Neurofilaments (NF), the major cytoskeletal component in neuronal cells, are one of the most highly phosphorylated proteins expressed in brain. Apart from the structural role NF play in maintaining neuronal architecture, little else is known of their function. I describe here evidence suggesting that NF may support many other proteins in the neuronal axoplasm including protein kinases. In order to isolate proteins that bind NF, I first expressed the carboxyl-terminal tail domain of the mouse heavy-molecular-weight neurofilament subunit (NF-H) as a fusion protein in bacteria and then used this portion of NF-H as a ligand in affinity chromatography. A number of different proteins were isolated, from mouse brain lysate, that specifically bound to the NF-H column and which did not bind to a control column to which BSA was bound. The proteins eluted from the NF-H column contained kinases able to efficiently phosphorylate NF proteins in vitro. I characterized these kinases further by separating proteins on denaturing polyacrylamide gels and reconstituting kinase activity in situ. Using this assay I identified a number of individual kinases including a 115 kDa polypeptide which showed a significant preference for NF proteins as substrate. Native NF was found to be the best substrate for the 115 kDa kinase, followed by a bacterially expressed NF-H non-fusion protein, and NF-H fusion protein. However, NF-L was a poor substrate. Two different NF monoclonal antibodies, SMI31 and SMI32 (Sternberger Monoclonal Inc.) were used to further demonstrate that the 115 kDa kinase is associated with NF in vivo. The kinase was co-immunoprecipitated along with NF by the two NF monoclonal antibodies but appeared to be preferentially associated with phosphorylated forms of NF. I discuss here some of the novel properties of the 115 kDa NF-associated kinase I have termed NAK115 (for NF-associated kinase with a molecular mass of 115 kDa). Other biochemical properties of NAK115 were also studied. It appears to be a peripheral membrane protein with a pI of 5.4-6.2, and it is expressed at different levels in a variety of mouse tissues. NAK115 exists as a large 570 protein complex in vivo. Purification of NAK115 was also explored. After four steps of purification, NAK115 was enriched around 20 fold.;Alzheimer's disease (AD) is characterized pathologically by two distinguishable deposits in brain, namely senile plaques and neurofibrillary tangles (NFT). Senile plaques are composed of fragments of the amyloid precursor protein, whereas NFT are composed primarily of paired-helical filaments (PHF). The latter are in turn composed principally of the microtubule-associated tau protein. Tau in PHF is highly and unusually phosphorylated. However, neither the mechanism nor the identity of the protein kinases or phosphatases that govern this unusual phosphorylation is known. Using a combination of immunoblotting and kinase assays, I demonstrate that a discreet set of kinases co-purify with PHF. One of these kinases was found by immunoblotting to be {dollar}\alpha{dollar}-calcium-calmodulin dependent kinase II {dollar}(\alpha{dollar}-CaM kinase). Immunogold labeling revealed that {dollar}\alpha{dollar}-CaM kinase was localized to a novel globular structure found at the ends of PHF. Since previous studies have shown {dollar}\alpha{dollar}-CaM kinase to be involved in memory, its association with PHF may have important implications in understanding memory loss in AD. I also discuss the possibility that the association of {dollar}\alpha{dollar}-CaM kinase with PHF may indicate sites where tau protein is converted into PHF.

