Carbonic Anhydrase Inhibition Ameliorates Inflammation and Experimental Pulmonary Hypertension.

Inflammation and vascular smooth muscle cell (VSMC) phenotypic switching are causally linked to pulmonary arterial hypertension (PAH) pathogenesis. Carbonic anhydrase inhibition induces mild metabolic acidosis and exerts protective effects in hypoxic pulmonary hypertension. Carbonic anhydrases and metabolic acidosis are further known to modulate immune cell activation. To evaluate if carbonic anhydrase inhibition modulates macrophage activation, inflammation, and VSMC phenotypic switching in severe experimental pulmonary hypertension, pulmonary hypertension was assessed in Sugen 5416/hypoxia (SU/Hx) rats after treatment with acetazolamide or ammonium chloride (NH4Cl). We evaluated pulmonary and systemic inflammation and characterized the effect of carbonic anhydrase inhibition and metabolic acidosis in alveolar macrophages and bone marrow-derived macrophages (BMDMs). We further evaluated the treatment effects on VSMC phenotypic switching in pulmonary arteries and pulmonary artery smooth muscle cells (PASMCs) and corroborated some of our findings in lungs and pulmonary arteries of patients with PAH. Both patients with idiopathic PAH and SU/Hx rats had increased expression of lung inflammatory markers and signs of PASMC dedifferentiation in pulmonary arteries. Acetazolamide and NH4Cl ameliorated SU/Hx-induced pulmonary hypertension and blunted pulmonary and systemic inflammation. Expression of carbonic anhydrase isoform 2 was increased in alveolar macrophages from SU/Hx animals, classically (M1) and alternatively (M2) activated BMDMs, and lungs of patients with PAH. Carbonic anhydrase inhibition and acidosis had distinct effects on M1 and M2 markers in BMDMs. Inflammatory cytokines drove PASMC dedifferentiation, and this was inhibited by acetazolamide and acidosis. The protective antiinflammatory effect of acetazolamide in pulmonary hypertension is mediated by a dual mechanism of macrophage carbonic anhydrase inhibition and systemic metabolic acidosis.

Dose-Dependent Effects of L-Arginine on PROP Bitterness Intensity and Latency and Characteristics of the Chemical Interaction between PROP and L-Arginine.

Genetic variation in the ability to taste the bitterness of 6-n-propylthiouracil (PROP) is a complex trait that has been used to predict food preferences and eating habits. PROP tasting is primarily controlled by polymorphisms in the TAS2R38 gene. However, a variety of factors are known to modify the phenotype. Principle among them is the salivary protein Ps-1 belonging to the basic proline-rich protein family (bPRP). Recently, we showed that oral supplementation with Ps-1 as well as its related free amino acids (L-Arg and L-Lys) enhances PROP bitterness perception, especially for PROP non-tasters who have low salivary levels of Ps-1. Here, we show that salivary L-Arg levels are higher in PROP super-tasters compared to medium tasters and non-tasters, and that oral supplementation with free L-Arg enhances PROP bitterness intensity as well as reduces bitterness latency in a dose-dependent manner, particularly in individuals with low salivary levels of both free L-Arg and Ps-1 protein. Supplementation with L-Arg also enhanced the bitterness of caffeine. We also used 1H-NMR spectroscopy and quantum-mechanical calculations carried out by Density Functional Theory (DFT) to characterize the chemical interaction between free L-Arg and the PROP molecule. Results showed that the -NH2 terminal group of the L-ArgH+ side chain interacts with the carbonyl or thiocarbonyl groups of PROP by forming two hydrogen bonds with the resulting charged adduct. The formation of this PROP•ArgH+ hydrogen-bonded adduct could enhance bitterness intensity by increasing the solubility of PROP in saliva and its availability to receptor sites. Our data suggest that L-Arg could act as a 'carrier' of various bitter molecules in saliva.

Measurement of carbonic anhydrase isozyme VI (CA-VI) in swine sera, colostrums, saliva, bile, seminal plasma and tissues.

Swine secretory carbonic anhydrase VI (CA-VI) was purified from swine saliva and an antibody to CA-VI was generated. A specific and sensitive enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of swine CA-VI. The assay can detect as little as 5 ng/mL of swine CA-VI. Typical standard curves were determined for a range of CA-VI solutions (7.8 to 500 ng/mL). The coefficients of variation for these solutions were less than 5%. When 500, 250 or 100 ng/mL of swine CA-VI was added to swine sera, the recoveries were 102.0%, 109.7% and 100.2%, respectively. The concentrations of CA-VI in the saliva (26.2 ± 30.4 µg/mL), sera (3.3 ± 4.9 ng/mL), bile (153.0 ± 114.0 ng/mL), seminal plasma (124.0 ± 39.0 ng/mL) and parotid gland (441.3 ± 90.0 µg/g wet tissue), submaxillary gland (88.1 ± 124.4 µg/g wet tissue), sublingual gland (58.6 ± 24.6 µg/g wet tissue) and gallbladder (2.4 ± 1.3 µg/1g wet tissue) were determined by ELISA. The concentration of CA-VI in colostrum was 163.3 ± 101.4 ng/mL and did not decrease within 10 days following parturition. An immunohistochemical reaction to anti-CA-VI antiserum was observed in the columnar epithelial cells lining the gallbladder. These data suggest that secretory CA-VI plays various roles in pH regulation and the maintenance of ion and fluid balance.

