Toll-like Receptor 3 Agonist, Polyinosinic-polycytidylic Acid, Upregulates Carbonic Anhydrase II in Human Keratinocytes.

Carbonic anhydrases are ubiquitously expressed enzymes that reversibly hydrate carbon dioxide to bicarbonate and protons. While the main function of carbonic anhydrases is to regulate pH and osmotic balance, their involvement in other physiological processes remains to be explored. This study analysed changes in mRNA and protein levels of carbonic anhydrase II in human primary keratinocytes treated with various toll-like receptor agonists and cytokines. A significant upregulation of carbonic anhydrase II at the mRNA and protein levels was observed upon treatment with polyinosinic-polycytidylic acid, a toll-like receptor 3 agonist. Furthermore, in agreement with the increased expression of carbonic anhydrase II in atopic dermatitis skin, carbonic anhydrase II was upregulated by the Th2 cytokines interleukins -4 and -13. In conclusion, these results suggest a potential role of carbonic anhydrase II in Th2-dependent and toll-like receptor 3-induced pathways in inflammatory skin conditions.

Chronic opioids regulate KATP channel subunit Kir6.2 and carbonic anhydrase I and II expression in rat adrenal chromaffin cells via HIF-2α and protein kinase A.

At birth, asphyxial stressors such as hypoxia and hypercapnia are important physiological stimuli for adrenal catecholamine release that is critical for the proper transition to extrauterine life. We recently showed that chronic opioids blunt chemosensitivity of neonatal rat adrenomedullary chromaffin cells (AMCs) to hypoxia and hypercapnia. This blunting was attributable to increased ATP-sensitive K(+) (KATP) channel and decreased carbonic anhydrase (CA) I and II expression, respectively, and involved µ- and δ-opioid receptor signaling pathways. To address underlying molecular mechanisms, we first exposed an O2- and CO2-sensitive, immortalized rat chromaffin cell line (MAH cells) to combined µ {[d-Arg(2),Ly(4)]dermorphin-(1-4)-amide}- and δ ([d-Pen(2),5,P-Cl-Phe(4)]enkephalin)-opioid agonists (2 µM) for ∼7 days. Western blot and quantitative real-time PCR analysis revealed that chronic opioids increased KATP channel subunit Kir6.2 and decreased CAII expression; both effects were blocked by naloxone and were absent in hypoxia-inducible factor (HIF)-2α-deficient MAH cells. Chronic opioids also stimulated HIF-2α accumulation along a time course similar to Kir6.2. Chromatin immunoprecipitation assays on opioid-treated cells revealed the binding of HIF-2α to a hypoxia response element in the promoter region of the Kir6.2 gene. The opioid-induced regulation of Kir6.2 and CAII was dependent on protein kinase A, but not protein kinase C or calmodulin kinase, activity. Interestingly, a similar pattern of HIF-2α, Kir6.2, and CAII regulation (including downregulation of CAI) was replicated in chromaffin tissue obtained from rat pups born to dams exposed to morphine throughout gestation. Collectively, these data reveal novel mechanisms by which chronic opioids blunt asphyxial chemosensitivity in AMCs, thereby contributing to abnormal arousal responses in the offspring of opiate-addicted mothers.

The effect of rotative stress on CAII, FAS, FASL, OSCAR, and TRAP gene expression in osteoclasts.

This study was designed to explore the effects of rotative stress on carbonic anhydrase II (CAII), TNF receptor superfamily member 6 (FAS), FAS ligand (FASL), osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP) gene expression in osteoclasts. Osteoclasts were induced from RAW264.7 cells cultured in medium containing recombinant murine soluble receptor activator of NF-Kß ligand (sRANKL). The mRNA and protein expression of CAII, FAS, FASL, OSCAR, and TRAP genes in osteoclasts was detected by RT-PCR and Western blot, respectively, after osteoclasts were loaded at various rotative stress strengths and times. No significant differences in mRNA and protein expression were observed between any of the control groups (P > 0.05). Importantly, rotative stress had a significant effect on the mRNA and protein expression of these genes (P < 0.05). We found a negative relationship between rotative stress strength and prolonged loading time and the expression of FAS/FASL genes in osteoclasts. In addition, there was a positive relationship between rotative stress strength and prolonged loading time and the expression of CAII, OSCAR, or TRAP genes in osteoclasts. Based on these results, rotative stress has a significant effect on CAII, FAS, FASL, OSCAR, and TRAP gene expression in osteoclasts.

