The effect of cholera toxin on choroid plexus carbonic anhydrase activity in vitro

The effects of the adenylate cyclase agonists cholera toxin and prostaglandin E2 on carbonic anhydrase activity in vitro was measured in choroid plexuses isolated from Sprague-Dawley rats. Choroid plexuses were incubated in buffer at 38 degrees C (pH 7.4) with either cholera toxin or prostaglandin E2 (PGE2) at a concentration and for a time period that had been shown in earlier studies to result in maximal stimulation of cyclic AMP production. Cholera toxin (10 micrograms/ml) caused a twofold increase (p < .001) in choroid plexus carbonic anhydrase activity when cholera toxin treated plexuses [20.92 +/- .46 mol CO2/(min)(mg protein X 10(-8)] were compared with plexuses exposed to heat inactivated cholera toxin (10.92 +/- .43). When choroid plexuses were homogenized and separated into a 10,000 g pellet and a supernatant fraction, the supernatant carbonic anhydrase was unresponsive to cholera toxin stimulation. In the pellet fraction, which contained all the cellular adenylate cyclase, challenge with cholera toxin produced a significant increase in carbonic anhydrase activity (p < .01). Control activity was 10.9 +/- 1.2 mol CO2/(min)(mg protein X 10(-8), while carbonic anhydrase activity in fractions exposed to cholera toxin was 28.4 +/- 0.8. PGE2 had no effect, however, upon choroid plexus carbonic anhydrase activity. Since both PGE2 and cholera toxin stimulate cyclic AMP production in vitro, a compartmental model of secretory control is proposed.     

Serum carbonic anhydrase III in myotonic dystrophy

Serum carbonic anhydrase III (CAIII) levels were determined by means of an enzyme immunoassay method and were compared with serum creatine kinase (CK) and muscle-specific enolase (MSE) levels in 33 patients with myotonic dystrophy. Serum CAIII levels were elevated in all 33 patients, whereas serum CK and MSE levels were elevated in 12 and 10 patients, respectively. Serum CAIII levels showed a good correlation with CK levels, but a poor one with MSE levels. There was no obvious correlation between the serum CAIII level and the duration of illness or the age of the patient. These results suggest that serum CAIII is probably a more sensitive marker than CK and MSE in myotonic dystrophy and may also reflect the type 1 fiber abnormality more predominantly observed in myotonic dystrophy.     

Serotonergic effects on carbonic anhydrase activity in the choroid plexus

The carbonic anhydrase (CA) activities in the choroid plexus of dogs were investigated by electron microscopy, and the effects of 5-hydroxytryptophan (5-HTP) on them were examined to elucidate the participation of serotonin in the production of cerebrospinal fluid. The reaction products yielded by the method employed proved to be CA activity by elimination tests using acetazolamide (Diamox). Following administration of 5-HTP, the CA activities fell to 43.3% of the control value, that is, approximately 56% of the CA activities in the choroid plexus were affected by serotonin. When tetrabenazine (TBZ) was administered, the CA activities in the choroid plexus decreased to 22.4% of the control value. These results suggest that the CA activity in the choroid plexus is remarkably suppressed when nervous control of the choroid plexus is disturbed by the administration of a monoamine denervator such as TBZ. The present data indicate that the serotonergic inhibitory effect on the CA activity in the choroid plexus may be less predominant than that of TBZ and acetazolamide.     

Serum carbonic anhydrase III in progressive muscular dystrophy

Serum carbonic anhydrase III (CA-III) levels were determined by means of an enzyme immunoassay method and compared with serum creatine kinase (CK) and muscle-specific enolase (MSE) levels in 143 patients with four types of progressive muscular dystrophy (PMD), namely, Duchenne muscular dystrophy (DMD), limb-girdle dystrophy, facioscapulohumeral dystrophy and congenital dystrophy. Serum CA-III levels were raised in the majority of patients, especially in those with DMD. In DMD patients, the gradual decline in the CA-III level was observed with age. High correlations were found between CA-III, CK and MSE levels. The frequency of cases with elevated CA-III levels was the same as or greater than that of elevated CK or MSE levels in four types of PMD. These results suggest that serum CA-III may be a useful marker of muscle disease.     

Functional demonstration of surface carbonic anhydrase IV activity on rat astrocytes

Buffering of the brain extracellular fluid is catalyzed by carbonic anhydrase (CA) activity. Whereas the extracellular isoform CA XIV has been localized exclusively to neurons in the brain, and to glial cells in the retina, there has been uncertainty regarding the form or forms of CA on the surface of brain astrocytes. We addressed this issue using physiological methods on cultured and acutely dissociated rat astrocytes. Prior work showed that the intracellular lactate-induced acidification (LIA) of astrocytes is diminished by benzolamide, a poorly permeant, nonspecific CA inhibitor. We demonstrate that pretreatment of astrocytes with phosphatidylinositol-specific phospholipase C (PI-PLC) results in a similar inhibition of the mean LIA (by 66 +/- 3%), suggesting that the glycosylphosphatidylinositol-anchored CA IV was responsible. Pretreatment of astrocytes with CA IV inhibitory antisera also markedly reduced the mean LIA in both cultured cortical (by 46 +/- 4%) and acutely dissociated hippocampal astrocytes (by 54 +/- 8%). Pre-immune sera had no effect. The inhibition produced by PIPLC or CA IV antisera was not significantly less than that by benzolamide, suggesting that the majority of detectable surface CA activity was attributable to CA IV. Thus, our data collectively document the presence of CAIV on the surface of brain astrocytes, and suggest that this is the predominant CA isoform on these cells.     

