Carbon Dioxide Sensing by Immune Cells Occurs through Carbonic Anhydrase 2-Dependent Changes in Intracellular pH.

CO2, the primary gaseous product of respiration, is a major physiologic gas, the biology of which is poorly understood. Elevated CO2 is a feature of the microenvironment in multiple inflammatory diseases that suppresses immune cell activity. However, little is known about the CO2-sensing mechanisms and downstream pathways involved. We found that elevated CO2 correlates with reduced monocyte and macrophage migration in patients undergoing gastrointestinal surgery and that elevated CO2 reduces migration in vitro. Mechanistically, CO2 reduces autocrine inflammatory gene expression, thereby inhibiting macrophage activation in a manner dependent on decreased intracellular pH. Pharmacologic or genetic inhibition of carbonic anhydrases (CAs) uncouples a CO2-elicited intracellular pH response and attenuates CO2 sensitivity in immune cells. Conversely, CRISPR-driven upregulation of the isoenzyme CA2 confers CO2 sensitivity in nonimmune cells. Of interest, we found that patients with chronic lung diseases associated with elevated systemic CO2 (hypercapnia) display a greater risk of developing anastomotic leakage following gastrointestinal surgery, indicating impaired wound healing. Furthermore, low intraoperative pH levels in these patients correlate with reduced intestinal macrophage infiltration. In conclusion, CO2 is an immunomodulatory gas sensed by immune cells through a CA2-coupled change in intracellular pH.

Hypercapnia Induces Inositol-Requiring Enzyme 1α-Driven Endoplasmic Reticulum-associated Degradation of the Na,K-ATPase ß-Subunit.

Acute respiratory distress syndrome is often associated with elevated levels of CO2 (hypercapnia) and impaired alveolar fluid clearance. Misfolding of the Na,K-ATPase (NKA), a key molecule involved in both alveolar epithelial barrier tightness and resolution of alveolar edema, in the endoplasmic reticulum (ER) may decrease plasma membrane abundance of the transporter. Here, we investigated how hypercapnia affects the NKA ß-subunit (NKA-ß) in the ER. Exposing murine precision-cut lung slices and human alveolar epithelial A549 cells to elevated CO2 levels led to a rapid decrease of NKA-ß abundance in the ER and at the cell surface. Knockdown of ER mannosidase α class 1B member 1 and ER degradation-enhancing α-mannosidase like protein 1 by siRNA or treatment with the mannosidase α class 1B member 1 inhibitor kifunensine rescued loss of NKA-ß in the ER, suggesting ER-associated degradation (ERAD) of the enzyme. Furthermore, hypercapnia activated the unfolded protein response by promoting phosphorylation of inositol-requiring enzyme 1α (IRE1α), and treatment with an siRNA against IRE1α prevented the decrease of NKA-ß in the ER. Of note, the hypercapnia-induced phosphorylation of IRE1α was triggered by a Ca2+-dependent mechanism. In addition, inhibition of the inositol trisphosphate receptor decreased phosphorylation levels of IRE1α in precision-cut lung slices and A549 cells, suggesting that Ca2+ efflux from the ER might be responsible for IRE1α activation and ERAD of NKA-ß. In conclusion, here we provide evidence that hypercapnia attenuates maturation of the regulatory subunit of NKA by activating IRE1α and promoting ERAD, which may contribute to impaired alveolar epithelial integrity in patients with acute respiratory distress syndrome and hypercapnia.

Hypercapnia Impairs Na,K-ATPase Function by Inducing Endoplasmic Reticulum Retention of the ß-Subunit of the Enzyme in Alveolar Epithelial Cells.

