Carbonic anhydrases maintain tissue acid-base balance and gas transport by catalyzing the reversible conversion of carbon dioxide and bicarbonate. Abnormal expression of different isoenzymes can contribute to tumor acidification, glaucoma, abnormal neuronal excitability, and metabolic disorders.

Keywords: carbonic anhydrase; CA IX; CA XII; acid-base homeostasis; carbon dioxide transport; tumor microenvironment; hypoxia; glaucoma; carbonic anhydrase inhibitor; targeted therapy

1 Molecular Characteristics and Isoenzyme Classification of the Carbonic Anhydrase Family

1.1 Catalytic Structure of Carbonic Anhydrases

Carbonic anhydrases (CAs) are metalloenzymes that catalyze the reversible conversion of carbon dioxide and water into bicarbonate and protons. The active site of human α-carbonic anhydrases generally contains a Zn²⁺ ion coordinated by three histidine residues and catalyzes the reaction through a bound water molecule or hydroxide ion. Hydrophilic and hydrophobic regions surrounding the active-site entrance participate in proton transfer and carbon dioxide positioning, respectively, thereby determining the catalytic efficiency and inhibitor-binding characteristics of different isoenzymes.

1.2 Catalytic Reaction and Proton Transfer

The CA catalytic cycle includes deprotonation of Zn²⁺-bound water, nucleophilic attack of hydroxide on carbon dioxide, formation and release of bicarbonate, and regeneration of the active site. A histidine proton-shuttle residue transfers protons from the active site to the external buffer system, allowing the reaction to proceed much faster than spontaneous carbon dioxide hydration. Buffering capacity, ionic conditions, and substrate concentrations in different tissues influence the actual catalytic rate.

1.3 Classification of Human Carbonic Anhydrase Isoenzymes

Multiple human α-carbonic anhydrase isoenzymes have been identified and can be classified according to subcellular localization as cytosolic, membrane-associated, mitochondrial, and secreted forms. CA I, CA II, CA III, CA VII, and CA XIII are mainly cytosolic; CA IV, CA IX, CA XII, and CA XIV are membrane-associated; CA VA and CA VB are mitochondrial; and CA VI is a secreted isoenzyme. CA VIII, CA X, and CA XI lack typical catalytic activity and are generally referred to as carbonic anhydrase-related proteins.

1.4 Functional Differences Among Isoenzymes

Different CA isoenzymes vary in catalytic efficiency, tissue expression, and cellular localization. CA II has high catalytic activity and participates broadly in erythrocyte gas transport, renal acid-base regulation, and aqueous humor production. CA IX and CA XII are mainly oriented toward the extracellular surface and are therefore suited to regulating the acid-base environment surrounding tumor cells. Mitochondrial CA VA and CA VB provide bicarbonate for enzymes such as pyruvate carboxylase and carbamoyl phosphate synthetase.

Table 1 Classification and Functions of Major Human Carbonic Anhydrase Isoenzymes

2 Carbonic Anhydrase-Mediated Carbon Dioxide Transport and Acid-Base Homeostasis

2.1 Carbon Dioxide Transport in Tissues and Erythrocytes

After carbon dioxide produced by tissue metabolism enters erythrocytes, CA I and CA II rapidly convert it into bicarbonate and protons. Bicarbonate exits erythrocytes through an anion exchanger in exchange for plasma chloride, whereas protons are mainly buffered by hemoglobin. This process increases the carbon dioxide-carrying capacity of blood and limits local tissue acidification.

2.2 Carbon Dioxide Release in the Lungs

When venous blood reaches the pulmonary capillaries, plasma bicarbonate re-enters erythrocytes and combines with protons to form carbon dioxide and water. CA II accelerates this reverse reaction, allowing carbon dioxide to diffuse rapidly into the alveoli and be eliminated by exhalation. Hemoglobin oxygenation also promotes proton release, further driving the conversion of bicarbonate to carbon dioxide.

2.3 Renal Bicarbonate Reabsorption and Acid Secretion

Renal tubular epithelial cells use CA II and membrane-associated CA IV to catalyze carbon dioxide-bicarbonate conversion in the tubular lumen and cytoplasm. In coordination with Na⁺/H⁺ exchange, proton pumps, and bicarbonate transporters, this reaction enables reabsorption of filtered bicarbonate and acidification of urine. Inhibition of renal CAs increases bicarbonate excretion and may cause metabolic acidosis and electrolyte disturbances.