Histochemical localization of carbonic anhydrase in vertebrate nervous tissue

The histochemical method of H?usler was employed to demonstrate carbonic anhydrase (CA, carbonate hydrolase, 4.2.1.1.) in tissue sections. The CA reaction was inhibited in the presence of 5 mM acetazolamide. In the frog and fish retinas the CA activity was positive in the Müller fiber, the laminated segment of the cones and the myelin of the axons, and was negative in the laminated segment of the rods, the photoreceptor ellipsoid and myoid, the horizontal, bipolar, amacrine and ganglion cells, and the pigment epithelium. In the spinal cord of cat and fish the CA activity was positive in the myelin of the axons, the perineuronal oligodendroglia and the protoplasmic astrocytes, and was negative in the cell body, dendrite, and axon of the neurons and in the fibrous astrocytes. In the dorsal root ganglion of the cat CA reaction was positive in the satellite cells and the myelin, and was negative in the neuronal cell body and its processes. The capillaries in central nervous tissue show no CA reaction. The erythrocyte is CA positive while the mitochondria are CA negative.     

Surface carbonic anhydrase activity on astrocytes and neurons facilitates lactate transport

A number of studies have provided physiological evidence for extracellular carbonic anhydrase (CA) in brain. Association of extracellular CA with glia has been limited to functional studies of gliotic slices and retinal Muller cells. While astrocytes contain intracellular CA, there has been no direct evidence for surface CA on these cells. In fact, some morphological studies suggest that the extracellular CA in brain parenchyma resides on neurons, not glia. There has been no functional demonstration of extracellular CA activity on CNS neurons, however. Here we capitalized on the H(+) dependence of inward lactate transport to reveal functional extracellular CA activity on cultured astrocytes and acutely isolated hippocampal pyramidal neurons. Exposure to 20 mM L-lactate produced a rapid acidification of astrocytes that was reversibly blocked by 10 microM benzolamide. The lactate-induced acidification (LIA) was also blocked by a dextran-conjugated CA inhibitor. In CO(2)/HCO(3) (-)-free, HEPES-buffered media, the LIA was largely unaffected. Acutely dissociated hippocampal pyramidal neurons underwent a similar LIA that was reversibly blocked by benzolamide. Surface CA is likely to facilitate lactate transport by enabling rapid replenishment (i.e., buffering) of surface H(+) required for inward lactate-H(+) cotransport. These results demonstrate functional surface CA for the first time on individual mammalian astrocytes and neurons and suggest that this enzyme may play a role in the utilization of monocarboxylate substrates such as lactate and pyruvate by the brain.     

Distribution of carbonic anhydrase activity in neurons of the rat

Certain neurons of dorsal root ganglia (DRG) and some fibers of the sciatic nerve contain histochemically demonstrable carbonic anhydrase activity. Since the distribution of this enzyme throughout the nervous system has not yet been evaluated systematically, we conducted a comprehensive histochemical survey focusing particularly on structures derived from the neural crest and nonneural crest ectoderm. In the peripheral nervous system, we observed carbonic anhydrase activity in some, but not all, neurons of dorsal root, trigeminal, celiac, and myenteric ganglia as well as in glial cells throughout the CNS. Some neurons of the nodose ganglion also showed carbonic anhydrase activity. In all first order sensory ganglia that were studied, the enzyme was found only in large (50 micron or above) and medium (20-50 micron) size neurons; in the case of spinal ganglia, the reactive neurons constituted approximately 30% of the total neuronal population. Of these reactive neurons, 56% were heavily stained and 44% were moderately stained. Several possible roles for neuronal carbonic anhydrase are considered.     

Differential expression of carbonic anhydrase isozymes in microglial cell types

Microglial cells have been shown to express carbonic anhydrase. Using carbonic anhydrase histochemistry and immunohistochemistry, different types of cen-tral nervous system microglial cells were detected, which expressed two main carbonic anhydrase (CA) isozymes during the early postnatal stage of development and after peripheral nerve injury in the spinal cord of adult rats. Amoeboid and reactive microglial cells were heavily immunostained for CA-II and CA-III and showed colocalization with complement receptor type 3 and Griffonia Simplicifolia B₄ isolectin. Resting microglial cells in the brain and spinal cord showed faint CA-III staining and were negative for CA-II. These results show that not only CA-II, but also CA-III isozyme is represented in the central nervous system and carbonic anhydrase activity may correlate with metabolic and immunological changes of microglial cells. These data also further strengthen the idea of the mesodermal origin of central nervous system macrophages. © 1993 Wiley-Liss, Inc.     