Neurofilaments (NF) constitute the predominant intermediate filament components of the neuronal cytoskeleton. While the structural properties of NF are well understood, less is known about their function. Protein phosphorylation appears to play a key role in regulating aspects of NF behavior, such as subunit polymerization and axonal transport. Local regulation of NF phosphorylation may govern variations in the size and morphology of the NF matrix in different regions of the neuron. However, few kinases responsible for these modifications have been identified. This dissertation aimed to identify and characterize proteins which interact with NF, with particular emphasis on protein kinases. Several approaches were used. Firstly, a panel of monoclonal antibodies (MAbs) specific for NF-associated proteins was characterized. MAbs were analyzed by coimmunoprecipitation and immunocytochemical assays. The ligand of one MAb, which coprecipitated with NF, was found to be synapsin I. This synaptic vesicle-associated protein modulates synaptic vesicle function by association with the cytoskeleton. The association of synapsin I with NF may also contribute to this function. The second approach used coimmunoprecipitation assays to assess whether various neuronal kinases interact with NF in brain tissue. An association between a novel cdc2-related kinase and NF was detected. In vitro kinase assays demonstrated that immunoprecipitated cdc2-related kinase phosphorylates NF. The expression of a cdc2-like protein in mature brain tissue suggests that the role of this protein differs from that of other cdc2-related proteins. Previous studies have suggested that NF phosphorylation is regulated by myelination of neurons. This study tested the effect of myelination on NF phosphorylation by examining co-cultures of dorsal root ganglion neurons and Schwann cells. NF were shown to be more highly phosphorylated in myelinated cultures compared to unmyelinated, thus demonstrating that myelination alone is sufficient to induce changes in NF phosphorylation. The activities of individual NF kinases were compared in myelinated and unmyelinated cultures, and candidate kinases which may phosphorylate NF in response to myelination were identified. Collectively, this study has demonstrated the feasibility of these approaches for the identification of proteins that bind or modify NF. What remains to be discovered is how these proteins affect NF function and dynamics in neurons.

Water channels and fluid-phase markers are retrieved from the apical membrane of the toad bladder upon removal of antidiuretic hormone (ADH). These cells maintain high ADH responses even after repeated stimulation, suggesting a high recycling rate of vesicles carrying water channel proteins. Alternatively, there could be a large population of vesicles with only a small fraction responding. The number of retrieved labeled vesicles with single or multiple markers were counted by custom image analysis programs that extracted novel information for statistical analysis. The programs included median and brightness thresholds and an algorithm that counted vesicles by color and size. Recycling was evaluated by loading the retrieved vesicles with Texas Red dextran or biotin/avidin-fluorescein and evaluating the loss of these vesicles due to ADH restimulation. Tissue was stimulated with ADH and then surface proteins were covalently labeled with NHS-SS-Biotin followed by avidin-fluorescein. The tissue was then exposed to Texas Red dextran for 10 min during washout of ADH. ADH restimulation caused a significant reduction in vesicle number with a maximal drop of 22%. Biotin-labeled vesicles were also significantly reduced by 25% after ADH restimulation but did not show preferential sorting and recycling back to the membrane in comparison to vesicles with fluid-phase marker. To study marker retrieved from both the apical and basolateral membranes, samples were exposed to FITC dextran in the serosal bath for 60 min, followed by addition of ADH and Texas Red dextran to the mucosal bath. Cells were examined for colocalization of both markers at various time points following ADH washout. No significant colocalization was seen at the 0 time point but colocalization increased by 60 and 105 mins. After ADH restimulation, vesicles with marker only from the basolateral or apical bath were reduced but vesicles with both markers remained unchanged. Additionally, the basolateral marker uptake was stimulated by a transepithelial osmotic gradient compared to ADH stimulation alone. This work provides evidence that in native ADH-responsive tissue the majority of vesicles labeled from the apical membrane do not respond to ADH. Additionally, apical marker can enter ADH-insensitive vesicles with marker retrieved from the basolateral membrane.

