In vivo analysis of glial cell phenotypes during a viral demyelinating disease in mice

C57 BL/6N mice injected intracranially with the A59 strain of mouse hepatitis virus exhibit extensive viral replication in glial cells of the spinal cord and develop demyelinating lesions followed by virus clearing and remyelination. To study how different glial cell types are affected by the disease process, we combine three-color immunofluorescence labeling with tritiated thymidine autoradiography on 1-micron frozen sections of spinal cord. We use three different glial cell specific antibodies (a) to 2',3' cyclic-nucleotide 3' phosphohydrolase (CNP) expressed by oligodendrocytes, (b) to glial fibrillary acidic protein (GFAP) expressed by astrocytes, and (c) the O4 antibody which binds to O-2A progenitor cells in the rat. These progenitor cells, which give rise to oligodendrocytes and type 2 astrocytes and react with the O4 antibody in the adult central nervous system, were present but rare in the spinal cord of uninfected mice. In contrast, cells with the O-2A progenitor phenotype (O4 + only) were increased in number at one week post viral inoculation (1 WPI) and were the only immunostained cells labeled at that time by a 2-h in vivo pulse of tritiated thymidine. Both GFAP+ only and GFAP+, O4+ astrocytes were also increased in the spinal cord at 1 WPI. Between two and four WPI, the infected spinal cord was characterized by the loss of (CNP+, O4+) oligodendrocytes within demyelinating lesions and the presence of O-2A progenitor cells and O4+, GFAP+ astrocytes, both of which could be labeled with thymidine. As remyelination proceeded, CNP immunostaining returned to near normal and tritiated thymidine injected previously during the demyelinating phase now appeared in CNP+ oligodendrocytes. Thus O4 positive O-2A progenitor cells proliferate early in the course of the demyelinating disease, while CNP positive oligodendrocytes do not. The timing of events suggests that the O-2A progenitors may give rise to new oligodendrocytes and to type 2 astrocytes, both of which are likely to be instrumental in the remyelination process.     

Sodium/calcium exchange in smooth-muscle microsomal fractions.

The existence of Na+ -dependent Ca2+ transport was investigated in microsomal fractions from the longitudinal smooth muscle of the guinea-pig ileum and from the rat aorta, and its activity was compared with that of the plasmalemmal ATP-dependent Ca2+ pump previously identified in these preparations. The rate of Ca2+ release from plasmalemmal vesicles previously loaded with Ca2+ through the ATP-dependent Ca2+ pump was transiently faster in the presence of 150 mM-NaCl in the medium than in the presence of 150 mM-KCl or -LiCl or 300 mM-sucrose. Na+-loaded vesicles took up Ca2+ when an outwardly directed Na+ gradient was formed across the membrane. The Ca ionophore A23187 induced a rapid release of 85% of the sequestered Ca2+, whereas only 15% was displaced by La3+. Ca2+ accumulated by the Na+-induced Ca2+ transport was released by the addition of NaCl, but not KCl, to the medium. Ca2+ uptake in Na+-loaded vesicles was inhibited in the presence of increasing NaCl concentration in the medium. Half-maximum inhibition was observed with 28 mM-NaCl. Data fitted the Hill equation, with a Hill coefficient (h) of 1.9. Na+-induced Ca2+ uptake was a saturable function of Ca2+ concentration in the medium. Half-maximum activity was obtained with 18 microM-Ca2+ in intestinal-smooth-muscle microsomal fraction and with 50 microM-Ca2+ in aortic microsomal fraction. The results suggest that in these membrane preparations a transmembrane movement of Ca2+ can be driven by a Na+ gradient. However, the Na+-induced Ca2+ transport had a lower capacity, a lower affinity and a slower rate than the ATP-dependent Ca2+ pump.     

Differentiation of Ca2+ pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle

ATP promotes ⁴⁵Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca²⁺-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the ⁴⁵Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5′-nucleotidase. In contrast, the additional Ca²⁺-transport activity elicited by oxalate behaved like NADH-cytochrome $\text{c}$ reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca²⁺-storage capacity was not detectable in the absence of Ca²⁺-trapping agent. Low digitonin concentrations selectively increased the Ca²⁺ permeability of the plasmalemmal vesicles. The two Ca²⁺-transport activities were further differentiated by their distinct sensitivities to K⁺, vanadate and calmodulin. In this respect, the oxalte-insensitive and oxalate-stimulated Ca²⁺-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca²⁺ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.     

Calcium incorporation by smooth muscle microsomes.

The purpose of the present work was to study the factors influencing calcium incorporation into a microsomal fraction prepared from the longitudinal smooth muscle of the guinea-pig ileum. Calcium incorporation required the presence of both ATP and Mg2+ and was unaffected by azide. It was enhanced by oxalate; this effect was pH dependent and it was maximal at pH 6.6. The relation between calcium uptake with oxalate and free Ca2+ concentration in the medium was represented by a curve with an optimum for Ca2+ equal to 3-10-5 M. The threshold concentration was comprised between 5-10-7 and 10-6 7. The optimum calcium uptake rate was 4.5 nmol Ca2+/mg protein per min. In the absence of oxalate, two distinct groups of binding sites were identified. Low affinity sites had a binding constant of 7-104 M-1 and a maximum binding capacity of 0.6-106 M-1 and a binding capacity of 33 nmol Ca2+/mg protein; their capacity was sensitive to pH changes. In the absence of oxalate, Ca2+ binding was depressed by Na+ with respect to K+ or choline. When the medium was supplemented with oxalate, the stimulation of 45Ca incorporation was barely detectable in the presence of choline+ and it was lower in a medium containing Na+ instead of K+. The subcellular distribution profiles of calcium incorporation with and without oxalate indicate the microsomal location of both activities. However, the oxalate-stimulated calcium uptake activity sedimented faster than the calcium binding activity. The subcellular distribution of marker enzyme actvities has been examined. The present results indicate that Ca2+ incorporations with and without oxalate are the result of two processes likely related to two different structures. The role of microsomal calcium uptake in excitation-contraction coupling and its modification by the activity of the sodium pump is discussed.     

Antioxidant effects and the therapeutic mode of action of calcium channel blockers in hypertension and atherosclerosis

Drugs currently known as calcium channel blockers (CCB) were initially called calcium antagonists because of their ability to inhibit calcium-evoked contractions in depolarized smooth muscles. Blocking the entry of calcium reduces the active tone of vascular smooth muscle and produces vasodilatation. This pharmacological property has been the basis for the use of CCBs in the management of hypertension and coronary heart disease. A major question is whether drugs reducing blood pressure have other effects that help prevent the main complications of hypertension, such as atherosclerosis, stroke, peripheral arterial disease, heart failure and end-state renal disease. Experimental studies that focus on this question are reviewed in the present paper.