Carbonic anhydrase II-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth.

The regenerative process in the pancreas is of particular interest because diabetes results from an inadequate number of insulin-producing beta cells and pancreatic cancer may arise from the uncontrolled growth of progenitor/stem cells. Continued and substantial growth of islet tissue occurs after birth in rodents and humans, with additional compensatory growth in response to increased demand. In rodents there is clear evidence of pancreatic regeneration after some types of injury, with proliferation of preexisting differentiated cell types accounting for some replacement. Additionally, neogenesis or the budding of new islet cells from pancreatic ducts has been reported, but the existence and identity of a progenitor cell have been debated. We hypothesized that the progenitor cells are duct epithelial cells that after replication undergo a regression to a less differentiated state and then can form new endocrine and exocrine pancreas. To directly test whether ductal cells serve as pancreatic progenitors after birth and give rise to new islets, we generated transgenic mice expressing human carbonic anhydrase II (CAII) promoter: Cre recombinase (Cre) or inducible CreER(TM) to cross with ROSA26 loxP-Stop-loxP LacZ reporter mice. We show that CAII-expressing cells within the pancreas act as progenitors that give rise to both new islets and acini normally after birth and after injury (ductal ligation). This identification of a differentiated pancreatic cell type as an in vivo progenitor of all differentiated pancreatic cell types has implications for a potential expandable source for new islets for replenishment therapy for diabetes.

In vivo monitoring of carbonic anhydrase expression during the growth of larval zebrafish: a new environment-sensitive fluorophore for responsive turn-on fluorescence.

This study monitors the dynamic progress of a newly developed background-free, target responsive strategy; 2,3-dihydroquinolin-4-imine (DQI) that can instantly respond to environmental changes with fluorescence enhancement, revealing a comprehensive platform for in vivo fluorescence bioimaging of mebrane-bound carbonic anhydrase II in HeLa cells and its expression during the growth of larval zebrafish.

Mapping proton wires in proteins: carbonic anhydrase and GFP chromophore biosynthesis.

We have developed an algorithm for mapping proton wires in proteins and applied it to the X-ray structures of human carbonic anhydrase II (CA-II), the green fluorescent protein (GFP), and some of their mutants. For both proteins, we find more extensive proton wires than typically reported. In CA-II the active site wire exits to the protein surface, and leads to Glu69 and Asp72, located on an electronegative patch on the rim of the active site cavity. One possible interpretation of this observation is that positively charged, protonated buffer molecules dock in that area, from which a proton is delivered to the active site when the enzyme works in the dehydration direction. In GFP we find a new internal proton wire, in addition to the previously reported wire involved in excited state proton transfer. The new wire is located on the other face of the chromophore, and we conjecture that it plays a role in chromophore biosynthesis that occurs following protein folding. In the last step of this process, transient carbanion formation was suggested to occur on the bridge carbon [Pouwels et al. Biochemistry 2008, 47, 10111]. Residues on the new wire (Thr62, His181, Arg96) may participate in proton abstraction from this bridge carbon atom. A possible mechanism involves a rotation of the Thr62 side chain and completion of a short wire by which the proton is transported to His181, while the negative charge is transferred to the imidazolone carbonyl, producing a homoenolate intermediate that is stabilized by Arg96. Finally, comparison of the proton wires in the two proteins reveals common motifs, such as short internalized Ser/Thr-Glu hydrogen-bonded pairs for ultrafast proton abstraction, and threonine side chain rotation functioning as a proton wire switch.

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.

Low levels of Na, K-ATPase and carbonic anhydrase II during choroid plexus development suggest limited involvement in early CSF secretion.