Alveolar edema, impaired alveolar fluid clearance, and elevated CO2 levels (hypercapnia) are hallmarks of the acute respiratory distress syndrome (ARDS). This study investigated how hypercapnia affects maturation of the Na,K-ATPase (NKA), a key membrane transporter, and a cell adhesion molecule involved in the resolution of alveolar edema in the endoplasmic reticulum (ER). Exposure of human alveolar epithelial cells to elevated CO2 concentrations caused a significant retention of NKA-ß in the ER and, thus, decreased levels of the transporter in the Golgi apparatus. These effects were associated with a marked reduction of the plasma membrane (PM) abundance of the NKA-α/ß complex as well as a decreased total and ouabain-sensitive ATPase activity. Furthermore, our study revealed that the ER-retained NKA-ß subunits were only partially assembled with NKA α-subunits, which suggests that hypercapnia modifies the ER folding environment. Moreover, we observed that elevated CO2 levels decreased intracellular ATP production and increased ER protein and, particularly, NKA-ß oxidation. Treatment with α-ketoglutaric acid (α-KG), which is a metabolite that has been shown to increase ATP levels and rescue mitochondrial function in hypercapnia-exposed cells, attenuated the deleterious effects of elevated CO2 concentrations and restored NKA PM abundance and function. Taken together, our findings provide new insights into the regulation of NKA in alveolar epithelial cells by elevated CO2 levels, which may lead to the development of new therapeutic approaches for patients with ARDS and hypercapnia.

Nitric oxide-sensitive guanylyl cyclase signaling affects CO2-dependent but not pressure-dependent regulation of cerebral blood flow.

Cerebrovascular CO2 reactivity is affected by nitric oxide (NO). We tested the hypothesis that sildenafil selectively potentiates NO-cGMP signaling, which affects CO2 reactivity. Fourteen healthy males (34 ± 2 yr) were enrolled in the study. Blood pressure (BP), ECG, velocity of cerebral blood flow (CBF; measured by transcranial Doppler), and end-tidal CO2 (EtCO2) were assessed at baseline (CO2 ~39 mmHg), during hyperventilation (CO2 ~24 mmHg), during hypercapnia (CO2 ~46 mmHg), during boluses of phenylephrine (25-200 µg), and during graded head-up tilting (HUT). Measurements were repeated 1 h after 100 mg sildenafil were taken. Results showed that sildenafil did not affect resting BP, heart rate, CBF peak and mean velocities, estimated regional cerebrovascular resistance (eCVR; mean BP/mean CBF), breath/min, and EtCO2: 117 ± 2/67 ± 3 mmHg, 69 ± 3 beats/min, 84 ± 5 and 57 ± 4 cm/s, 1.56 ± 0.1 mmHg·cm-1·s-1, 14 ± 0.5 breaths/min, and 39 ± 0.9 mmHg, respectively. Sildenafil increased and decreased the hypercapnia induced in CBF and eCVR, respectively. Sildenafil also attenuated the decrease in peak velocity of CBF, 25 ± 2 vs. 20 ± 2% (P < 0.05) and increased the eCVR, 2.5 ± 0.2 vs. 2 ± 0.2% (P < 0.03) during hyperventilation. Sildenafil did not affect CBF despite significant increases in the eCVRs that were elicited by phenylephrine and HUT. This investigation suggests that sildenafil, which potentiates the NO-cGMP signaling, seems to affect the cerebrovascular CO2 reactivity without affecting the static and dynamic pressure-dependent mechanisms of cerebrovascular autoregulation.

Intermittent reductions in respiratory neural activity elicit spinal TNF-α-independent, atypical PKC-dependent inactivity-induced phrenic motor facilitation.

In many neural networks, mechanisms of compensatory plasticity respond to prolonged reductions in neural activity by increasing cellular excitability or synaptic strength. In the respiratory control system, a prolonged reduction in synaptic inputs to the phrenic motor pool elicits a TNF-α- and atypical PKC-dependent form of spinal plasticity known as inactivity-induced phrenic motor facilitation (iPMF). Although iPMF may be elicited by a prolonged reduction in respiratory neural activity, iPMF is more efficiently induced when reduced respiratory neural activity (neural apnea) occurs intermittently. Mechanisms giving rise to iPMF following intermittent neural apnea are unknown. The purpose of this study was to test the hypothesis that iPMF following intermittent reductions in respiratory neural activity requires spinal TNF-α and aPKC. Phrenic motor output was recorded in anesthetized and ventilated rats exposed to brief intermittent (5, ∼1.25 min), brief sustained (∼6.25 min), or prolonged sustained (30 min) neural apnea. iPMF was elicited following brief intermittent and prolonged sustained neural apnea, but not following brief sustained neural apnea. Unlike iPMF following prolonged neural apnea, spinal TNF-α was not required to initiate iPMF during intermittent neural apnea; however, aPKC was still required for its stabilization. These results suggest that different patterns of respiratory neural activity induce iPMF through distinct cellular mechanisms but ultimately converge on a similar downstream pathway. Understanding the diverse cellular mechanisms that give rise to inactivity-induced respiratory plasticity may lead to development of novel therapeutic strategies to treat devastating respiratory control disorders when endogenous compensatory mechanisms fail.

