Carbonic Anhydrase VI in Skin Wound Healing Study on Car6 Knockout Mice.

Carbonic anhydrases (CAs) contribute to tumor cell migration by generating an acidic environment through the conversion of carbon dioxide to bicarbonate and a proton. CA VI is secreted to milk and saliva, and it could contribute to wound closure, as a potential trophic factor, in animals that typically lick their wounds. Our aim was to investigate whether human CA VI improves skin-wound healing in full-thickness skin-wound models. The effect was studied in Car6 -/- knockout mice and wild type littermates. Half of both mice strains were given topically administered, milk-derived CA VI after wounding and eight hours later. The amount of topically given CA VI exceeded the predicted amount of natural saliva-delivered CA VI. The healing was followed for seven days and studied from photographs and histological sections. Our results showed no significant differences between the treatment groups in wound closure, re-epithelization, or granulation tissue formation, nor did the Car6 genotype affect the healing. Our results demonstrate that CA VI does not play a major role in skin-wound healing and also suggest that saliva-derived CA VI is not responsible for the licking-associated improved wound healing in animals.

Lowering of elevated tissue PCO2 in a hemorrhagic shock rat model after reinfusion of a novel nanobiotechnological polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase that is an oxygen and a carbon dioxide carrier with enhanced antioxidant properties.

Even though erythrocytes transport both oxygen and carbon dioxide, research on blood substitutes has concentrated on the transport of oxygen and its vasoactivity and oxidative effects. Recent study in a hemorrhagic shock animal model shows that the degree of tissue PCO(2) elevation is directly related to mortality rates. We therefore prepared a novel nanobiotechnological carrier for both O(2) and CO(2) with enhanced antioxidant properties. This is based on the use of glutaraldehyde to crosslink stroma free hemoglobin (SFHb), superoxide dismutase (SOD), catalase (CAT) and carbonic anhydrase (CA) to form a soluble PolySFHb-SOD-CAT-CA. It was compared to blood and different resuscitation fluids on the ability to lower elevated tissue PCO(2) in a 2/3 blood volume loss rat hemorrhagic shock model. Sixty minutes of sustained hemorrhagic shock at 30 mm Hg resulted in the increase of tissue PCO(2) to 95 mm ± 3 mmHg from the control level of 55 mm Hg. Reinfusion of whole blood (Hb 15 g/dL with its RBC enzymes) lowered the tissue PCO2 to 72 ± 4.5 mmHg 60 minutes after reinfusion. PolySFHb-SOD-CAT-CA (SFHb 10 g/dL plus additional enzymes) was more effective than whole blood in lowering PCO(2) lowering this to 66.2 ± 3.5 mmHg. Ringer's Lactated solution or polyhemoglobin lowered the elevated PCO2 only slightly to 87 ± 4.5 mmHg and 84.8 ± 1.5 mmHg, respectively. Moreover, ST-elevation for whole blood (Hb 15 g/dL) and PolySFHb-SOD-CAT-CA (Hb 10 g/dL) was respectively 12.8% ± 4% and 13.0% ± 2% of the control 60 minutes after reinfusion. Both are significantly better than those in the Ringer's lactated group and the PolyHb group. In conclusion, this novel approach for blood substitute design has resulted in a novel nanobiotechnological carrier for both O(2) and CO(2) with enhanced antioxidant properties.

Hypoxia-inducible carbonic anhydrase IX expression is insufficient to alleviate intracellular metabolic acidosis in the muscle of zebrafish, Danio rerio.

Recent evidence suggests that carbonic anhydrase (CA) IX in humans is under the regulatory control of hypoxia-inducible factor and is overexpressed in certain cancers. However, little is known of its presence in nonmammalian vertebrates or its physiological function in any vertebrate. The objective of this study was to examine and characterize the presence, distribution, induction by hypoxia, and physiological function of CA IX in the zebrafish. Zebrafish CA IX was highly expressed in the eye, brain, and gastrointestinal tract and showed increased expression in the eye, brain, and muscle in response to hypoxia (water Po(2) = 24 mmHg). The hypothesis that increased CA IX expression during hypoxia would act to attenuate intracellular acidosis was then examined. Muscle intracellular pH (pH(i)) decreased after 4 h of hypoxic exposure (from 7.15 +/- 0.02 to 7.06 +/- 0.01 pH units) and did not recover by 24 h. Manipulation of extracellular CA activity via intraperitoneal injection of either bovine CA or the selective extracellular CA inhibitor F3500 revealed that although increased CA activity could fully restore pH(i), removal of extracellular activity did not result in further acidosis. An exercise-induced acidosis was also attenuated in fish treated with bovine CA; however, the increased extracellular CA expression resulting from hypoxia had no affect. These data suggest that although extracellular CA can potentially minimize the impact of hypoxia on muscle pH(i), the actual level of extracellular CA activity is likely insufficient to achieve this goal, even when enhanced by hypoxia-induced increases in CA IX expression.

In vitro phagocytosis of carrier mouse red blood cells is increased by Band 3 cross-linking or diamide treatment.

