Carbon dioxide detection in biological systems

Arterial CO₂ tensions (PaCO₂) represents a balance between CO₂ production and elimination via the lungs, and in health is maintained within a tight range (3.5 – 4.5 kPa). Traditional approaches to CO₂ management in the critically ill emphasized the use of higher tidal and minute ventilation to avoid hypercapnia and its associated the risks. The demonstration that high lung stretch directly injures the lungs heralded the use of more protective ventilatory strategies that reduce lung stretch, and have been proven to improve survival in patients with ARDS. Consequently, hypercapnia – and its associated hypercapnic acidosis (HCA) - is prevalent in the critically ill, ‘permitted’ order to realize the benefits of lower lung stretch. Experimental and clinical investigations have generated key advances in our understanding of the effects of hypercapnia. Hypercapnia to be a potent biologic agent, with the potential to exert both beneficial and potentially harmful effects. Hypercapnia modulates the innate immune response, with inhibition of nuclear factor kappa-B, is a key transcriptional protein in injury, inflammation and repair, mediating diverse effects of hypercapnia. Advances in extracorporeal technologies have made possible the direct removal of CO₂ while maintaining lung protective ventilation, a promising, though as yet unproven, approach. Consequently, it is important to understand the biology of hypercapnia, in order to best understand when hypercapnia should be encouraged, tolerated or avoided in patients with ARDS.

The detection and regulated excretion of CO2, via breathing, is fundamental for homeostatic control of blood pH and preservation of life. Breathing is highly sensitive to the partial pressure of CO2 (PCO2) in blood. This vital physiological function was previously thought to depend exclusively upon the indirect changes in pH that follow the accumulation of CO2. However, evidence suggests that CO2 can have direct effects on breathing in addition to those of pH.

Connexins are large-conductance hexameric plasma membrane channels. They can dock together to form a passageway between adjacent cells, a gap junction, to permit transfer of ions and small molecules. Connexin channels not docked to those in neighbouring cells form “hemichannels” and open to the extracellular space. Chemosensory cells at the surface of the medulla oblongata use hemichannels of connexin26 (Cx26) to detect CO2 and effect adaptive changes in breathing. Physiological levels CO2 cause hemichannels of Cx26 to open, permitting the release of the neurotransmitter ATP and excitation of the neural networks controlling breathing. CO2 most likely binds to Cx26 by carbamylating Lys125, which forms a salt bridge to Arg104 on a neighbouring subunit to open the hemichannel.

Understanding the interaction of CO2 with Cx26 has enabled the rational design of a dominant negative subunit, dnCx26, which coassembles with wild type Cx26 to remove its CO2 sensitivity. Transduction with dnCx26 of the chemosensory cells in the medulla oblongata greatly reduces the chemosensitivity of breathing, thus directly linking the functional motif of CO2-binding to the physiological function of Cx26.

The rate of photosynthesis is very sensitive to the level of CO2 inside the chloroplasts, where photosynthesis takes place and CO2 is fixed by rubisco. CO2 first diffuses from the atmosphere through stomatal pores into the substomatal space. The CO2 must then cross internal airspace, cell walls, plasmalemma, cytoplasm, chloroplast envelope and part of the chloroplast stroma before it is fixed in the first step of the Calvin-Benson cycle. The combined conductance to CO2 transfer from substomatal cavities to the site of fixation is termed mesophyll conductance. In order to estimate mesophyll conductance, the exchange of CO2 from atmosphere into the leaf, together with the isotopic composition of the CO2 is measured. By exploiting Rubisco’s natural preference for 12CO2 over 13CO2, we can calculate mesophyll conductance from carbon isotopic discrimination. Mesophyll conductance is an important photosynthetic parameter that influences the amount of CO2 available for fixation and is a target for improving crop productivity. Understanding variations in mesophyll conductance across leaves, and what drives these changes, is essential for modelling how the manipulation of photosynthetic pathways may alter plant productivity. Using Nicotiana tabacum var. Samsun, we have investigated how mesophyll conductance, and other photosynthetic and leaf anatomy parameters, vary across leaf ages and throughout the canopy, with the goal of better informing plant productivity models.

My laboratory uses aquatic animals as models to study the evolution of acid-base sensing mechanisms at the molecular, cellular, and organismal levels. Because the internal fluids of aquatic organisms have lower CO2 and HCO3- levels and higher pH (“acid-base” parameters) compared to air-breathing vertebrates, their underlying acid-base sensing mechanisms must be tuned to different and specific set points. Kinetic assays on the evolutionary conserved acid-sensing enzyme soluble adenylyl cyclase (sAC) demonstrate a species-specific responsiveness to [HCO3-] that in each case matches physiologically relevant levels. For example, sAC’s HCO3- half-maximal stimulation is ~5 mM in sharks, ~10 mM in bony fishes and coral, which are lower than the ~20 mM reported in mammals. Additionally, aquatic animals routinely experience metabolic and environmental acid-base disturbances that can span >1 pH unit and >10-fold changes in [HCO3-]. Physiologically, this implies acid-base sensing plays essential and multiple homeostatic roles. Experimentally, this is advantageous because it allows imposing extreme (but physiologically relevant) acid-base challenges (i.e. 0-100 mM bicarbonate, pH 6.0-9.0) that maximize the magnitude of physiological responses and facilitate their detection. These approaches have led to the discovery of several novel physiological functions under sAC modulation in aquatic animals, including base secretion in shark gill epithelial cells, salt and fluid absorption across fish intestine, heart beat rate in hagfish, and pHi regulation in corals. In addition to their implications for organismal, environmental, and evolutionary physiology, these results provide clues about similar processes that might be under sAC control in humans and therefore might have biomedical relevance.

