Caveolins in glial cell model systems: from detection to significance.

Glial cells prevail in number and in diversity of cellular phenotypes in the nervous system. They have also gained prominence due to their multiple physiological and pathophysiological roles. Our current knowledge of the asymmetry and heterogeneity of the plasma membrane demands an in depth analysis of the diverse array of membrane microdomains postulated to exist in the context of glial cells. This review focuses and analyzes the studies reported to date on the detection of caveolae membrane rafts and the caveolin family members in glial cell model systems, the conditions leading to changes in their level of expression, and their functional and clinical significance. Outstanding in this work emerge the ubiquitous expression of caveolins, including the typically regarded 'muscle-specific' cav3, in diverse glial cell model systems, their participation in reactive astrogliosis, cancer, and their key relevance to calcium signaling. The knowledge obtained to date demands incorporation of the caveolins and caveolae membrane rafts in our current models on the role of glial cells in heath and neurological disease.

Downregulation of Kir4.1 inward rectifying potassium channel subunits by RNAi impairs potassium transfer and glutamate uptake by cultured cortical astrocytes.

Glial cell-mediated potassium and glutamate homeostases play important roles in the regulation of neuronal excitability. Diminished potassium and glutamate buffering capabilities of astrocytes result in hyperexcitability of neurons and abnormal synaptic transmission. The role of the different K+ channels in maintaining the membrane potential and buffering capabilities of cortical astrocytes has not yet been definitively determined due to the lack of specific K+ channel blockers. The purpose of the present study was to assess the role of the inward-rectifying K+ channel subunit Kir4.1 on potassium fluxes, glutamate uptake and membrane potential in cultured rat cortical astrocytes using RNAi, whole-cell patch clamp and a colorimetric assay. The membrane potentials of control cortical astrocytes had a bimodal distribution with peaks at -68 and -41 mV. This distribution became unimodal after knockdown of Kir4.1, with the mean membrane potential being shifted in the depolarizing direction (peak at -45 mV). The ability of Kir4.1-suppressed cells to mediate transmembrane potassium flow, as measured by the current response to voltage ramps or sequential application of different extracellular [K+], was dramatically impaired. In addition, glutamate uptake was inhibited by knock-down of Kir4.1-containing channels by RNA interference as well as by blockade of Kir channels with barium (100 microM). Together, these data indicate that Kir4.1 channels are primarily responsible for significant hyperpolarization of cortical astrocytes and are likely to play a major role in potassium buffering. Significant inhibition of glutamate clearance in astrocytes with knock-down of Kir4.1 highlights the role of membrane hyperpolarization in this process.

Caveolin isoform expression during differentiation of C6 glioma cells.

Caveolae, a specialized form of lipid rafts, are cholesterol- and sphingolipid-rich membrane microdomains implicated in potocytosis, endocytosis, transcytosis, and as platforms for signal transduction. One of the major constituents of caveolae are three highly homologous caveolin isoforms (caveolin-1, caveolin-2, and caveolin-3). The present study expands the analysis of caveolin isoform expression in C6 glioma cells. Three complementary approaches were used to assess their differential expression during the dibutyryl-cyclic AMP-induced differentiation of C6 cells into an astrocyte-like phenotype. Immunoblotting, conventional RT-PCR, and real-time RT-PCR analysis established the expression of the caveolin-3 isoform in C6 cells, in addition to caveolin-1 and caveolin-2. Similar to the other isoforms, caveolin-3 was associated with light-density, detergent-insoluble caveolae membrane fractions obtained using sucrose-density gradient centrifugation. The three caveolin isoforms display different temporal patterns of mRNA/protein expression during the differentiation of C6 cells. Western blot and real-time RT-PCR analysis demonstrate that caveolin-1 and caveolin-2 are up-regulated during the late stages of the differentiation of C6 cells. Meanwhile, caveolin-3 is gradually down-regulated during the differentiation process. Indirect immunofluorescence analysis via laser-scanning confocal microscopy reveals that the three caveolin isoforms display similar subcellular distribution patterns. In addition, co-localization of caveolin-1/caveolin-2 and caveolin-1/caveolin-3 was detected in both C6 glioma phenotypes. The findings reveal a differential temporal pattern of caveolin gene expression during phenotypic differentiation of C6 glioma cells, with potential implications to developmental and degenerative events in the brain.