2.4 Coordination With Transmembrane Ion Transport

CAs can form metabolic complexes with monocarboxylate transporters, bicarbonate transporters, anion exchangers, and proton-transport systems. Bicarbonate and protons produced or consumed by CA catalysis can be rapidly utilized by adjacent transporters, thereby increasing the efficiency of lactate, proton, and bicarbonate transport across membranes.

2.5 Secretion of Digestive Fluids and Cerebrospinal Fluid

CAs in the gastrointestinal tract, pancreas, choroid plexus, and ciliary body participate in the production of gastric acid, pancreatic juice, cerebrospinal fluid, and aqueous humor by supplying bicarbonate or protons. Although the direction of ion transport differs among secretory epithelia, all depend on CAs to maintain sufficient substrate availability and osmotic gradients.

3 Tissue Distribution and Functional Specialization of Carbonic Anhydrase Isoenzymes

3.1 Cytosolic CA I and CA II

CA I and CA II jointly participate in carbon dioxide transport in erythrocytes. CA II has higher catalytic activity and is also widely expressed in the kidneys, eyes, bone, gastrointestinal tract, and central nervous system. CA II deficiency can impair renal tubular acidification, osteoclast function, and cranial development, demonstrating its roles in acid-base balance, bone remodeling, and ion secretion.

3.2 CA III and Oxidative Stress Regulation

CA III is mainly expressed in skeletal muscle and adipose tissue. Although its catalytic efficiency is lower than that of CA II, it contains cysteine residues that can undergo reversible oxidative modification. CA III may help muscle and adipose cells respond to oxidative stress through protein thiol modification and antioxidant reactions, and its function cannot be assessed solely from carbon dioxide hydration activity.

3.3 CA VII and CA XIV in the Nervous System

CA VII is mainly located in the neuronal cytosol, whereas CA XIV is present on neuronal and brain vascular-associated cell membranes. Both can regulate activity-induced changes in intracellular and extracellular pH and influence bicarbonate flux through GABA_A receptor channels. Excessive CA activity can enhance depolarization and network excitability in some neurons, making these enzymes potential research targets in epilepsy and related disorders.

3.4 Mitochondrial CA VA and CA VB

Mitochondrial CAs generate bicarbonate to support pyruvate carboxylation, fatty acid synthesis, gluconeogenesis, and the urea cycle. CA VA is enriched in the liver, whereas CA VB is more widely distributed. Inhibition of mitochondrial CAs can alter glucose, lipid, and amino acid metabolism but may also disrupt normal energy homeostasis.

3.5 Secreted CA VI

CA VI is secreted by the salivary glands and other tissues and participates in pH buffering at the surfaces of the oral cavity, esophagus, and upper gastrointestinal tract. Changes in its expression or activity may affect enamel protection, taste perception, and the mucosal environment, although its effects generally depend on salivary flow and other buffering systems.

3.6 Noncatalytic Carbonic Anhydrase-Related Proteins

CA VIII, CA X, and CA XI lack a complete Zn²⁺ catalytic center and mainly participate in nervous system development and signaling through protein interactions. These proteins are not suitable for evaluation using conventional sulfonamide CA inhibitors. Their functions should instead be studied through protein expression, interaction partners, and cellular localization.

4 Tumor Microenvironment Acidification Mediated by CA IX and CA XII

4.1 Hypoxia-Induced CA IX Expression

CA IX is encoded by the CA9 gene, whose promoter contains a hypoxia-response element. Tumor hypoxia stabilizes HIF-1α, which forms a transcriptional complex with HIF-1β and strongly upregulates CA IX. Because CA IX expression is limited in many normal tissues but elevated on the surface of hypoxic tumor cells, it has value as both a hypoxia biomarker and a membrane target.

4.2 Maintenance of Intracellular pH by CA IX

High glycolytic activity and mitochondrial metabolism continuously generate lactate, protons, and carbon dioxide. CA IX catalyzes extracellular carbon dioxide hydration to produce bicarbonate and protons. Bicarbonate can re-enter the cell through transporters and buffer the intracellular acid load, whereas protons remain in the extracellular space. This process allows tumor cells to maintain a relatively alkaline intracellular environment while producing an acidic extracellular microenvironment.

4.3 Acid-Base Regulation by CA XII

CA XII is also located on the extracellular surface of the plasma membrane and can coordinate with bicarbonate transporters to regulate intracellular and extracellular pH. Its expression occurs in some breast, renal, colorectal, and other solid tumors, but unlike CA IX, CA XII also has physiological expression in multiple normal epithelial tissues. Its biomarker value should therefore be interpreted according to tumor type, membrane localization, and coexpressed molecules.