Hypercapnia, or elevated PCO2 in blood and tissue, is an independent risk factor for mortality in patients with severe acute and advanced chronic lung disease. Bacterial and viral lung infections are often proximate events leading to poor clinical outcomes in such individuals. The Sporn group showed that exposure to elevated concentrations of CO2 inhibits innate immune gene expression and phagocytosis in macrophages, suggesting a causal role for hypercapnia in worse outcomes related to lung infection. Further, Sporn and colleagues showed that normoxic hypercapnia increased the mortality of bacterial pneumonia in mice. In addition, hypercapnia suppressed macrophage antiviral gene expression and increased the mortality of influenza A (IAV) infection in mice. Interestingly, hypercapnia also inhibited innate immune gene expression and increased the mortality of bacterial infection in Drosophila. A genome-wide RNAi screen then led to the identification of a transcription factor, zfh2, whose expression was required for CO2-induced immunosuppression in the fly in vitro and in vivo. The Sporn group generated a myeloid-specific mouse knockout of Zfhx3, the mammalian ortholog of zfh2, and finds that Zfhx3-deficient macrophages from the mutant are protected against hypercapnia-induced suppression of antiviral gene expression and increased growth of IAV in vitro. Further, the myeloid Zfhx3-deficient mouse is partially protected against IAV-induced lung injury. RNA-seq analysis of alveolar macrophages from IAV-infected mice reveals that host defense-related gene expression pathways downregulated by hypercapnia in the wild-type are upregulated in alveolar macrophages from Zfhx3-deficient mice. Future studies will further elucidate the molecular mechanism(s) by which hypercapnia suppresses lung host defense.

There has traditionally been a far greater emphasis in respiratory physiology and medicine on effects and roles of O2 than CO2 on the lung. This ‘oxycentric’ focus has relegated CO2 to that of the neglected step sister of the two gases, yet it has considerable influence on gas exchange efficiency and inflammation that are quite under-recognized.  When either regional ventilation (VA) or perfusion (Q) varies, both O2 and CO2-dependent mechanisms are evoked to restore the balance of local blood flow and local gas flow. Matching of Q to changes in VA is accomplished by both hypoxic pulmonary vasoconstriction (HPV) and hypercapnic pulmonary vasoconstriction (HCPV), acting equally to limit perfusion into poorly ventilated areas. HCPV may be more important since it is active over the entire range of PCO2 in normal and diseased lungs, whereas as HPV is only engaged below a PO2 of 60 mmHg. In contrast to perfusion regulation, regulation of regional ventilation has no dependence on PO2. The changes in alveolar PCO2 caused by changes in Q act to alter VA by actions at the bronchial level by hypercapnic bronchodilation and at the parenchymal level by hypercapnic pneumo-relaxation causing changes in tissue compliance. These CO2-mediated effects are largely the result of the accompanying pH change which is accelerated by carbonic anhydrase in the lung. Beyond the benefits of CO2 on gas exchange efficiency and VA/Q matching, it also has anti-inflammatory effects that have the potential to mitigate lung injury from ischemia-reperfusion damage, acute respiratory distress syndrome and infections. 

Applying either elevated atmospheric carbon dioxide concentrations or the drought hormone abscisic acid (ABA), to plant leaves brings about similar effects; they reduce the apertures of stomatal pores and reduce the number of stomata that form. These two stomatal responses allow plants to reduce their rate of transpiration and to conserve water. We therefore investigated whether the same signalling components might regulate stomatal responses to both [CO2] and ABA. Our results indicate that the stomatal [CO2] responses utilise components of the ABA signaling pathway including ABA biosynthesis and ABA receptors. Thus it appears possible that stomatal [CO2] responses are mediated by the ABA response pathway. However, we found no evidence for an increase in leaf ABA levels in response to elevated [CO2], suggesting that ABA either increases the sensitivity of the system to [CO2], or that any [CO2]-induced increase in ABA occurs specifically in the stomata. 

Carbon dioxide (CO₂) is a product of aerobic metabolism that is mainly exhaled via the lungs to maintain stable paCO₂ pressure (normocapnia) and blood pH. Hypercapnia (paCO₂ >50mmHg) is a feature of chronic lung diseases, and permissive hypercapnia occurs in the intensive care setting as a feature of a protective ventilation strategy. Several in vitro and in vivo animal based studies have implicated CO₂-dependent signaling in the suppression of immune and inflammatory signaling. Thus, these CO₂-dependent signaling events may be detrimental in the context of a bacterial infection but of benefit in the context of uncontrolled inflammation. However, the molecular mechanisms underpinning CO₂-dependent changes in gene expression are not well understood and are the focus of research in the Cummins lab. This presentation will initially outline investigations from the lab into how elevated CO₂ modulates signaling, particularly within the NFkB pathway. Hypercapnia causes a marked change in the nuclear localisation and processing of key NFkB family members and modulates the expression of NFkB target genes. Dr Cummins will outline some of the recent RNA-seq and proteomic approaches taken by the group to understand the molecular mechanisms underpinning CO₂-dependent regulation of inflammatory signaling. Finally, hypoxia and hypercapnia frequently co-exist given that O₂ and CO₂ are the substrate and product of aerobic metabolism respectively. Dr Cummins will describe important cross-talk between the O₂ and CO₂-sensing pathways and demonstrate that hypercapnia suppresses the cellular adaptive response to low oxygen in vitro and in vivo.