Chemical alteration of red blood cells (RBCs) can induce increased phagocytosis of modified cells by macrophages. In this study we have used different chemical treatments for the modification of the mouse red-blood-cell membrane surface, namely oxidant compounds, such as ascorbate/Fe(+2) and diamide [azodicarboxylic acid bis(dimethylamide)], or Band 3-cross-linking reagents. We monitored the phagocytosis of oxidized or Band 3-cross-linked mouse red blood cells by peritoneal macrophages. The extent of phagocytosis of RBCs is not affected by oxidation with ascorbate/Fe(3+), but it is increased (up to 10%) by oxidation with 2 mM diamide. Furthermore, phagocytosis is greatly increased (up to 40%) as a result of cross-linking with either of two Band 3 bifunctional reagents [bis(sulphosuccinimidyl) suberate (BS(3)) and 3,3'-dithiobis(sulphosuccinimidyl propionate) (DTSSP)]. To evaluate targeting towards macrophages of such modified RBCs for therapeutical purposes, we have determined the phagocytosis of Band 3 carrier RBCs loaded with carbonic anhydrase. In this case phagocytosis is high enough (25%) to deliver the enzyme into macrophages. We have also assayed the influence of serum components and IgG on the efficiency of phagocytosis and discuss the possible phagocytosis mechanisms. In the case of BS(3)-cross-linked carrier RBCs, phagocytosis is markedly enhanced (from 12% up to 25%) by serum components. This opens a way for therapeutic application of these carrier RBCs, with special relevance in short-term delivery to cells of the mononuclear phagocytic system.

Apparent diffusion limitations for CO(2) excretion in rainbow trout are relieved by injections of carbonic anhydrase.

Experiments were performed in vivo to elucidate the underlying mechanism(s) of apparent diffusion limitations for CO(2) excretion in rainbow trout (Oncorhynchus mykiss). Ligation of two gill arches and the associated expected reduction in gill surface area of 30% caused pronounced respiratory acidosis as indicated by elevated arterial blood P(CO(2)) (Pa(CO(2))) and reduced arterial blood pH. Under conditions of normoxia, arterial blood P(O(2)) (Pa(O(2))) was not significantly (statistically) reduced. However, during hypoxia (water P(O(2))=70-80 mmHg), the apparent trend for reduced Pa(O(2)) values became statistically significant in fish with 15% surface area reduction. To determine whether the elevated Pa(CO(2)) in fish with reduced surface area (30%) reflected true diffusion limitations or chemical equilibrium limitations imposed by the relatively slow rate of red blood cell Cl(-)/HCO(3)(-) exchange, fish were injected with carbonic anhydrase (CA) to permit catalysis of HCO(3)(-) dehydration within the plasma. Injection of CA caused a lowering of Pa(CO(2)) by 0.87+/-0.32 mmHg within 120 min and thus essentially eliminated the increase in Pa(CO(2)) (1.04+/-0.33 mmHg) that was caused by the reduction in surface area. These results clearly demonstrate that the elevation in Pa(CO(2)) evoked by gill surface area reduction is a consequence of chemical equilibrium limitations rather than true diffusion limitations, per se.

Protein release from poly(lactic-co-glycolic acid) microspheres: protein stability problems.

The enzyme, carbonic anhydrase, has been incorporated within poly(lactic-co-glycolic acid) microspheres using a double emulsion and solvent evaporation technique. The protein stability problems during the microsphere formulation procedure and during the release period were examined in relation to the protein release kinetics over a 2 month period. Different protein release profiles could be obtained depending on the polymers used. The protein release kinetics exhibited an initial fast release followed by a slow release, resulting in an incomplete protein release although the microspheres degraded significantly. The very slow release kinetics were attributed to the protein aggregation and non-specific adsorption within the microspheres. It was found that the protein was significantly denatured and aggregated during the double emulsion formulation step. Several excipients such as albumin, poly(ethylene oxide), Pluronic F-127, and gelatin, which were loaded along with the protein within microspheres, demonstrated better protein release kinetics partly due to an increase in the protein stability. The released protein from these fast degrading microspheres, however, was severely hydrolyzed and lost its catalytic activity, caused by the accumulation of degradation products in the medium.

Inibiçäo da anidrase carbônica no tratamento de edema macular crônico associado a gliose macular idiopática / Inhibition of carbonic anhydrase in the treatment of chronic macular edema associated to idipatic gliosis

Durante 1989 e 1990 foram documentados 14 pacientes com edema macular crônico associado à gliose idiopática pré-macular. Destes, 9 aceitaram submeter-se a um teste terapêutico com acetazolamida oral. Seis casos (66,6//) mostraram resoluçäo angiográfica parcial ou completa no curso do tratamento. Nenhum dos 5 pacientes do grupo-controle mostrou esta evoluçäo. Este resultado sugere que os inibidores da anidrase carbônica podem oferecer algum auxílio terapêutico nesta situaçäo

The additive effect of timolol on open angle glaucoma patients on maximal medical therapy.

Thirty-one eyes of 20 glaucomatous patients on maximum medication were treated with timolol drops for a mean period of 9 months. Adequate levels of control of intraocular pressures were obtained in 21 eyes. No significant ocular or systemic side effects were noted. Timolol has an additive effect in further lowering intraocular pressures in patients being treated with a combination of carbonic anhydrase inhibitors, pilocarpine, epinephrine, and the anticholinesterases.