Protein kinase A-Iα regulates Na,K-ATPase endocytosis in alveolar epithelial cells exposed to high CO(2) concentrations.

Elevated concentrations of CO2 (hypercapnia) lead to alveolar epithelial dysfunction by promoting Na,K-ATPase endocytosis. In the present report, we investigated whether the CO2/HCO3(-) activated soluble adenylyl cyclase (sAC) regulates this process. We found that hypercapnia increased the production of cyclic adenosine monophosphate (cAMP) and stimulated protein kinase A (PKA) activity via sAC, which was necessary for Na,K-ATPase endocytosis. During hypercapnia, cAMP was mainly produced in specific microdomains in the proximity of the plasma membrane, leading to PKA Type Iα activation. In alveolar epithelial cells exposed to high CO2 concentrations, PKA Type Iα regulated the time-dependent phosphorylation of the actin cytoskeleton component α-adducin at serine 726. Cells expressing small hairpin RNA for PKAc, dominant-negative PKA Type Iα, small interfering RNA for α-adducin, and α-adducin with serine 726 mutated to alanine prevented Na,K-ATPase endocytosis. In conclusion, we provide evidence for a new mechanism by which hypercapnia via sAC, cAMP, PKA Type Iα, and α-adducin regulates Na,K-ATPase endocytosis in alveolar epithelial cells.

Dependence on NIRS source-detector spacing of cytochrome c oxidase response to hypoxia and hypercapnia in the adult brain.

Transcranial near-infrared spectroscopy (NIRS) provides an assessment of cerebral oxygen metabolism by monitoring concentration changes in oxidised cytochrome c oxidase Δ[oxCCO]. We investigated the response of Δ[oxCCO] to global changes in cerebral oxygen delivery at different source-detector separations in 16 healthy adults. Hypoxaemia was induced by delivery of a hypoxic inspired gas mix and hypercapnia by addition of 6 % CO2 to the inspired gases. A hybrid optical spectrometer was used to measure frontal cortex light absorption and scattering at discrete wavelengths and broadband light attenuation at 20, 25, 30 and 35 mm. Without optical scattering changes, a decrease in cerebral oxygen delivery, resulting from the reduction in arterial oxygen saturation during hypoxia, led to a decrease in Δ[oxCCO]. In contrast, Δ[oxCCO] increased when cerebral oxygen delivery increased due to increased cerebral blood flow during hypercapnia. In both cases the magnitude of the Δ[oxCCO] response increased from the detectors proximal (measuring superficial tissue layers) to the detectors distal (measuring deep tissue layers) to the broadband light source. We conclude that the Δ[oxCCO] response to hypoxia and hypercapnia appears to be dependent on penetration depth, possibly reflecting differences between the intra- and extracerebral tissue concentration of cytochrome c oxidase.

[Ornithine decarboxylase in mammalian organs and tissues at hibernation and artificial hypobiosis].