Thromboxane synthase (TS) catalyzes the conversion of prostaglandin H{dollar}\sb2{dollar} into thromboxane A{dollar}\sb2{dollar} (TxA{dollar}\sb2),{dollar} which is a potent mediator of platelet aggregation and vasoconstriction. A deficiency of platelet TS or mutations in the TxA{dollar}\sb2{dollar} receptor gene have been shown to result in bleeding disorders, while an elevated level of TxA{dollar}\sb2{dollar} may be associated with cardiovascular and renal diseases. Several post-transcriptional events have been demonstrated to curtail the level of TS in vivo, presumably for preventing over-production of the autacoid. At present, little is known about the transcriptional regulation of TS gene expression. To address this, a genomic DNA containing the human TS promoter was cloned and characterized. 5{dollar}\sp\prime{dollar}-RACE (rapid amplification of cDNA ends) and ribonuclease (RNase) A/T1 protection assays revealed multiple transcription initiation sites. Deletion analysis indicated that TS transcription is mainly TATA-independent. A proximal positive regulatory sequence (PPRS, {dollar}-{dollar}90 to {dollar}-{dollar}25 bp) and several distal repressive elements, including a silencer, were also identified in the promoter. The PPRS worked in an orientation-independent but position-dependent manner, and could be further divided into two independent elements, PPRS{dollar}\sb1{dollar} ({dollar}-{dollar}90 to {dollar}-{dollar}50 bp) and PPRS{dollar}\sb2{dollar} ({dollar}-{dollar}50 to {dollar}-{dollar}25 bp). While similar amounts of nuclear factor(s) from different cell types may interact with PPRS{dollar}\sb2,{dollar} those interacting with PPRS{dollar}\sb1{dollar} exhibit cell specificity. Internal sequence deletion and oligonucleotide competition established that a binding sequence for NF-E2 in PPRS{dollar}\sb1{dollar} ({dollar}-{dollar}60 tgctgattcat {dollar}-{dollar}50) was important for enhancing TS promoter activity in HL-60 cells. The presence of NF-E2 mRNA in HL-60 cells was demonstrated by RT-PCR amplification of the cDNA and Northern analysis. A 9-fold trans-activation of luciferase (luc) reporter gene expression was detected when NF-E2 cDNA was co-expressed with a TS promoter/luc construct. Despite that NF-E2 and the cis-elements could alter the level of TS transcription, they were not sufficient for restricting cell-specific TS expression. Analysis of the methylation status of the TS promoter in several human cell lines revealed cell-specific patterns of methylation that might correlate with TS expression. Taken together, these results suggest that the expression of human TS gene is modulated by multiple factors including cis-elements, trans-activator(s), and possibly genomic methylation.

The emergence of drug-resistance in cancer cells during chemotherapy remains a major obstacle in the treatment of neoplasia. Multidrug resistance (MDR) to a group of unrelated cytotoxic compounds can be conferred to eucaryotic cells by the expression of P-glycoprotein (Pgp), a putative plasma membrane transporter believed to mediate the efflux of these agents out of cells. A variety of agents are able to reverse this MDR phenotype by inhibiting the Pgp transporter. Blocking the action of this protein increases the effectiveness of cancer chemotherapeutic agents and, hence, has significant clinical implications. A mutant Pgp1 cDNA containing the substitution (Gly{dollar}\sp{lcub}338{rcub}{dollar}Ala{dollar}\sp{lcub}339{rcub}{dollar} to Ala{dollar}\sp{lcub}338{rcub}{dollar}Pro{dollar}\sp{lcub}339{rcub}{dollar}) within the sixth transmembrane domain (tm6) has been cloned. The expression of this mutant confers an MDR phenotype preferentially resistant to actinomycin D. In this thesis we report that this MDR phenotype also has a decreased sensitivity toward reversal by cyclosporin A, while the sensitivity toward verapamil is unaltered. The accumulation of {dollar}\rm\lbrack\sp3H\rbrack{dollar} vincristine in cells expressing the wild-type Pgp1, not the mutant, increases dramatically in the presence of cyclosporin A, which correlates well with the reversal profile. We have altered only one amino acid residue at this location (Gly{dollar}\sp{lcub}338{rcub}{dollar} to Ala{dollar}\sp{lcub}338{rcub}{dollar} or Ala{dollar}\sp{lcub}339{rcub}{dollar} to Pro{dollar}\sp{lcub}339{rcub}{dollar}). The transfectants expressing the Pgp1 containing the proline substitution, rather than the alanine, demonstrate an MDR phenotype which is preferentially resistant to actinomycin D, and insensitive to reversal by cyclosporin A. Modeling the whole tm6 domain (with the Quanta modeling program and energy minimization by the CHARMm program) reveals that the proline substitution at position 339 rather than the alanine at 338 drastically changes the local {dollar}\alpha{dollar}-helice conformation, especially the polar side chain alignment along the hydrophilic side of this amphipathic {dollar}\alpha{dollar}-helice. We hypothesize that the Ala{dollar}\sp{lcub}339{rcub}{dollar} to Pro{dollar}\sp{lcub}339{rcub}{dollar} substitution, rather than the Gly{dollar}\sp{lcub}338{rcub}{dollar} to Ala{dollar}\sp{lcub}338{rcub}{dollar}, is the primary contributor to the aforementioned altered phenotype. We have also attempted to determine the functions of two spliced variants of Pgp1, previously cloned in this laboratory, by expressing them in an in vitro system. The biogenesis of one of the variants, ADX124, has also been investigated. We conclude that it is derived from a splicing event that involves the internal splicing signals that are maintained in the mature full-length Pgp1 transcript.

