Rat carbonic anhydrase (CA) IV was purified by affinity chromatography and used to produce a specific antiserum in rabbits for immunolocalization studies in rat kidney. CA IV was localized in apical plasma membranes of the proximal convoluted tubule and the thick ascending limb of Henle. Both of these segments are involved in bicarbonate reabsorption in the rat. Immunofluorescent staining of the brush border was faint in the S1 segment, greatest in the S2 segment, and absent from the S3 segment of the proximal tubule. CA IV was also detected in the basolateral plasma membrane of proximal-tubule and thick-ascending-limb epithelial cells by immunofluorescence and immunoelectron microscopy. In the proximal tubule, an extracellular membrane CA had been previously suggested on the basis of electrophysiological studies. CA IV was not detected in intercalated cells of the collecting ducts. These cells contain, in contrast, abundant cytosolic CA II. Thus, the distribution of CA IV is quite distinct from that of CA II; it corresponds with the localization of an isoenzyme(s) that did not stain with antibodies against CA II but that was revealed by histochemical-staining procedures. We conclude that the apical CA IV is the luminal CA responsible for bicarbonate reabsorption in the proximal tubule and the thick ascending limb in the rat kidney. These studies also suggest that CA IV plays a role in bicarbonate transport across the basolateral plasma membrane in these two segments of the rat nephron.

Carbonic anhydrase (CA) activity plays an important role in controlling cerebrospinal fluid production and also influences neuroexcitation and susceptibility to seizures. Until recently, CA II was the only CA demonstrated in brain. Its distribution is limited to the epithelial cells of the choroid plexus and to the myelin-forming cells, the oligodendrocytes. In this report, we present immunoblots, using an antibody raised to CA IV from rat lung, that show that CA IV is also present in rat and mouse brain. Results of immunohistochemistry and immunoelectron microscopy on sections from rat and mouse brain are presented that show the distribution of CA IV to be quite distinct from that of CA II. CA IV is expressed on and is limited to the luminal surface of endothelial cells of cerebral capillaries. These results establish CA IV as a cytochemical marker associated with the blood-brain barrier and suggest an important role for CA IV in CO2 and HCO3- homeostasis in brain.

Carbonic anhydrase (CA) activity plays an important role in controlling aqueous humor production in the eye and in regulating intraocular pressure. Prior studies identified the soluble isozymes CA II and CA I in the human eye and also suggested a distinct membrane-associated CA. We used an antibody to CA IV, the membrane-anchored isozyme from human lung, to study CA IV in eye tissues and to compare its distribution with that of CA II. We found intense immunostaining for CA IV associated with endothelial cells of one specific uveal capillary bed, the choriocapillaris. CA IV was not detected in endothelial cells of the contiguous capillaries of the iris or in endothelial cells of other vessels. Immunoreactivity for CA IV was also intense in epithelial and fiber cells of the lens but was not detectable in the neuroretina, the ciliary process (except for capillaries), and the cornea, all sites where immunostaining with anti-CA II antibody was intense. These studies indicate that the membrane-associated CA in human eye, which was suspected from histochemical studies, is CA IV. Defining the physiological role of this ocular isozyme remains a challenge.

We have isolated a full-length cDNA for human carbonic anhydrase IV (CA IV) from a AgtlO human kidney cDNA library. The 1105-base-pair (bp) cDNA contains a 47-bp 5' untranslated region, a 936-bp open reading frame, and a 122-bp 3' untranslated region. The deduced amino acid sequence is colinear with the N-terminal sequence and the sequence of several tryptic peptides of human lung CA IN. It includes an 18-amino acid signal sequence, a 260-amino acid region that shows 30-36% similarity with the 29-kDa cytoplas-

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