Ornithine decarboxylase (ODC, EC 4.1.1.17.) is a short-lived and dynamically regulated enzyme of polyamines biosynthesis. Regulation of functional, metabolic and proliferative state of organs and tissues involves the modifications of the ODC enzymatic activity. The organ-specific changes in ODC activity were revealed in organs and tissues (liver, spleen, bone marrow, kidney, and intestinal mucosa) of hibernating mammals - squirrels Spermophilus undulates - during the hibernating season. At that, a positive correlation was detected between the decline and recovery of the specialized functions of organs and tissues and the respective modifications of ODC activity during hibernation bouts. Investigation of changes in ODC activity in organs and tissues of non-hibernating mammals under artificial hypobiosis showed that in Wistar rats immediately after exposure to hypothermia-hypoxia-hypercapnia (hypobiosis) the level of ODC activity was low in thymus, spleen, small intestine mucosa, neocortex, and liver. The most marked reduction in enzyme activity was observed in actively proliferating tissues: thymus, spleen, small intestine mucosa. In bone marrow of squirrels, while in a state of torpor, as well as in thymus of rats after exposure to hypothermia-hypoxia-hypercapnia, changes in the ODC activity correlated with changes in the rate of cell proliferation (by the criterion of cells distribution over cell cycle). The results obtained, along with the critical analysis of published data, indicate that the ODC enzyme is involved in biochemical adaptation of mammals to natural and artificial hypobiosis. A decline in the ODC enzymatic activity indicates a decline in proliferative, functional, and metabolic activity of organs and tissues of mammals (bone marrow, mucosa of small intestine, thymus, spleen, neocortex, liver, kidneys) when entering the state of hypobiosis.

Salvinorin A pretreatment preserves cerebrovascular autoregulation after brain hypoxic/ischemic injury via extracellular signal-regulated kinase/mitogen-activated protein kinase in piglets.

BACKGROUND: Cerebral hypoxia/ischemia during infant congenital heart surgery is not uncommon and may induce devastating neurologic disabilities persistent over the lifespan. Hypoxia/ischemia-induced cerebrovascular dysfunction is thought to be an important contributor to neurological damage. No pharmacological agents have been found to prevent this. Mitogen activated protein kinase (MAPK), including extracellular signal regulated kinase (ERK), c-Jun-N-terminal kinase, and p38, is thought to contribute to ischemic preconditioning. We investigated whether pretreatment with salvinorin A, the only natural nonopioid κ receptor agonist, could preserve autoregulation of the pial artery via MAPK. METHODS: The response of the pial artery to hypotension and hypercapnia was monitored in piglets equipped with a closed cranial window before and after hypoxia and ischemia in the presence or absence of U0126, an inhibitor for the protein kinase upstream of ERK, sp600125, an inhibitor of c-Jun-N-terminal kinase or sb203580, an inhibitor of p38. Salvinorin A (10 µg/kg IV) was administered 30 minutes before hypoxia/ischemia in salvinorin-treated animals. Cerebrospinal fluid samples were collected before and 30 minutes after salvinorin A administration for the measurement of MAPK. Data (n = 5) were analyzed by repeated-measures analysis of variance. RESULTS: Pial artery dilation to hypercapnia and hypotension was blunted after hypoxia/ ischemia but preserved well by pretreatment with salvinorin A. U0126, but not sp600125 or sb203580, abolished the preservative effects of salvinorin A on cerebral vascular autoregulation to hypotension and hypercapnia. The ratio of pERK/ERK in cerebrospinal fluid increased significantly in salvinorin-treated animals, which was inhibited by U0126. CONCLUSIONS: Salvinorin A pretreatment preserves autoregulation of the pial artery to hypotension and hypercapnia after hypoxia/ischemia via ERK in a piglet model.

AMP-activated protein kinase regulates CO2-induced alveolar epithelial dysfunction in rats and human cells by promoting Na,K-ATPase endocytosis.

