both rhodamine and LY. Our studies show: extensive connections among chemosensitive brainstem regions; HA‐induced increased FR of cNTS neurons results in increased action potentials (APs) to the LC; and HA‐induced decreased FR of LC neurons results in reduced APs to cNTS neurons. Complex interactions clearly exist among chemosensitive regions within the central respiratory control network.Supported by FAPESP, CAPES, CNPq, NIH R01‐HL‐56683 and WSU Research Incentive Grant