UNLABELLED: In the adult, cerebrospinal fluid (CSF) is produced by the actions of numerous transporters and enzymes creating ion gradients that drive the entry of water into the ventricles via the aquaporin-1 water channels (AQP1). It is not known when in development CSF secretion starts but, in the rat, it has been postulated to occur around the time of birth. However, recent evidence suggests that the secretion may start much earlier, as soon as the lateral choroid plexuses first appear (around E14). PURPOSE: To investigate the developmental profiles of two major enzymes responsible for CSF secretion in the adult, Na, K-ATPase (NKA) and carbonic anhydrase II (CAII). METHODS: The developmental profiles of both enzymes were investigated using immunohistochemistry and Western Blot analysis in tissue from embryonic day (E) 15, 18, postnatal day (P) 0, 9 and adult rats. RESULT: Western Blot analysis showed low levels of NKA at E15 followed by a progressive increase with age. Immunohistochemistry confirmed the presence of NKA on the apical membrane of the lateral ventricular choroid plexus epithelium from E15 onwards. Western Blot analysis of CAII was complicated by its presence in blood, but the amount of protein increased with age. Immunohistochemically, CAII appeared in the lateral ventricular choroid plexus between P0 and P9. CONCLUSIONS: The low levels of NKA and CAII during early choroid plexus development indicate that other mechanisms, such as the previously described specific protein transfer across epithelial cells, may be involved in early CSF secretion and movement of water into the cerebral ventricles.

Cloning, polymorphism, and inhibition of beta-carbonic anhydrase of Helicobacter pylori.

BACKGROUND: Carbonic anhydrase (CA) catalyzes the reversible hydration of CO(2) to bicarbonate and a proton, and alpha-class CA has been reported to facilitate the acid acclimation of Helicobacter pylori (hpalphaCA). The purpose of this study was to characterize the beta-class CA of H. pylori (hpbetaCA) and elucidate the role of this enzyme as a possible drug target for eradication therapy. METHODS: We isolated DNA clones of independent H. pylori strains obtained from patients with gastritis (n = 15), gastric ulcer (n = 6), or gastric cancer (n = 16), and then studied genetic polymorphisms. In addition, the susceptibility of H. pylori to sulpiride, an antiulcer drug and efficient inhibitor of both hpalphaCA and hpbetaCA, was studied with an in vitro killing assay. RESULTS: DNA sequences of all 37 hpbetaCA clones encoded a 221 amino acid polypeptide with a variety of polymorphisms (57 types of amino acid substitution at 48 residue positions). There was no polymorphism functionally relevant to the gastric lesion type. One strain included unique residues that were not seen in the other 36 clones from Japanese patients but which were found in a strain obtained from the United Kingdom. Sulpiride had killing effects at concentrations greater than 200 microg/ml for H. pylori, including strains resistant to clarithromycin, metronidazole, or ampicillin. CONCLUSIONS: Helicobacter pylori might have evolved independently in the Caucasian and Japanese populations. Dual inhibition of alpha-and beta-class CAs could be applied as alternative therapy for eradication of H. pylori.

Inhibition on angiotensin-converting enzyme exerts beneficial effects on trabecular bone in orchidectomized mice.

BACKGROUND: This study aimed to study the osteo-preservative effects of captopril, an inhibitor on angiotensin-converting enzyme (ACE), on bone mass, micro-architecture and histomorphology as well as the modulation of captopril on skeletal renin-angiotensin system (RAS) and regulators for bone metabolism in mice with bilateral orchidectomy. METHODS: The orchidectomized (ORX) mice were orally administered with vehicle or captopril at low dose (10mg/kg) and high dose (50mg/kg) for six weeks. The distal femoral end, the proximal tibial head and the lumbar vertebra (LV) were stained by hematoxylin and eosin, Safranin O/Fast Green and masson-trichrome. Micro-computed tomography was performed to measure bone mineral density (BMD). RESULTS: Treatment with captopril increased trabecular bone area at distal metaphysis of femur, proximal metaphysis of tibia and LV-4, moreover, high dose of captopril significantly elevated trabecular BMD of LV-2 and LV-5. The mRNA expressions of renin receptor, angiotensinogen, carbonic anhydrase II, matrix metalloproteinase-9, and tumor necrosis factor-alpha were significantly decreased in tibia of ORX mice following treatment with captopril. The administration with captopril enhanced the ratio of OPG/RANKL mRNA expression, the mRNA expression of transforming growth factor-beta and the protein expression of bradykinin receptor-1. CONCLUSIONS: The inhibition on ACE by captopril exerts beneficial effects on trabecular bone of ORX mice. The therapeutic efficacy may be attributed to the regulation of captopril on local RAS and cytokines in bone.

Carbonic anhydrase II in the endothelium of glial tumors: a potential target for therapy.

