John R. Dedman

Immunohistochemical Determination of Calcium-Calmodulin Binding Predicts Neuronal Damage After Global Ischemia

Since ionic Ca2+ binds with intracellular calmodulin (CaM) before activating proteases, kinases, and phospholipases, demonstration of persistent Ca2+ –CaM binding in neurons destined to show ischemic cellular injury would support the concept that elevated intracellular Ca2+ plays a causative role in ischemic neuronal damage. In order to characterize Ca2+ –CaM binding, we used a sheep anti-CaM antibody (CaM-Ab) which recognizes CaM that is not bound to Ca2+ or brain target proteins. Therefore, immunohistochemical staining of brain sections by labeled CaM-Ab represented only unbound CaM. Six normal rats were compared to 15 animals rendered ischemic for 30 min by a modification of the four-vessel occlusion model. Animals were killed immediately after ischemia, and after 2 and 24 h of reperfusion. Brain sections through hippocampus were incubated in CaM-Ab, and a diaminobenzadiene labeled anti-sheep secondary antibody was added to stain the CaM-Ab. Staining in the endal limb of dentate, dorsal CA1, lateral CA3, and parietal cortex was graded on a 4-point scale. All normal animals had grade 4 staining indicating the presence of unbound CaM in all four brain regions. Ischemic animals demonstrated reduced (grade 0 to 2) staining in the CA1 and CA3 regions immediately and 2 and 24 h after ischemia (p < 0.01 for both regions at all three time intervals) indicating persistent binding of CaM with Ca2+ and target proteins in these regions. Staining decreased in dentate and cortex up to 2 h after ischemia (p = 0.02 for both regions) but returned toward normal by 24 h. We conclude that while most brain regions demonstrate increased Ca2+ –CaM binding immediately after ischemia, this binding returns to normal in brain regions destined to recover, such as cortex and dentate, but persists beyond 24 h in selectively vulnerable CA1 and partially vulnerable CA3, which are destined to undergo irreversible damage. These findings support the hypothesis that calcium entry into neurons and consequent persistent activation of Ca2+ -dependent enzyme systems leads to irreversible cell damage.

Mediation of intracellular calcium: variances on a common theme

The discovery of troponin C and calmodulin set the tenor for understanding the intracellular mechanism of action of calcium. These proteins represent cellular receptors and distinct mediators of calcium. More recently, additional calcium-binding proteins have been identified. Immunological and sequence data suggest that these proteins represent a novel family of calcium mediators. The precise pathways in which these proteins are involved are not known. However, function by inference, the mediation of intracellular calcium, provides new avenues in which to better understand the complex cellular role calcium plays in regulating cell function.

Calcium binding proteins and cellular regulation

An important feature of cellular regulation is the precise control of intracellular calcium levels. This is accomplished both by dynamic organelle release and sequestration of calcium and by specific calcium active transport mechanisms located in the plasma membrane. The actual calcium signal for mediation of a cellular response is carried out by specific intracellular proteins, the most widely studied examples are calmodulin and troponin C. The recent discovery of phospholipid protein kinase and calcimedins suggests receptor mediation via several independent proteins. The physiological importance of a particular protein as a calcium messenger rests on several features: 1) calcium binding is of the order of 1-10 microns, 2) the protein is known to be localized at the site of proposed action, 3) if translocation occurs upon activation, the time required is consistent with the time course of the physiologic response and 4) substrates or effectors at the next level of action when isolated can be demonstrated to have similar activation kinetics as in situ.

Differential localization of annexins in ram germ cells: a biochemical and immunocytochemical study.

We used antibodies that specifically bind annexins on Western blots to determine the distribution and abundance of these proteins in ram spermatids and sperm by immunogold electron microscopy. Annexins I and II were found essentially within the entire acrosome of spermatids. During epididymal maturation, they concentrated in the postacrosomal region or the acrosomal equatorial segment, respectively. They were also present in sperm flagellum, on the surface of the coarse fibers and fibrous sheath. These findings show that during ram germ cell maturation, annexins I and II are exported from the spermatid acrosome towards structurally and functionally defined parts of the sperm. Annexins III, IV, and V were not found in ram germ cells. Annexin VI was isolated from testis and sperm. In spermatids, it was found to be associated with endoplasmic reticulum and the mitochondria but was absent from the acrosome. In sperm, it was confined to the flagellum, the mitochondria, and on the coarse fibers and fibrous sheath. The presence of three annexins, in addition to calmodulin, in functional areas may indicate differential ways for sperm to control and regulate events that are known to be calcium dependent, such as flagellar motility, acrosome reaction, and fertilization.

The role of calmodulin in cell transformation.