Identification of caveolae and caveolin in C6 glioma cells.

Caveolae (CAV) constitute a novel subcellular transport vesicle that has received special attention based on its proven and postulated participation in transcytosis, potocytosis, and in cell signaling events. One of the principal components of CAV are caveolin protein isoforms. Here, we have undertaken the immunochemical identification of CAV and the known caveolin isoforms (1alpha, 1beta, 2 and 3) in cultured rat C6 glioma cells. Immunoblot analysis revealed that particulate fractions from rat C6 glioma cells express caveolin-1 and caveolin-2. The relative detergent-insolubility of these caveolin isoforms was also determined by Western blot analysis. Indirect immunofluorescence analysis with caveolin-1 and -2 antibodies revealed staining patterns typical of CAV's known subcellular distribution and localization. For both caveolin isoforms immunocytochemical staining was characterized by intensely fluorescent puncta throughout the cytoplasm and diffuse micropatches at the level of the plasmalemma. Perinuclear staining was also detected, consistent and suggestive of caveolin's localization in the trans Golgi region. The caveolin-1 and -2 immunoreactivity seen in Western blots and immunocytochemically is related to structurally relevant CAV as supported by the isolation of caveolin-enriched membrane complexes using two different methods. Light-density, Triton X-100-insoluble caveolin-1- and caveolin-2-enriched fractions were obtained after fractionation of rat C6 glioma cells and their separation over 5-40% discontinuous sucrose-density gradients. Similar fractions were obtained using a detergent-free, sodium carbonate-based fractionation method. These results further support the localization of CAV and caveolins in glial cells. In addition, they demonstrate that cultured C6 glioma cells can be useful as a model system to study the role of CAV and caveolins in subcellular transport and signal transduction events in glial cells and the brain.

Labeling of the Amphiuma erythrocyte K+/H+ exchanger with H2DIDS.

Subsequent to swelling, the Amphiuma red blood cells lose K+, Cl-, and water until normal cell volume is restored. Net solute loss is the result of K+/H+ and Cl-/HCO3- exchangers functionally coupled through changes in pH and therefore HCO3-. Whereas the Cl-/HCO3- exchanger is constitutively active, K+/H+ actively is induced by cell swelling. The constitutive Cl-/HCO3- exchanger is inhibited by low concentrations (< 1 microM) of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or H2DIDS, yet the concentration of H2DIDS > 25 microM irreversibly modifies the K+/H+ exchanger in swollen cells. We exploited the volume-dependent irreversible low-affinity reaction between H2DIDS and the K+/H+ to identify the protein(s) associated with K+/H+ exchange activity. Labeling of the membrane proteins of intact cells with 3H2DIDS results in high-affinity labeling of a broad 100-kDa band, thought to be the anion exchanger. Additional swelling-dependent low-affinity labeling at 110 kDa suggests the possibility of a volume-induced population of anion exchangers. Finally, the correlation between volume-sensitive K+/H+ modification and low-affinity labeling suggests that transport activity is associated with a protein of approximately 85 kDa. Although a 55-kDa protein is also labeled, it is a less likely candidate, since label incorporation and transport modification are less well correlated than that of the 85- and 110-kDa proteins.

pH regulatory Na/H exchange by Amphiuma red blood cells.