Carbonic anhydrase isozyme II (CA II) is a cytosolic enzyme that is highly expressed in most organs, including the brain, where it is mainly located in the oligodendrocytes. Recent studies have shown that its expression is induced in the endothelium of neovessels in melanoma and esophageal, renal, and lung cancer. Immunological studies further indicate that CA II represents a major target antigen stimulating an autoantibody response in melanoma patients. These results prompted us to investigate endothelial CA II expression in two types of brain cancer: oligodendrogliomas and astrocytomas. A series of 255 astrocytoma and 71 oligodendroglial tumor specimens was immunostained for CA II. The staining results were correlated with a number of different clinicopathological factors and survival data. CA II showed weak or no expression in low-grade tumors, while grade 3 mixed oligoastrocytoma and glioblastoma multiforme were the most positively stained tumor types. Survival analysis indicated that endothelial CA II staining is significantly associated with a poor prognosis in patients with astrocytomas. About 17% of patients with CA II-negative tumors (weak or no endothelial signal) were still alive at the end of the follow-up period of five years. The presence of CA II in the tumor endothelium suggests that it may play an important functional role in tumor metabolism. From a clinical perspective, the results also open new avenues for selecting tumor types for dendritic cell therapy trials.

Carbonic anhydrase II: a novel biomarker for pseudomyxoma peritonei.

Altered expression of carbonic anhydrase (CA) II is associated with human carcinogenesis. We analysed CA II protein expression in 89 patients with pseudomyxoma peritonei (PMP) and correlated its association against survival. We determined the expression of CA II by immunohistochemistry and then scored the staining results. The correlations of CA II expression with Peritoneal Cancer Index (PCI) and tumour grade were examined. The effect of CA II and tumour grade on survival was investigated. Positive CA II expression was found in 58 patients (65%) and absent in 31 patients (35%). High-grade (HG) morphology was associated with a loss of CA II expression (p = 0.048). The mean CA II immunostaining intensity score was 1.00 ± 1.1 (median 1, range 0-3) for HG morphology and 1.54 ± 1.1 (median 2, range 0-3) for low-grade (LG) morphology. The 5-year overall survival (OS) for those patients with CA II expression was 80% and 59% for those without (p < 0.001). The 5-year OS rates for those patients with HG morphology and positive CA II expression was 72% and 31% for those with negative CA II expression (p = 0.044). This study suggests that the expression of CA II acts as independent prognostic biomarker for survival in PMP.

Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II.

Human carbonic anhydrase II (HCA II) is a zinc metalloenzyme that catalyzes the reversible hydration and dehydration of carbon dioxide and bicarbonate, respectively. The rate-limiting step in catalysis is the intramolecular transfer of a proton between the zinc-bound solvent (H2O/OH-) and the proton-shuttling residue His64. This distance (approximately 7.5 A) is spanned by a well defined active-site solvent network stabilized by amino-acid side chains (Tyr7, Asn62, Asn67, Thr199 and Thr200). Despite the availability of high-resolution (approximately 1.0 A) X-ray crystal structures of HCA II, there is currently no definitive information available on the positions and orientations of the H atoms of the solvent network or active-site amino acids and their ionization states. In preparation for neutron diffraction studies to elucidate this hydrogen-bonding network, perdeuterated HCA II has been expressed, purified, crystallized and its X-ray structure determined to 1.5 A resolution. The refined structure is highly isomorphous with hydrogenated HCA II, especially with regard to the active-site architecture and solvent network. This work demonstrates the suitability of these crystals for neutron macromolecular crystallography.

Primary glial tumor of the retina with features of myxopapillary ependymoma.

We report a primary retinal tumor with features of myxopapillary ependymoma. The lesion occurred in a 33-year-old man with a long history of phthisis bulbi and a more recent history of pain to the right eye. Enucleated ocular globe revealed a lesion occupying most of the retinal surface. Histologically, the retina was replaced by a tumor composed of spindle cells with fibrillary cytoplasm and round to ovoid nuclei forming fascicles, perivascular pseudorosettes, microcysts, and deposition of extracellular mucins. Calcifications, metaplastic bone, and lymphoplasmacytic inflammatory infiltrate were also seen. Tumor cells expressed GFAP and S-100 and to lesser extent carbonic anhydrase II. The immunoreaction for EMA showed diffuse granular positivity, decorated a few extracellular lumina, and highlighted intracytoplasmic lumina in a few cells. Ultrastructurally, there was accumulation of extracellular material between cells and around capillaries, long interdigitating cytoplasmic processes, extracellular lumina packed with microvilli, a few junctions evident around lumina, and some ciliary basal bodies and ciliary basal rootlets. As control cases, we also investigated expression of EMA and carbonic anhydrase II in an ocular globe with retinal gliosis and three cases of myxopapillary ependymoma of the cauda equina. The lesion described here represents the first example of retinal tumor with features of myxopapillary ependymoma. Pathologic features and particularly expression of carbonic anhydrase II suggest a derivation from intrinsic glial cells of retina otherwise known as Muller cells.