Two cell lines transformed with temperature sensitive retroviruses were examined for: their ability to grow in low Ca2+ medium, their calmodulin levels and changes in calmodulin acceptor proteins. Both cell lines grow in low Ca2+ medium at the permissive temperature 34 degrees C while both lines did not replicate at the non-permissive temperature 39 degrees C. The NRKLA23 cells have nearly twice as much calmodulin at the permissive temperature than they do at the non-permissive temperature while the 6M2 cells have an equal amount of calmodulin at both temperatures. Both cell lines exhibit changes in the calmodulin acceptor proteins going from the permissive to the non-permissive temperature. We suspect that the changes in the calmodulin acceptor proteins may be involved in the altered Ca2+-sensitivity of growth in the cells going from the permissive to non-permissive temperature.

Calcium-calmodulin binding in ischemic rat neurons after calcium channel blocker therapy.

Calcium channel blockers such as nicardipine improve outcome after global cerebral ischemia and may attenuate ischemic neuronal injury by preventing calcium influx and binding to calmodulin. We followed the temporal and regional sequence of neuronal calcium-calmodulin binding in normal rats (n = 6), untreated ischemic rats (n = 15), and ischemic rats treated with 0.05 mg/kg/hr s.c. nicardipine (n = 13). After 30 minutes of four-vessel occlusion, 40-microns brain sections were incubated in an anti-calmodulin antibody specific for calmodulin not bound to calcium and brain protein. Light-microscopic sections were examined immediately after ischemia and after 2 and 24 hours of reperfusion. Extensive staining of unbound calmodulin was seen in all hippocampal regions and in the cortex in normal rats. In untreated ischemic control rats, staining was lost, indicating calcium-calmodulin binding immediately after ischemia in all regions. However, after 24 hours, staining returned to normal in the cortex and dentate, and minimal staining returned in CA1 and CA3. Nicardipine-treated animals had significantly less calcium-calmodulin binding in CA1 and in the dentate after 2 hours of reperfusion. This study demonstrates that in clinically relevant doses nicardipine has a limited effect on calcium-calmodulin binding in selectively vulnerable regions after severe ischemia.

Affinity-purified site-directed antibody recognizes the entire annexin protein family

Annexins are a family of calcium/phospholipid binding proteins sharing strong sequence similarities. A site-directed affinity-purified antibody was produced against the consensus peptide, K A M K G L G T D E. This antibody recognizes all six annexin proteins in the purified state and in total-protein tissue extracts. The antibody should prove useful in identifying functional domains of this protein family.

Epitope mapping of alpha-transforming growth factor: Evidence of an immunodominant region

Antisera were produced in rabbits and sheep against both full-length synthetic rat alpha-transforming growth factor and peptides corresponding to the carboxy-terminal 17 amino acids. These antisera were used to develop a peptide based radioimmunoassay of alpha-TGF. All antisera reacted only with a restricted region of the alpha-TGF corresponding to the 8 residues (43-50) at the carboxy-terminus: (formula; see text) A series of synthetic peptides representing deletions or substitutions of amino acids in this carboxy-terminal region were tested for competition with 125I-alpha-TGF. All changes in the above peptide sequence resulted in a marked reduction in competition. All of the polyclonal antisera demonstrated similar specificity whether they were produced against the 50 amino acid, full-length alpha-TGF, against shorter 17 amino acid and 8 amino acid carboxy-terminal sequences.

Reserpine-induced alterations in mucus production and calmodulin-binding proteins in a human epithelial cell line

The characterization of a mucus-producing human cell line (HC-84) derived from a colon carcinoma and its response to in vitro reserpine treatment is reported. Mucous granules were demonstrated within these cells on the basis of electron microscopic examination and incorporation of [3H]glucosamine with subsequent autoradiographic analysis. Fluorographic analysis of total HC-84 cell protein after incubation with [3H]glucosamine indicated that the majority of tritium was incorporated into two proteins with molecular weights of 115 and 120 kD. When total HC-84 protein was subjected to immuno-blot analysis utilizing rabbit antibody against human intestinal mucus, only these two proteins (115K and 120K) reacted positively, indicating a direct correlation between [3H]glucosamine incorporation and mucus production. Immunofluorescence localization of mucus within HC-84 cells utilizing this same antibody resulted in a punctate pattern of fluorescence within the cytoplasm. Treatment of HC-84 cells with 30 microM reserpine for 7 days resulted in a three-fold increase in mucus production compared with controls. There was also a concomitant loss of a 30K calmodulin-binding protein in cells treated with reserpine. These cells represent a useful system for studying the effect of reserpine on the processes of mucus synthesis and secretion.

Cellular Localization of Calmodulin and Calmodulin-Acceptor Proteins

Calcium plays a pivotal role in the regulation of numerous cellular functions. Cell growth, motility, exocytosis, and endocytosis are all examples of processes which are triggered by an increase in the level of intracellular free calcium. The importance of calcium in cellular regulation was initially demonstrated in skeletal muscle. Heilbrunn and Wiercinski (1947) reported that injection of calcium into muscle fibers induced contraction of the muscle. It has since been well demonstrated that the effect of increased intracellular free calcium on skeletal muscle contraction is mediated by the calcium-binding protein troponin C. The interaction of actin with myosin and concomitant force transduction in skeletal muscle is sterically precluded unless troponin C binds calcium.