Inositol 1,4,5-trisphosphate is not effective in releasing calcium from skeletal sarcoplasmic reticulum microsomes

Regulation of many cell systems has been shown to be mediated by Inositol 1,4,5-trisphosphate which causes a release of calcium from intracellular sites. We have shown that release of Ca2+ from sarcoplasmic reticulum microsomes was not stimulated by IP3. The phorbol ester, TPA, also had no effect on Ca2+ release or Ca2+ ATPase activity. Thus, it is unlikely that the breakdown of polyphosphatidylinositides serves as a second messenger to mediate release of Ca2+ in skeletal muscle.     

Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels

Small conductance Ca²⁺-sensitive potassium (SK2) channels are voltage-independent, Ca²⁺-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca²⁺ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca²⁺ and Ca²⁺-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca²⁺ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.     

Verapamil as a co-mutagen in the Salmonella/mammalian microsome mutagenicity test

Verapamil is a calcium-channel blocking agent, commonly used for chronic treatment of heart conditions. We have previously demonstrated that verapamil acts as a co-mutagen in a bacterial mutagenicity test for some experimental anilinoacridine antitumour drugs. Within the anilinoacridines series there are several compounds which are apparently non-mutagenic (or very weak mutagens) in the absence of verapamil, but strong mutagens in its presence. We have now tested a wider range of materials for verapamil enhancement of mutagenicity, to include some of those to which persons on verapamil therapy might be exposed through life-style or occupation. Some verapamil enhancement of mutagenicity was seen with most mutagenic compounds including anticancer drugs, antiparasitic agents, one biological stain and one hair dye. A number of tricyclic antidepressants and biological stains were tested and found to be non-mutagenic. If these results extrapolate to mammalian cells, long-term verapamil therapy could potentially increase the effects of certain environmental mutagens.     

Mutagenic and clastogenic activity of nitracrine analogues in cultured V79 Chinese hamster cells

Nitracrine is used clinically as an antitumour agent, and analogues are actively being developed in some laboratories. The mutagenic activity of 9-[(3-dimethylaminopropyl)amino]-acridine and its 1-nitro (nitracrine), 2-, 3- and 4-nitro derivatives was evaluated at the 6-thioguanine and ouabain resistance loci in cultured Chinese hamster fibroblasts (V79-171b cell line). The des-nitro, 2- and 3-nitro caused no statistically significant mutagenic activity at either locus. Each of these 3 compounds weakly increased (approximately 2-fold) the incidence of micronuclei in the same cell line when tested at cytotoxic doses. Both the 1- and 4-nitro compounds increased the incidence of 6-thioguanine resistant cells from around 1 in 10(-6) to approximately 1 in 10(-4). The former compound significantly increased the frequency of ouabain-resistant cells. Both of these compounds were potent inducers of micronuclei in V79-171b cells, indicating high clastogenic activity. It would appear prudent to regard both of these compounds as potential human carcinogens.     

Clonal populations of the mouse mammary cell line,COMMA-D,which retain capability of morphogenesis in vivo

Summary Clonal populations were isolated from the mouse mammary cell line, COMMA-D, by transfection with a dominant-selectable gene, pSV2Neo, which confers resistance to the antibiotic, G418. Seven of twenty-four clones isolated retained the ability of the parental line to repopulate cleared mammary fat pads in vivo as ductal-alveolar hyperplasias. Two sublines designated CDNR2 and CDNR4 retained hyperplastic growth potential after multiple passages in vitro with low incidence of tumor formation. A third subpopulation, CDNR1, contained a single integration site for the pSV2Neo plasmid indicating a bonafide clonal origin for this subline. CDNR1 cells displayed heterogeneous growth phenotypes in vivo including hyperplasia, adenocarcinoma, and bone formation. Functional differentiation of CDNR1 cells organized as alveolarlike structures in vivo or on floating collagen gels in vitro was observed as determined by immunoperoxidase staining for the milk-specific protein, casein. Overall, the results indicate that a subset of cells from the COMMA-D cell line may be functionally analogous to stem cells existing in the mammary gland. Supported by NCI research grants CA-38650, CA-33369, CA-39017, and CA-25215.