Hypercapnia (elevated CO(2) levels) occurs as a consequence of poor alveolar ventilation and impairs alveolar fluid reabsorption (AFR) by promoting Na,K-ATPase endocytosis. We studied the mechanisms regulating CO(2)-induced Na,K-ATPase endocytosis in alveolar epithelial cells (AECs) and alveolar epithelial dysfunction in rats. Elevated CO(2) levels caused a rapid activation of AMP-activated protein kinase (AMPK) in AECs, a key regulator of metabolic homeostasis. Activation of AMPK was mediated by a CO(2)-triggered increase in intracellular Ca(2+) concentration and Ca(2+)/calmodulin-dependent kinase kinase-beta (CaMKK-beta). Chelating intracellular Ca(2+) or abrogating CaMKK-beta function by gene silencing or chemical inhibition prevented the CO(2)-induced AMPK activation in AECs. Activation of AMPK or overexpression of constitutively active AMPK was sufficient to activate PKC-zeta and promote Na,K-ATPase endocytosis. Inhibition or downregulation of AMPK via adenoviral delivery of dominant-negative AMPK-alpha(1) prevented CO(2)-induced Na,K-ATPase endocytosis. The hypercapnia effects were independent of intracellular ROS. Exposure of rats to hypercapnia for up to 7 days caused a sustained decrease in AFR. Pretreatment with a beta-adrenergic agonist, isoproterenol, or a cAMP analog ameliorated the hypercapnia-induced impairment of AFR. Accordingly, we provide evidence that elevated CO(2) levels are sensed by AECs and that AMPK mediates CO(2)-induced Na,K-ATPase endocytosis and alveolar epithelial dysfunction, which can be prevented with beta-adrenergic agonists and cAMP.

Elevated seawater PCO2 differentially affects branchial acid-base transporters over the course of development in the cephalopod Sepia officinalis.

The specific transporters involved in maintenance of blood pH homeostasis in cephalopod molluscs have not been identified to date. Using in situ hybridization and immunohistochemical methods, we demonstrate that Na(+)/K(+)-ATPase (soNKA), a V-type H(+)-ATPase (soV-HA), and Na(+)/HCO(3)(-) cotransporter (soNBC) are colocalized in NKA-rich cells in the gills of Sepia officinalis. mRNA expression patterns of these transporters and selected metabolic genes were examined in response to moderately elevated seawater Pco(2) (0.16 and 0.35 kPa) over a time course of 6 wk in different ontogenetic stages. The applied CO(2) concentrations are relevant for ocean acidification scenarios projected for the coming decades. We determined strong expression changes in late-stage embryos and hatchlings, with one to three log2-fold reductions in soNKA, soNBCe, socCAII, and COX. In contrast, no hypercapnia-induced changes in mRNA expression were observed in juveniles during both short- and long-term exposure. However, a transiently increased ion regulatory demand was evident during the initial acclimation reaction to elevated seawater Pco(2). Gill Na(+)/K(+)-ATPase activity and protein concentration were increased by ~15% during short (2-11 days) but not long-term (42-days) exposure. Our findings support the hypothesis that the energy budget of adult cephalopods is not significantly compromised during long-term exposure to moderate environmental hypercapnia. However, the downregulation of ion regulatory and metabolic genes in late-stage embryos, taken together with a significant reduction in somatic growth, indicates that cephalopod early life stages are challenged by elevated seawater Pco(2).

Analysis of the changes in the oxidation of brain tissue cytochrome-c-oxidase in traumatic brain injury patients during hypercapnoea: a broadband NIRS study.

Using broadband near-infrared spectroscopy (NIRS) and cerebral microdialysis (MD),we investigated cerebral cellular metabolism and mitochondrial redox states, following hypercapnoea in 6 patients with traumatic brain injury (TBI). In all patients hypercapnoea increased intracranial pressure and cerebral blood flow velocity measured with transcranial Doppler. Despite the likely increase in cerebral oxygen delivery, we did not see an increase in the oxidation status of cytochrome-c-oxidase [oxCCO] in every patient. Analysis of the NIRS data demonstrated two patterns of the changes; Group A (n = 4) showed an increase in [oxCCO] of 0.34(± 0.34)µM and Group B (n = 2) a decrease of 0.40(± 0.41)µM. Although no obvious association was seen between the Δ[oxCCO] and the MD, measured changes in lactate and pyruvate concentrations. Further work using model informed data interpretation may be helpful in understanding the multimodal signals acquired in this heterogeneous patient group.

PAI-1-derived peptide EEIIMD prevents impairment of cerebrovasodilation by augmenting p38 MAPK upregulation after cerebral hypoxia/ischemia.