Immunohistochemical localization of carbonic anhydrase isoenzyme C in human brain

Carbonic anhydrase isoenzymes of human brain were examined by the immunoperoxidase method. Only the catalytically highly active isoenzyme C was found in normal cerebral and cerebellar tissues, being located in a limited number of non-neuronal cells interpreted as oligodendrocytes and in myelinated nerve fibres. The enzyme was not evident in the glial cells of astrocytomas.     

Putative effects of nerve extract on carbonic anhydrase III expression in rat muscles

Carbonic anhydrase III (CA III), the predominant CA isoform in skeletal muscle is very sensitive to neuronal influences. We aimed to determine whether CA III expression could be influenced by neurotrophic factor(s) present in sciatic nerve extract (SNE). Intact muscles were thus compared with denervated soleus (SOL), extensor digitorum longus (EDL), and tibialis anterior (TA) muscles injected daily for 7 days with saline solution (SS) or with SNE. CA III activity was significantly increased in SS-treated EDL and TA muscles compared to control (CTR), while SNE injections partially prevented this increase. There was no significant difference for CA III activity in the SOL between CTR, SS, and SNE groups. The CA III mRNA increase observed in response to denervation was reduced by 40% in SNE-treated EDL and TA muscles. While SOL CA III mRNA level was not affected by denervation, a 52% decrease was observed with SNE. We concluded that neuronal modulation of CA III expression in type II fibers may involve a neurotrophic component. © 1994 John Wiley & Sons, Inc.     

Oligodendrocytes express a normal phenotype in carbonic anhydrase II-deficient mice

The nervous system of a mouse mutant characterized by a carbonic anhydrase II (CA II) deficiency was examined with light and electron microscopy and with immunocytochemistry using different glial cell markers. No major morphologic abnormalities at either the cellular or subcellular level are detectable in the brains of CAII-deficient mice, even though CAII is the main isozyme of CA in the brain. The oligodendrocytes, which characteristically express high levels of CA II, do not exhibit signs of degeneration or abnormalities even in 1-year-old CA II-deficient mice. Similarly, neurons and astrocytes have a normal structure and distribution. Oligodendrocytes show a normal staining pattern and distribution for galactocerebroside (GC), 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), and myelin basic protein (MBP). Astrocytes have a normal morphology and distribution when stained for GFAP and S100 protein. The lack of major degeneration in the brain due to a CA II deficiency suggests these mice utilize other enzymatic or physiological pathways to compensate for the enzyme absence.     

Histochemical localization of carbonic anhydrase in normal and diseased human muscle

A method is described for histological localization of carbonic anhydrase (CA) in sections of frozen human muscle using the rapid and inexpensive histochemical technique of Hansson. Results obtained in normal subjects indicate clearly that CA reactive fibers are of type 1. Similarly, abnormalities seen with CA in the muscle biopsy of a patient presenting with type 1 fiber hypotrophy and preponderance duplicated almost exactly those observed with the actinomyosine adenosine triphosphatase and the reduced nicotinamide adenine dinucleotide dehydrogenase reactions. Observations of grouped CA-positive muscle fibers in a case of chronic neurogenic atrophy suggest that, like other enzymes, CA expression in muscle is under neurogenic control.     

Peripheral nerve carbonic anhydrase activity and chronic acetazolamide treatment of rats

Examination of cranial nerves shows that the sensory infraorbital branch of the trigeminal nerve contains many carbonic anhydrase-reactive axons whereas axons of the motor facial nerve are non-reactive. This motor/sensory axon staining difference holds for both cranial and spinal nerves. Chronic treatment with acetazolamide produced no apparent changes in carbonic anhydrase histochemical activity or the structure of peripheral nerve fibers.     

Developmental regulation of carbonic anhydrase expression in mouse dorsal root ganglia

The development of proprioceptive neurons in mammalian dorsal root ganglia (DRG) remains poorly documented since few specific markers for these neurons are known. Recent studies suggest that carbonic anhydrase (CA) is a specific marker of this functionally defined neuronal population. The present study was designed to investigate the development of CA staining in sensory neurons. We investigated CA reactivity in mouse lumbar DRGs from embryonic day 13 (E13) to postnatal day 100 (P100) using a modified cytoenzymatic Hansson method. Neuronal CA reactivity was first detected during the perinatal stage (1–3% of DRG neurons) and increased progressively from P0 to P60 when it reached a plateau (about 30–33% of DRG neurons). Statistical morphometric analysis was used to define whether CA staining identifies the same population(s) during development. The results demonstrated that, whatever the stage of development, reactive neuronal cells are included in the well-defined large type A population. The possibility that neuronal CA expression is a reliable marker of the ‘functional activity’ of the proprioceptive neurons in mammals is discussed. The late developmental expression of the enzyme (after target innervation) raises the possibility of a regulation of the CA phenotype by neuron-target interactions.