Higher plants share with animals a responsiveness to the Ca 2؉ mobilizing agents inositol 1,4,5-trisphosphate (InsP3) and cyclic ADP-ribose (cADPR). In this study, by using a vesicular 45 Ca 2؉ flux assay, we demonstrate that microsomal vesicles from red beet and cauliflower also respond to nicotinic acid adenine dinucleotide phosphate (NAADP), a Ca 2؉ -releasing molecule recently described in marine invertebrates. NAADP potently mobilizes Ca 2؉ with a K 1/2 ‫؍‬ 96 nM from microsomes of nonvacuolar origin in red beet. Analysis of sucrose gradient-separated cauliflower microsomes revealed that the NAADP-sensitive Ca 2؉ pool was derived from the endoplasmic reticulum. This exclusively nonvacuolar location of the NAADP-sensitive Ca 2؉ pathway distinguishes it from the InsP3-and cADPR-gated pathways. Desensitization experiments revealed that homogenates derived from cauliflower tissue contained low levels of NAADP (125 pmol͞mg) and were competent in NAADP synthesis when provided with the substrates NADP and nicotinic acid. NAADP-induced Ca 2؉ release is insensitive to heparin and 8-NH2-cADPR, specific inhibitors of the InsP3-and cADPR-controlled mechanisms, respectively. However, NAADP-induced Ca 2؉ release could be blocked by pretreatment with a subthreshold dose of NAADP, as previously observed in sea urchin eggs. Furthermore, the NAADP-gated Ca 2؉ release pathway is independent of cytosolic free Ca 2؉ and therefore incapable of operating Ca 2؉ -induced Ca 2؉ release. In contrast to the sea urchin system, the NAADPgated Ca 2؉ release pathway in plants is not blocked by L-type channel antagonists. The existence of multiple Ca 2؉ mobilization pathways and Ca 2؉ release sites might contribute to the generation of stimulus-specific Ca 2؉ signals in plant cells.

Little is known about the mechanisms underlying calcium-induced Ca 2+ release (CICR) in plants. The slowactivating vacuolar (SV) channel is both permeable to, and activated by Ca 2+ , and is therefore a prime candidate for a role in CICR. Cytosol-side-out vacuolar membrane vesicles loaded with 45 Ca 2+ showed voltage-and Ca 2+ -dependent Ca 2+ release, which was sensitive to the SV channel modulators DIDS, protein phosphatase 2B and calmodulin. Significantly, voltage-dependent Ca 2+ release strongly depended on cytoplasmic Ca 2+ concentrations. The results support the notion that CICR occurs in plant cells and that the process can be catalysed by the SV channel on the vacuolar membrane.z 1999 Federation of European Biochemical Societies.

Ligand-gated Ca(2+) channels provide a possible mechanism for linking perception of stimuli to intracellular Ca(2+) mobilization. Evidence for ligand-gated Ca(2+) release in plant cells arises from radiolabeled ligand binding, microsomal ion flux, and electrophysiological approaches. Results from these diverse approaches demonstrate that two classes of ligand-gated channels are present at the plant cell vacuolar membrane. One class of channel is gated by inositol 1,4,5 trisphosphate (InsP(3)) and the second is gated by cyclic adenosine 5'-diphosphoribose (cADPR). Previous biochemical studies on plant InsP(3) binding sites have been hampered by low density of specific binding. The present work reports optimization of yield for solubilized InsP(3) binding sites with respect to detergent type and concentration, and the originating tissue. Further studies reveal a pharmacological similarity between cADPR-activated Ca(2+) release in plant and animal cells and demonstrate that the extent of cADPR-induced Ca(2+) release is dependent on the plant tissue type. In animal cells cADPR releases Ca(2+) through activation of at least one isoform of the so-called ryanodine receptor. It is shown here that ryanodine itself is able to activate single channel currents in vacuolar membranes. These observations are integrated into current models for ligand-gated Ca(2+) release in plant and animal cells and their role in Ca(2+)-based cell signalling.

The concept of engineered extracellular matrices that can aid in the repair of damaged tissues is a common theme in the tissue engineering field. Central to this theme is the rationale that matrices, which mimic the composition and architecture of native tissues, can support and signal appropriate host cell repair responses. The design of such matrices draws upon concepts emerging from the cell biology, biomaterial and bioengineering fields. We have incorporated expertise from each of these areas into the development of matrices and matrix/growth factor combinations for the repair of bone and cartilage tissue using biomaterials such as type I collagen and hyaluronan. Type I collagen fibers coated with resorbable hydroxyapatite and crosslinked form an osteoconductive matrix that performs equivalent to autogenous bone when combined with a bone marrow aspirate or a recombinant human bone morphogenetic protein (GDF-5). The combination of recombinant human basic fibroblast grorth factor (FGF-2) with a hyaluronan gel results in a percutaneously injectable formulation that enhances fracture repair. More recent matrix formulations utilize a modified hyaluronan to coat and crosslink type I collagen fibers into a 3-D matrix that supports both osteogenesis and chondrogenesis in combination with growth factors such as GDF-5. The studies to be presented highlight the progress made in our understanding of cell, matrix, and growth factor interactions as they relate to skeletal tissue repair.Elevation of cytosolic free Ca plays a central role in transducing many metabolic and developmental stimuli in plants. Pathways for mobilisation of Ca from vacuoles include channels which are gated by inositol 1,4,5trisphosphate (IP3) and cyclic ADP-ribose (cADPR). By contrast much less is known about the properties of calcium-permable channels at the ER.We investigated the possibility that ligand-gated Ca release occurs in ERenriched vesicles derived from cauliflower inflorescences. cADPR-gated calcium release co-migrated with two ER markers, calnexin and antimycin A-insensitive NADH-cyt c reductase activity. Calcium release could also be evoked by the NADP metabolite nicotinic acid adenine dinucleotide phosphate (NAADP). This pathway, not previously described in plants, was insensitive to inhibitors of IP3-and cADPR-elicited calcium release, and, unlike the IP3-and cADPR-gated pathways, was located exclusively at the ER. Production of NAADP from N A D P by homogenates of cauliflower was detected using a sea urchin microsome bioassay. The existence of multiple Ca mobilisation pathways might contribute to the generation of stimulus-specific calcium signals in plants. In yeast, the alpha-factor induced mating response requires external C a and Ca uptake is mediated by interaction of two proteins. Cchl and Midl. The phenotype of deletion mutants suggest roles for these Ca channel forming subunits in a variety of additional physiological processes, including (1) cell viability at low Ca; (2) the adaptation of yeast growth to temperature; (3) th...