Babies are frequently exposed to cerebral hypoxia and ischemia (H/I) during the perinatal period as a result of stroke, problems with delivery, or postdelivery respiratory management. The sole approved treatment for acute stroke is tissue type plasminogen activator. H/I impairs pial artery dilation (PAD) induced by hypercapnia and hypotension, the impairment aggravated by type plasminogen activator and attenuated by the plasminogen activator inhibitor-1-derived peptide EEIIMD. Mitogen-activated protein kinase (MAPK), a family of at least three kinases, ERK, p38, and JNK, is upregulated after H/I and ERK contribute to impaired cerebrovasodilation. This study determined the roles of p38 and JNK MAPK in the impairment of dilation post-H/I in pigs equipped with a closed cranial window and the relationship between alterations in MAPK isoforms and EEIIMD-mediated cerebrovascular protection. Cerebrospinal fluid-phosphorylated (activated) p38 MAPK, but not JNK MAPK, was increased after H/I, an effect potentiated by intravenous EEIIMD administered 1 h postinjury. PAD in response to hypercapnia and hypotension was blunted by H/I, but dilation was maintained by EEIIMD. PAD was further impaired by the p38 antagonist SB-203580 but unchanged by the JNK antagonist SP-600125. Isoproterenol-induced PAD was unchanged by H/I, EEIIMD, SB-203580, and SP-600125. These data indicate that postinjury treatment with EEIIMD attenuated impaired cerebrovasodilation post-H/I by upregulating p38 but not JNK. These data suggest that plasminogen activator inhibitor-1-based peptides and other approaches to upregulate p38 may offer a novel approach to increase the benefit-to-risk ratio of thrombolytic therapy for diverse central nervous system disorders associated with H/I.

Modification of the hepatic hemodynamic response to acute changes in PaCO2 by nitric oxide synthase inhibition in rabbits.

BACKGROUND: Hypercapnia has been reported to modify liver circulation. The vascular regulations implicated in this response remain partly unknown. METHODS: Using anesthetized and ventilated rabbits, we designed this study to evaluate the hepatic artery and portal vein blood flow velocity adjustments (20 MHz pulsed Doppler) after changes in PaCO2 (by varying the inspiratory fraction of CO2 and to assess the proper role of pH, independent of PaCO2 changes, the role of portal vein CO2, and the effect of nitric oxide synthase inhibition on CO2-induced modifications of hepatic hemodynamics. RESULTS: Increasing PaCO2 from 30.9 +/- 5 mm Hg to 77 +/- 11 mm Hg increased arterial blood pressure by 20% (P < 0.01) and hepatic artery blood flow velocity by 90% (P < 0.05) and decreased aortic blood flow velocity by 15% and portal vein blood flow velocity by 40% (both P < 0.05). Changes in pH (1 mL of 0.1 N hydrochloric acid infusion) or isolated changes in portal vein CO2 at constant PaCO2 induced by CO2 insufflation in an open abdomen had no effect on hepatic hemodynamics. Pretreatment with a nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine (2.5 mg/kg), blunted the systemic response to hypercapnia, whereas the portal modifications persisted, with a largely attenuated hepatic artery blood flow increase. CONCLUSIONS: CO2 per se acts on hepatic blood flow by its systemic effect, probably via chemoreflexes. Nitric oxide does not mediate hepatosplanchnic hemodynamic modifications to acute changes in PaCO2 but may play a permissive role by regulating the amplitude of hepatic vascular response.

Carbonic anhydrase II and alveolar fluid reabsorption during hypercapnia.

Carbonic anhydrase II (CAII) plays an important role in carbon dioxide metabolism and intracellular pH regulation. In this study, we provide evidence that CAII is expressed in both type I (AECI) and type II (AECII) alveolar epithelial cells by RT-PCR and Western blotting in freshly isolated rat cells. These results were further confirmed by double immunostaining with CAII antibodies and AECI- or AECII-specific markers in freshly isolated alveolar epithelial cells and rat lung tissues. Inhibition of CAII by acetazolamide or methazolamide delayed the decrease in the intracellular pH observed during hypercapnia in cultured AECI, AECII, and AECI-like cells. In an isolated-perfused rat lung model, alveolar fluid reabsorption significantly decreased during high CO(2) exposure, which was not prevented by carbonic anhydrase inhibition. Thus, we provide evidence that CAII is expressed in rat alveolar epithelial cells and does not regulate lung alveolar fluid reabsorption during hypercapnia.