4.4 Formation of the Acidic Microenvironment

CA IX and CA XII are not the sole sources of tumor acidification. They form part of an acid-export network together with lactate production, MCT1/MCT4-mediated lactate transport, Na⁺/H⁺ exchange, proton pumps, and bicarbonate transporters. Measurement of CA expression alone cannot fully represent tumor acidity and should be integrated with lactate, extracellular pH, and hypoxia indicators.

Figure 1. CA IX cooperates with lactate, proton, and bicarbonate transport systems to maintain acid–base homeostasis in tumor cells

4.5 Effects of an Acidic Environment on Immunity and the Extracellular Matrix

Low extracellular pH can suppress cytotoxic T-cell and natural killer-cell functions, promote the development of immunosuppressive myeloid cells and tumor-associated macrophages, and alter extracellular matrix degradation and collagen remodeling. Inhibition of CA IX or CA XII may weaken the acidic barrier, but restoration of antitumor immunity also depends on lactate concentration, the degree of hypoxia, and other immunosuppressive pathways.

Table 2 Differences Between CA IX and CA XII in Tumor Acid-Base Regulation

5 Carbonic Anhydrases in Tumor Growth, Invasion, and Treatment Resistance

5.1 Tumor Cell Proliferation and Survival

Tumor cells must maintain stable intracellular pH under highly metabolic and hypoxic conditions. By promoting bicarbonate reuptake, CA IX and CA XII reduce the inhibitory effects of intracellular acidification on glycolytic enzymes, DNA synthesis, and protein function, thereby supporting continued tumor-cell proliferation and survival.

5.2 Invasion and Extracellular Matrix Degradation

An acidic extracellular environment can enhance the activity of some proteases, weaken intercellular adhesion, and promote extracellular matrix remodeling. CA IX can also form complexes with proteins associated with adhesion and migration, altering cellular polarity and motility and allowing hypoxic tumor cells to invade surrounding tissues more readily.

5.3 Tumor Stemness and Metastasis

Hypoxia, acidification, and HIF signaling can jointly maintain a tumor stem cell-like phenotype and increase tolerance to nutrient limitation and oxidative stress. CA IX-high cells often exhibit stronger clonogenicity, migration, and distant colonization. However, CA IX may be an important component of the hypoxia-adaptation network rather than the sole driver.

5.4 Chemotherapy Resistance

Weakly basic drugs can become protonated in an acidic extracellular environment, reducing their ability to cross the plasma membrane and enter tumor cells. CA-mediated pH gradients can also affect vesicular sequestration, drug efflux, and cell-death signaling, reducing the activity of some chemotherapeutic agents. Inhibition of CA IX or CA XII may improve drug entry, but the effect depends on the physicochemical properties of the drug and tumor blood flow.

5.5 Radiotherapy and Hypoxia Tolerance

Hypoxia reduces radiation-induced oxygen fixation and limits stabilization of DNA radical damage. High CA IX expression can identify hypoxic and radioresistant tumor regions. CA IX inhibition may increase cellular stress by disrupting acid-base adaptation but cannot substitute for improved tumor oxygenation or treatments that directly increase DNA damage.

5.6 Resistance to Targeted Therapy and Immunotherapy

An acidic microenvironment can reduce the metabolic capacity of immune cells, alter receptor signaling, and promote slow-proliferating or stem-like tumor states, thereby reducing sensitivity to some targeted agents. CA inhibitors may show mechanistic complementarity with immune checkpoint inhibitors, radiotherapy, or metabolic inhibitors, but resistance should not be attributed entirely to a single CA pathway.

6 Roles of Carbonic Anhydrases in Glaucoma, Neurological Disorders, and Metabolic Diseases

6.1 Glaucoma and Aqueous Humor Production

CA II and CA IV in the ciliary epithelium catalytically support bicarbonate production, driving sodium and water transport into the posterior chamber and contributing to aqueous humor formation. CA inhibition reduces aqueous humor production and lowers intraocular pressure. Topical administration reduces systemic acid-base and electrolyte effects and represents an important application of CA inhibitors in glaucoma.

6.2 Epilepsy and Neuronal Excitability

Neuronal CA II, CA VII, and CA XIV accelerate carbon dioxide-bicarbonate equilibration and influence bicarbonate efflux through GABA_A receptor channels. CA inhibition can reduce the depolarizing component of some GABAergic signals and alter intracellular pH. Accordingly, drugs with CA-inhibitory activity, such as Topiramate and Zonisamide, can contribute to the control of neuronal excitability, although their antiepileptic effects also involve other ion channels and neurotransmitter systems.