Treatment of hypokalemic periodic paralysis with topiramate.

Hypokalemic periodic paralysis (hypoPP), the most common form of periodic paralysis, is a disorder characterized by attacks of transient muscle weakness associated with a drop in serum potassium level.The mainstay of treatment is potassium supplementation and drugs that inhibit the enzyme carbonic anhydrase. In this report we describe 11-year-old twins with hypoPP who were treated with topiramate, an anti-epileptic drug known to have carbonic anhydrase inhibitory properties. The patients experienced a decrease in the severity of their attacks upon initiation of treatment. Topiramate may warrant further investigation as a treatment option in hypoPP.

Effect of vitamin D status on the activity of carbonic anhydrase in chicken epiphysis and kidney.

Chickens were raised for 6 weeks from the date of hatch under red light on a vitamin D-free diet; controls were given an oral vitamin D supplement. Vitamin D-deficient animals showed decreased total serum calcium concentration and decreased DNA content in epiphysis and kidney homogenates. In calcifying epiphysis, total carbonic anhydrase (CA) activity was decreased, but activity per microgram DNA was slightly increased and specific activity was double that of the controls. Polyacrylamide gel isoelectric focusing after preparation of the enzyme showed a picture similar to that seen after parathyroid hormone (PTH) administration in chicks; therefore, this could be considered a secondary hyperparathyroidism. The CA activation was not seen in the kidney which can be explained by induction of an endogenous inhibitor protein of the cyclic AMP-dependent protein kinase exclusively in the kidney in vitamin D deficiency. In an additional experiment, chickens were raised for 3 weeks from the date of hatch under red light on a vitamin D-free diet. Daily oral substitution by different vitamin D metabolites (1,25(OH)2D3, 25OHD3, 24,25(OH)2D3) over 7 days led to CA activation compared with controls probably by restoring protein kinase activity in the kidney. Our results show that CA activity is inversely correlated with serum calcium concentrations which is in agreement with a regulatory mechanism recently proposed by us.

Role of carbonic anhydrase in bone resorption induced by 1,25 dihydroxyvitamin D3 in vitro.

The present investigation was undertaken to study the role of carbonic anhydrase in 1,25 dihydroxyvitamin D3-induced bone resorption. Calvaria were removed from 5- to 6-day-old mice and cultured for periods up to 96 h in Dulbecco's Modified Eagle Medium (high glucose, 4,500 mg/dl) supplemented with antibiotics and either heat-inactivated horse and fetal calf sera or bovine serum albumin. The experimental cultures contained 1 X 10(-8) M 1,25 dihydroxyvitamin D3 (1,25(OH)2D3). All cultures were incubated at 37 degrees C in 5% CO2/95% air. Bone resorption was assessed by release of stable calcium into the medium. Bone enzymes (acid and alkaline phosphatases and carbonic anhydrase) were determined following homogenization in 0.25 M sucrose. The effects of 1,25(OH)2D3 were studied in the presence and absence of the carbonic anhydrase inhibitor acetazolamide and its analogue (CL 13,850), which lacks inhibitory activity. Acetazolamide inhibited 1,25(OH)2D3-induced calcium release in a dose-dependent fashion from 10(-5)-10(-4)M. When added to the cultures at a concentration of 1 X 10(-4)M, acetazolamide completely blocked the 1,25(OH)2D3-induced calcium release, a phenomenon not seen with an equimolar concentration of CL 13,850. The most significant finding was that 1,25(OH)2D3-induced calcium release was accompanied by a significant increase in the carbonic anhydrase activity of bone at both 48 (treated/control ratio = 2.05) and 96 (treated/control ratio = 2.59) hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of carbonic anhydrase in bone: plasma acetazolamide concentrations associated with inhibition of bone loss.

Earlier reports from our laboratory have indicated that the carbonic anhydrase inhibitor acetazolamide blocks the hypercalcemic response to parathyroid hormone. In addition, we have reported that acetazolamide when administered by several routes partially prevents denervation-induced bone loss in a rat model of disuse osteoporosis. Continuous subcutaneous infusion required the least daily dose (8 mg/kg). The present study extends these earlier findings in several ways. It was found that in partially preventing denervation-induced bone loss: (1) incorporation of 1 M THAM [tris(hydroxymethyl)aminoethane] enhanced the potency of acetazolamide such that it was effective at daily doses of 0.6 mg/kg; (2) acetazolamide in the presence of 1 M THAM was effective at plasma concentrations as low as 50 ng/ml which are more than 500-fold less than peak plasma concentrations normally encountered in the human when acetazolamide is being used as a therapeutic agent; and (3) another inhibitor, benzolamide, was also effective by continuous subcutaneous infusion.

Proximal renal tubular acidosis in vitamin D deficiency rickets.

A proximal renal tubular acidosis (RTA) is the mechanism underlying the systemic acidosis found in vitamin D deficiency rickets. Acidotic subjects have high levels of PTH. In non-acidotic subjects proximal bicarbonate wasting can be induced by exogenous PTH injection. Carbonic anhydrase activity is not involved in this process. Calcium infusion is able to suppress both the spontaneous and the PTH-induced bicarbonate leak. The development of RTA in vitamin D deficiency is related to a particular equilibrium between two antagonizing factors at tubular level, parathyroid hormone and calcium.