Widespread attenuation of the cerebrovascular reactivity to hypercapnia following inhibition of nitric oxide synthase in the conscious rat.

Despite the increasing number of publications devoted to the cerebrovascular role of NO, its precise influence in awake animals is still poorly characterized. The effect of nitric oxide synthase (NOS) inhibition on the cerebrovascular CO2 reactivity was therefore studied in conscious rats. Regional CBF was measured using the [14C]iodoantipyrine technique and brain tissue sampling. The CO2 reactivity was determined 60 min after administration of 30 mg kg-1 N omega-nitro-L-arginine methyl ester (L-NAME). Blockade of NOS by L-NAME significantly decreased CBF in all 11 brain regions studied (-17 to -49%) and increased arterial pressure from 117 +/- 12 to 147 +/- 11 mn Hg. In control conditions, CO2 responsiveness ranged from 1.3 +/- 0.4 in the hypophysis to 6.4 +/- 0.6 ml 100 g-1 min-1 mm Hg-1 in the parietal cortex. Following L-NAME injection, the reactivity to hypercapnia was significantly attenuated in all structures, the magnitude of the reduction ranging from 57% in the medulla to 74% in the cerebellum. This result shows that NO is an important mediator of the hypercapnic vasodilation in the conscious rat.

PGE(2)-mediated eNOS induction in prolonged hypercapnia.

PURPOSE: Because prostaglandins (PGs) are implicated in acute hypercapnia-induced hyperemia, this study was conducted to test the hypothesis that prolonged hypercapnia may cause a sustained increase in retinal blood flow (RBF) through a PG-dependent induction of endothelial nitric oxide synthase (eNOS). METHODS: Time-dependent RBF (microsphere technique), PGE(2), nitrite (NO(2)(-)), and NOS protein (reduced nicotinamide adenine dinucleotide phosphate [NADPH]-diaphorase staining) production were measured in hypercapnia (6% CO(2))-treated piglets. From the same species, PGE(2), eNOS mRNA, NOS protein, and vasomotor responses were measured in eyecup preparations, as were Ca(2+) transients in neuroretinovascular endothelial cells. RESULTS: Hypercapnia caused biphasic (at 0.5 hours and 6-8 hours) increases in RBF that were abolished with normalization of the pH. The early phase (0.5 hour) was associated with an increase in PGE(2) levels and the latter phase (6-8 hours) with an increase in NO(2)(-) and NOS protein. Inhibition of cyclooxygenase by diclofenac prevented the early and late increase in RBF. NOS inhibitor L-nitro-arginine prevented only the latter. Hypercapnic acidosis increased retinal PGE(2) levels and eNOS-dependent vasorelaxation ex vivo. The ex vivo time course of eNOS mRNA expression corresponded with the late-phase increase in RBF and was blocked by the transcription inhibitor actinomycin D and the receptor-operated Ca(2+) channel blocker SK&F96365. In neuroretinovascular cells, acidosis increased Ca(2+) transients, which were inhibited by SK&F96365, but not diclofenac. CONCLUSIONS: This study discloses a previously unexplored mechanism for late retinal hyperemia during sustained hypercapnia that appears secondary to the induced expression of eNOS mediated by PGE(2).

Cardiorespiratory responses to systemic administration of a protein kinase C inhibitor in conscious rats.