6.3 Cerebrospinal Fluid Production and Intracranial Pressure

CAs in the choroid plexus epithelium participate in bicarbonate and cerebrospinal fluid secretion. Systemic CA inhibitors such as Acetazolamide can reduce cerebrospinal fluid production and are used to study or manage conditions involving elevated intracranial pressure. Treatment requires monitoring for metabolic acidosis, hypokalemia, kidney stones, paresthesia, and related effects.

6.4 Renal and Bone Metabolic Abnormalities

CA II is an important enzyme in renal tubular acid secretion and osteoclast-mediated bone resorption. CA II deficiency can simultaneously cause impaired renal tubular acidification and abnormal bone remodeling. Pharmacological inhibition increases bicarbonate excretion and may affect calcium salt solubility and the risk of stone formation during long-term use.

6.5 Obesity and Glucose-Lipid Metabolism

Bicarbonate generated by mitochondrial CA VA and CA VB supports pyruvate carboxylation, fatty acid synthesis, and the urea cycle. Inhibition of mitochondrial CAs may reduce lipid synthesis and alter appetite and energy metabolism. However, these metabolic effects are also influenced by the central nervous system, ion channels, and other pharmacological targets, and CA dependence cannot be inferred from body-weight changes alone.

6.6 High-Altitude Adaptation and Respiratory Regulation

CA inhibition induces mild metabolic acidosis, stimulates ventilation, and improves the respiratory response under hypoxic conditions. This action also alters renal bicarbonate excretion and systemic acid-base balance. Excessive dosing may therefore aggravate dehydration and electrolyte imbalance.

7 Mechanisms of Carbonic Anhydrase Inhibitors and Targeted Therapy Research

7.1 Sulfonamide Inhibitors

After deprotonation, sulfonamide compounds directly coordinate the active-site Zn²⁺ ion, displace the catalytic water molecule, and block carbon dioxide hydration. Acetazolamide, Methazolamide, Dorzolamide, and Brinzolamide belong to this class but differ substantially in tissue distribution, membrane permeability, and isoenzyme selectivity.

7.2 Nonclassical Zn²⁺-Binding Inhibitors

Sulfamates, sulfamides, dithiocarbamates, and other metal-binding groups can also interact with the active-site Zn²⁺ of CAs. Modification of their tail structures can alter hydrophobicity, cellular permeability, and isoenzyme selectivity. However, strong Zn²⁺ binding does not necessarily confer tumor targeting.

7.3 Coumarins and Blockade of the Active-Site Entrance

Some coumarins undergo CA-catalyzed lactone hydrolysis, and the resulting products bind at the entrance of the active site rather than directly coordinating Zn²⁺. Because the structures of active-site entrances differ markedly among isoenzymes, these inhibitors have potential for isoenzyme-selective inhibition. Their onset of action and metabolic stability must be evaluated separately.

7.4 Selective Inhibition of CA IX and CA XII

Tumor-associated CA inhibitors often contain hydrophilic or charged tails that restrict cellular entry, allowing preferential action on extracellular CA IX and CA XII while reducing inhibition of cytosolic CA I and CA II. U-104 (SLC-0111) is a research compound in this category and can be used to evaluate the effects of CA IX/XII inhibition on tumor acidification, invasion, and treatment resistance.

7.5 Antibody Targeting of CA IX

The extracellular domain of CA IX can serve as an antibody-recognition site. Anti-CA IX antibodies such as Girentuximab can be used in tumor localization, immune effector, and radionuclide-delivery studies. Antibody strategies do not require entry into the catalytic pocket, but efficacy is influenced by membrane expression, target heterogeneity, internalization efficiency, and expression in normal tissues.

7.6 Combination Treatment Strategies

CA IX or CA XII inhibition can be combined with chemotherapy, radiotherapy, immunotherapy, lactate transport inhibition, or hypoxia-targeted therapy. The key to evaluating such combinations is to confirm that CA inhibition truly alters extracellular tumor pH or intracellular acid load and to exclude apparent synergy caused by pharmacokinetic interactions between drugs.

Table 3 Characteristics of Carbonic Anhydrase Inhibition Strategies

8 Biomarker Value and Drug Evaluation Methods for Carbonic Anhydrases

8.1 Tissue Expression of CA IX

CA IX immunohistochemistry can reveal tumor-cell membrane expression and the distribution of hypoxic regions. Evaluation should distinguish complete membrane staining from nonspecific cytoplasmic signals and focal expression surrounding necrosis and should integrate HIF-1α, hypoxia tracing, and tumor type to determine pathological significance.