In Amphiuma red blood cells, the Na/H exchanger has been shown to play a central role in the regulation of cell volume following cell shrinkage (Cala, P. M. 1980. Journal of General Physiology. 76:683-708.) The present study was designed to evaluate the existence of pH regulatory Na/H exchange in the Amphiuma red blood cell. The data illustrate that when the intracellular pHi was decreased below the normal value of 7.00, Na/H exchange was activated in proportion to the degree of acidification. Once activated, net Na/H exchange flux persisted until normal intracellular pH (6.9-7.0) was restored, with a half time of approximately 5 min. These observations established a pHi set point of 7.00 for the pH-activated Na/H exchange of Amphiuma red blood cell. This is in contrast to the behavior of osmotically shrunken Amphiuma red blood cells in which no pHi set point could be demonstrated. That is, when activated by cell shrinkage the Na/H exchange mediated net Na flux persisted until normal volume was restored regardless of pHi. In contrast, when activated by cell acidification, the Na/H exchanger functioned until pHi was restored to normal and cell volume appeared to have no effect on pH-activated Na/H exchange. Studies evaluating the kinetic and inferentially, the molecular equivalence of the volume and pHi-induced Amphiuma erythrocyte Na/H exchanger(s), indicated that the apparent Na affinity of the pH activated cells is four times greater than that of shrunken cells. The apparent Vmax is also higher (two times) in the pH activated cells, suggesting the involvement of two distinct populations of the transporter in pH and volume regulation. However, when analyzed in terms of a bisubstrate model, the same data are consistent with the conclusion that both pH and volume regulatory functions are mediated by the same transport protein. Taken together, these data support the conclusion that volume and pH are regulated by the same effector (Na/H exchanger) under the control of as yet unidentified, distinct and cross inhibitory volume and pH sensing mechanisms.

Juvenile myoclonic epilepsy (JME) may be linked to the BF and HLA loci on human chromosome 6.

Although certain forms of epilepsy have long been suspected to be inherited, heterogeneity has made it difficult to find the genes responsible for any subtypes. We found that families ascertained through patients with juvenile myoclonic epilepsy show linkage with the BF and HLA loci on human chromosome 6. There is some evidence that the locus may be outside the HLA complex and no evidence as yet of an association with any allele of the HLA complex.

Complex partial seizures of hippocampal and amygdalar origin.

We studied the first clinical manifestations of 72 complex partial seizures (CPS) in 17 drug-resistant patients. CPS were indicated to be of hippocampal-amygdalar origin by scalp and depth EEG. We asked: (a) Do all CPS of hippocampal-amygdalar origin start with an initial motionless stare and/or oroalimentary automatisms? (b) If not, what other clinical manifestations appear at onset of the CPS? Results showed that approximately 39% of CPS begin with motionless staring, 25% with nonfocal discrete movements, 21% with oroalimentary automatisms, 10% with perseverative stereotyped automatisms, and 6% with vocalizations. Nonfocal discrete movements and oroalimentary automatisms were identified as the most common second and third clinical sequential manifestations during a CPS. We conclude that although approximately 60% of CPS of hippocampal-amygdalar origin start with motionless staring or oroalimentary automatisms, 40% do not.

Segregation analysis of juvenile myoclonic epilepsy.

We examined the inheritance of juvenile myoclonic epilepsy (JME). We looked at both the trait of "epilepsy" and the trait of "epilepsy-plus-EEG abnormalities," since EEG abnormalities are frequently found in the clinically unaffected sibs of JME patients. We tested several modes of inheritance including the fully penetrant recessive and several two-locus models. We could reject all models tested (fully penetrant single-locus and two-locus models) when abnormal EEGs were classified as "unaffected." We could also reject the fully penetrant single locus models when family members with abnormal EEGs were considered "affected." We also rejected the two-locus model where the inheritance at both loci was dominant. The two-locus model where both loci showed recessive inheritance could not be rejected, nor could the model where one locus was dominant and the other recessive. Our results suggest that the underlying predisposition for JME is genetically determined and is partially reflected in the abnormal EEGs found in clinically unaffected family members.

Unit activity related to head and eye movements in central thalamus of cats.

Single-unit activity was recorded with stereotaxically guided microelectrodes in the central thalamus of five alert cats. The animals were studied with the head either fixed or free to move in a horizontal plane. They were trained to make eye and/or head movements toward discrete visual targets presented on a screen. Unit activity was analyzed in relation to triggered and spontaneous gaze displacements with head fixed and free successively. Four groups of cells were found, all within the thalamic internal medullary lamina: 20 cells were active with eye but not head movements, 49 with head but not eye movements, 36 with head or eye movements, and 17 responding to visual stimuli in the absence of movement. The patterns of firing during gaze shifts are described. It is hypothesized that eye- or head-related units carry a signal representing gaze driving.