Although protein kinase C (PKC) is an essential component of multiple neurally mediated events, its role in respiratory control remains undefined. The ventilatory effects of a systemically active PKC inhibitor (Ro-32-0432; 100 mg/kg i.p.) were assessed by whole body plethysmography during normoxia, hypoxia (10% O2), and hyperoxia (100% O2) in unrestrained Sprague-Dawley rats. A sustained expiratory time increase occurred within 8-10 min of injection in room air[mean 44.8 +/- 5.2 (SE) % ], was similar to expiratory time prolongations after Ro-32-0432 administration during 100% O2 (45.5 +/- 8.1%; not significant), and was associated with mild minute ventilation (VE) decreases. Hypercapnic ventilatory responses (5% CO2) remained unchanged after Ro-32-0432. During 10% O2, VE increased from 122.6 +/- 15.6 to 195.7 +/- 10.1 ml/min in vehicle-treated rats (P < 0.001). In contrast, marked attenuation of VE hypoxic responses occurred after Ro-32-0432 [86.2 +/- 6.2 ml/min in room air to 104.1 +/- 7.1 ml/min in 10% O2; pre- vs. post-Ro32-0432, P < 0.001 (analysis of variance)]. Overall, PKC activity was reduced and increases with hypoxia were abolished in the particulate subcellular fraction of brain tissue after Ro-32-0432 treatment, indicating that this compound readily crosses the blood-brain barrier. We conclude that systemic PKC inhibition elicits significant centrally mediated expiratory prolongations and ventilatory reductions as well as blunted ventilatory responses to hypoxia but not to hypercapnia. We postulate that PKC plays an important role in signal transduction pathways within brain regions underlying respiratory control.

Cyclooxygenase-pathway participates in the regulation of regional cerebral blood flow in response to neuronal activation under normo- and hypercapnia.

The present study was designed to investigate whether cyclooxygenase products are involved in the regulation of the regional cerebral blood flow, evoked by somatosensory activation (evoked rCBF) under normo- and hypercapnia. Indomethacin (IMC) was used as cyclooxygenase inhibitor. It was applied intravenously (i.v., 10 mg/kg/h) in two experimental protocols-before hypercapnia (i) and after hypercapnia (ii). Somatosensory activation was induced by electrical hind paw stimulation (5 Hz frequency, 5 s duration, 1.5 mA). The evoked rCBF-response was measured in alpha -chloralose anesthetized rats using laser-Doppler flowmetry. IMC abolished completely the effect of hypercapnia on the baseline level of CBF. The drug reduced significantly evoked rCBF-response also. The inhibitory effect of IMC on evoked rCBF-response is better expressed under normocapnia (approximately 70%) than that under hypercapnia (approximately 40%). After IMC application, the normalized evoked rCBF curves peaked earlier as compared to that before its application (P<0.05), although the rise time of 0.5 s was nearly constant regardless of stimulus frequency. In conclusion, the results suggest a participation of IMC-sensitive and cyclooxygenase-dependent mechanisms in the regulation of evoked rCBF, induced by somatosensory stimulation.

Hyperglycemia and hypercapnia differently affect post-ischemic changes in protein kinases and protein phosphorylation in the rat cingulate cortex.

Hyperglycemia and hypercapnia aggravate intra-ischemic acidosis and subsequent brain damage. However, hyperglycemia causes more extensive post-ischemic damage than hypercapnia, particularly in the cingulate cortex. We investigated the changes in the subcellular distribution of protein kinase Cgamma (PKCgamma) and the Ca2+/calmodulin-dependent protein kinase II (CaMKII), as well as changes in protein tyrosine phosphorylation during and following 10 min normoglycemic, hyperglycemic (plasma glucose approximately 20 mM) and hypercapnic (paCO2) approximately 300 mm Hg) global cerebral ischemia. During reperfusion period, the translocation to cell membranes of PKCgamma, but not CaMKII, was prolonged by intra-ischemic hyperglycemia, while it was only marginally affected by hypercapnia. The tyrosine-phosphorylation of proteins in the synaptosomal membranes, as well as the extracellular signal-regulated kinase (ERK) in the cytosol, markedly increased during reperfusion following hyperglycemic ischemia, but to a lesser degree following hypercapnic ischemia. Our data suggest that PKCgamma, tyrosine kinase and ERK systems are involved in the process of ischemic damage in the cingulate cortex, where hyperglycemia may affect these kinases through an additional mechanism other than exaggerated acidosis.