8.2 CA XII Expression and Tumor Stratification

CA XII can be evaluated by immunohistochemistry, flow cytometry, Western blotting, and RNA analysis. Because CA XII is also expressed in normal epithelia, expression positivity alone does not establish tumor dependence. Membrane density, isoenzyme activity, extracellular pH, and drug sensitivity should be analyzed together.

8.3 Soluble CA IX and Body-Fluid Detection

The extracellular domain of CA IX can be shed into blood or culture supernatants. Soluble CA IX measurement can be used to evaluate tumor burden, hypoxic responses, and treatment-associated changes. However, its concentration is influenced by tumor volume, protease activity, renal clearance, and the epitope recognized by the detection antibody.

8.4 Evaluation of Enzyme Activity and Inhibitory Potency

CA inhibitors can be evaluated using stopped-flow, colorimetric, fluorescence, or mass spectrometry methods to measure carbon dioxide hydration and esterase-like activity. Major parameters include Ki, IC₅₀, association rate, and dissociation rate. Because catalytic efficiency differs substantially among CA isoenzymes, comparisons across experimental platforms require standardized enzyme concentration, buffer system, temperature, and substrate conditions.

8.5 Evaluation of Cellular Acid-Base Effects

Cellular studies should separately measure intracellular and extracellular pH and integrate lactate release, bicarbonate transport, proliferation, migration, and cell-death indicators. A reduction in cell viability alone does not demonstrate that a drug acts through CA inhibition. Target dependence should also be validated using CA knockdown, knockout, or selective antagonistic controls.

8.6 Evaluation of Antitumor Efficacy in Tumor Models

In vivo studies can assess tumor growth, hypoxic regions, CA IX or CA XII expression, lactate content, tissue pH, and drug distribution. Changes in tumor volume should be analyzed together with target occupancy and acid-base effects to avoid misinterpreting nonspecific toxicity as CA-targeted activity.

8.7 Evaluation of Non-Tumor Safety

Systemic CA inhibition may cause metabolic acidosis, electrolyte disturbances, kidney stones, paresthesia, and ocular or neurological reactions. Tumor-targeted agents directed against CA IX or CA XII also require evaluation of effects on the kidneys, gastrointestinal tract, and other tissues expressing related isoenzymes.

Table 4 Major Evaluation Indicators for Carbonic Anhydrase-Related Drugs

9 Products

9.1 Carbonic Anhydrase Proteins, Antibodies, and Detection Reagents

9.2 Carbonic Anhydrase Inhibitors and Functional Research Compounds

The carbonic anhydrase family maintains carbon dioxide transport, ion secretion, and acid-base homeostasis through tissue-specific expression and subcellular localization. CA IX and CA XII are important components of the tumor acidification network, whereas CA II, CA VII, and mitochondrial CAs participate in ocular, neurological, and metabolic functions.

References

[1] Lemon, N., Canepa, E., Ilies, M. A., & Fossati, S. Carbonic anhydrases as potential targets against neurovascular unit dysfunction in Alzheimer’s disease and stroke. Frontiers in Aging Neuroscience, 13, 772278 (2021).https://doi.org/10.3389/fnagi.2021.772278

[2] Waheed, A., & Sly, W. S. Carbonic anhydrase XII functions in health and disease. Gene, 623, 33–40 (2017).https://doi.org/10.1016/j.gene.2017.04.027

[3] Cholkar, K., Trinh, H. M., Pal, D., & Mitra, A. K. Discovery of novel inhibitors for the treatment of glaucoma. Expert Opinion on Drug Discovery, 10, 293–313 (2015).https://doi.org/10.1517/17460441.2015.1000857

[4] Zamanova, S., Shabana, A. M., Mondal, U. K., & Ilies, M. A. Carbonic anhydrases as disease markers. Expert Opinion on Therapeutic Patents, 29, 509–533 (2019).

[5] Singh, S., Lomelino, C. L., Mboge, M. Y., Frost, S. C., & McKenna, R. Cancer drug development of carbonic anhydrase inhibitors beyond the active site. Molecules, 23, 1045 (2018).https://doi.org/10.3390/molecules23051045

[6] Ismail, I. S., & Amodu, A. D. Carbonic anhydrase: A new therapeutic target for managing diabetes. Journal of Metabolic Syndrome, 5 (2016).

[7] Pastorekova, S., & Gillies, R. J. The role of carbonic anhydrase IX in cancer development: Links to hypoxia, acidosis, and beyond. Cancer and Metastasis Reviews, 38, 65–77 (2019)

[8] Supuran, C. T. Applications of carbonic anhydrase inhibitors in renal and central nervous system diseases. Expert Opinion on Therapeutic Patents, 28, 713–721 (2018).https://doi.org/10.1080/13543776.2018.1519023