Expression of a novel carbonic anhydrase, CA XIII, in normal and neoplastic colorectal mucosa.

BACKGROUND: Carbonic anhydrase (CA) isozymes may have an important role in cancer development. Some isozymes control pH homeostasis in tumors that appears to modulate the behaviour of cancer cells. CA XIII is the newest member of the CA gene family. It is a cytosolic isozyme which is expressed in a number of normal tissues. The present study was designed to investigate CA XIII expression in prospectively collected colorectal tumor samples. METHODS: Both neoplastic and normal tissue specimens were obtained from the same patients. The analyses were performed using CA XIII-specific antibodies and an immunohistochemical staining method. For comparison, the tissue sections were immunostained for other cytosolic isozymes, CA I and II. RESULTS: The results indicated that the expression of CA XIII is down-regulated in tumor cells compared to the normal tissue. The lowest signal was detected in carcinoma samples. This pattern of expression was quite parallel for CA I and II. CONCLUSION: The down-regulation of cytosolic CA I, II and XIII in colorectal cancer may result from reduced levels of a common transcription factor or loss of closely linked CA1, CA2 and CA13 alleles on chromosome 8. Their possible role as tumor suppressors should be further evaluated.

Co-expression of pendrin, vacuolar H+-ATPase alpha4-subunit and carbonic anhydrase II in epithelial cells of the murine endolymphatic sac.

The endolymph in the endolymphatic sac (ES) is acidic (pH 6.6-7). Maintaining this acidic lumen is believed to be important for the normal function of the ES. The acid-base regulation mechanisms of the ES are unknown. Here we investigated the expression patterns of acid-base regulators, including vacuolar (v)H+-ATPase (proton pump), carbonic anhydrase (CA) II, and pendrin in the murine ES epithelium by immunohistochemistry (IHC) and compared their expression patterns by double immunostaining. We found that pendrin and vH+-ATPase were co-localized in the apical membrane of a specific type of ES epithelial cell. Pendrin- and vH+-ATPase-positive cells also expressed cytoplasmic CA II. Co-expression of pendrin, vH+-ATPase, and CA II in the same subgroup of ES cells suggests that this specific type of ES cell is responsible for the acid-base balance processes in the ES and pendrin, vH+-ATPase, and CA II are involved in these processes.

In vitro effects of S12911-2 on osteoclast function and bone marrow macrophage differentiation.

In order to determine whether 5-[bis(carboxymethyl) amino]-2-carboxy4-cyano-3-thiopheneacetic acid distrontium salt (S12911-2) inhibits bone resorption by acting on the differentiation and/or function of osteoclasts, its effects were assessed on the 1,25-dihydroxyvitamin D(3)-induced expression of carbonic anhydrase II and vitronectin receptor in chicken bone marrow cells, and on the resorbing activity of authentic rat osteoclasts cultured on bone slices. S12911-2 dose-dependently inhibited, after a 6-day exposure, the expression of carbonic anhydrase II and vitronectin receptor in stimulated osteoclasts (46% and 40%, respectively, at 10(-3) M Sr(2+), P<0.05). A pre-incubation of bone slices with S12911-2 induced a dose-dependent inhibition of bone resorbing activity from 32% at 10(-4) M Sr(2+) to 66% at 10(-3) M Sr(2+) (P<0.05 in each case). A continuous incubation (10(-3) M Sr(2+)) induced a greater inhibition of bone resorbing activity (73%, P<0.05). The inhibition of bone resorption obtained specifically with S12911-2 is related to an inhibition of the differentiation and resorbing activity of the osteoclasts.

Interleukin-6 and soluble interleukin-6 receptor suppress osteoclastic differentiation by inducing PGE(2) production in chondrocytes.

This study examined how interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6r) influence osteoclastic differentiation through the function of chondrocytes. Chondrocytes were cultured with or without IL-6 and/or sIL-6r in the presence or absence of NS398, a specific inhibitor of cyclooxygenase (COX)-2, for up to 28 days. Chondrocytes were also cultured with or without IL-6 and sIL-6r for 28 days, and the conditioned medium from cells cultured without IL-6 and sIL-6r was used to induce differentiation of RAW264.7 cells into osteoclast precursors. Osteoclastic differentiation was assessed by tartrate-resistant acid phosphatase (TRAP) staining. Expression of osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), COX-2, and prostaglandin E(2) (PGE(2)) increased in cells exposed to IL-6 and sIL-6r, whereas expression of macrophage colony-stimulating factor (M-CSF) and bone resorption-related enzymes decreased. NS398 blocked the stimulatory/suppressive effects of IL-6 and sIL-6r on the expression of OPG, RANKL, and M-CSF. Fewer TRAP-positive multinucleated cells were detected after treatment with conditioned medium from IL-6- and sIL-6r-treated chondrocytes than after treatment with conditioned medium from untreated chondrocytes. These results suggest that IL-6 and sIL-6r interfere with osteoclast function through the involvement of chondrocytes. Specifically, they appear to suppress the differentiation of osteoclast precursors into osteoclasts by inducing chondrocytic PGE(2) production, which, in turn, increases OPG secretion and decreases M-CSF secretion by chondrocytes.

Immunohistolocalization and gene expression of the carbonic anhydrase isoenzymes (CA-II and CA-VI) in glands associated with the canine lacrimal apparatus.

Cytosolic and secretory carbonic anhydrase isoenzymes (CA-II and CA-VI, respectively) were detected by immunohistolocalization using specific canine CA-II and CA-VI antisera. CA-II and CA-VI were identified in glands associated with the canine lacrimal apparatus, such as lacrimal gland, superficial gland of the third eyelid (third eyelid gland) and tarsal gland. CA-II and CA-VI mRNA signals were also detected by reverse-transcriptase polymerase chain reaction in the same tissues. Some serous acinar cells and duct segments in the lacrimal gland and serous acinar cells in the third eyelid gland were immunopositive for anti-CA-II and CA-VI antisera. In particular, some immunopositive acini to CA-II and CA-VI on the edge of the third eyelid gland are histologically similar to sebaceous gland cells. Sebaceous gland cells in the tarsal and ciliary glands also showed immunopositivity to both CA antisera. CA-II and CA-VI gene transcripts were detected in the same regions. These results suggest that secreted CA-VI may form together with cytosolic CA-II, a high-activity isozyme mostly considered as a bicarbonate producer, in a mutually complementary system for the maintenance of bicarbonate levels to regulate pH in tear fluid and protect the corneal epithelia against injuries. In sebaceous gland cells in the lacrimal apparatus, CA-VI may be related to lipogenesis in an unknown function.

Carbonic anhydrase I, II, and VI, blood plasma, erythrocyte and saliva zinc and copper increase after repetitive transcranial magnetic stimulation.

INTRODUCTION: Repetitive transcranial magnetic stimulation (rTMS) has been used to treat symptoms from many disorders; biochemical changes occurred with this treatment. Preliminary studies with rTMS in patients with taste and smell dysfunction improved sensory function and increased salivary carbonic anhydrase (CA) VI and erythrocyte CA I, II. To obtain more information about these changes after rTMS, we measured changes in several CA enzymes, proteins, and trace metals in their blood plasma, erythrocytes, and saliva. METHODS: Ninety-three patients with taste and smell dysfunction were studied before and after rTMS in an open clinical trial. Before and after rTMS, we measured erythrocyte CA I, II and salivary CA VI, zinc and copper in parotid saliva, blood plasma, and erythrocytes, and appearance of novel salivary proteins by using mass spectrometry. RESULTS: After rTMS, CA I, II and CA VI activity and zinc and copper in saliva, plasma, and erythrocytes increased with significant sensory benefit. Novel salivary proteins were induced at an m/z value of 21.5K with a repetitive pattern at intervals of 5K m/z. CONCLUSIONS: rTMS induced biochemical changes in specific enzymatic activities, trace metal concentrations, and induction of novel salivary proteins, with sensory improvement in patients with taste and smell dysfunction. Because patients with several neurologic disorders exhibit taste and smell dysfunction, including Parkinson disease, Alzheimer disease, and multiple sclerosis, and because rTMS improved their clinical symptoms, the biochemical changes we observed may be relevant not only in our patients with taste and smell dysfunction but also in patients with neurologic disorders with these sensory abnormalities.