Angiotensin II (AngII) exerts many functional effects on the heart through the activation of protein kinase C (PKC) to affect contractility, and growth. It is now known that PKC is a family of 11 isoforms designated {dollar}\alpha{dollar}, {dollar}\beta{dollar}I, {dollar}\beta{dollar}II, {dollar}\gamma{dollar}, {dollar}\delta{dollar}, {dollar}\epsilon{dollar}, {dollar}\xi{dollar}, {dollar}\eta{dollar}, {dollar}\theta{dollar}, {dollar}\lambda{dollar}, and {dollar}\mu{dollar}. To examine the effects of PKC on the heart, it was first necessary to characterize which isoforms are expressed in this tissue. A RT-PCR approach was developed to identify isoforms that would amplify regions of the target cDNA of all the PKC isozymes in a single reaction. Cardiac cDNA was RT-PCR amplified and the products analyzed by a combination of restriction mapping and DNA sequencing which revealed the presence of only the {dollar}\alpha{dollar}, {dollar}\delta{dollar}, {dollar}\epsilon{dollar}, {dollar}\eta{dollar}, and {dollar}\xi{dollar} isoforms cardiac myocytes. Since many cardioactive hormones modulate intracellular pH (pH{dollar}\sb{lcub}\rm i{rcub}{dollar}), the goal of this study was to determine if AngII and PKC altered pH{dollar}\sb{lcub}\rm i{rcub}{dollar} in cultured neonatal rat ventricular myocytes. pH{dollar}\sb{lcub}\rm i{rcub}{dollar} was monitored in single cells loaded with the fluorescent indicator c-SNARF-1 or BCECF. Superfusion with 100 nM TPA, a direct activator of PKC, induces an alkalinization of 0.06 {dollar}\pm{dollar} 0.01 pH unit and increased the initial rate of recovery from an imposed acid load by 2.20 {dollar}\pm{dollar} 0.36 fold. The alkalinization and transporter activation are HCO{dollar}\sb3\sp-{dollar}-independent and amiloride-sensitive indicating the involvement of the Na{dollar}\sp+{dollar}/H{dollar}\sp+{dollar} exchanger. Furthermore, Cl{dollar}\sp-{dollar} removal experiments revealed a TPA-stimulated 1.31 {dollar}\pm{dollar} 0.11 fold enhancement of the acid-loading HCO{dollar}\sb3\sp-{dollar}-/Cl{dollar}\sp-{dollar} exchanger. The increase in the Na{dollar}\sp+{dollar}/H{dollar}\sp+{dollar} activity compared to that of the HCO{dollar}\sb3\sp-{dollar}/Cl{dollar}\sp-{dollar} exchanger is consistent with the alkalinization observed. Stimulation of the myocytes with 100 nM AngII resulted in a rapid HCO{dollar}\sb3\sp-{dollar}-dependent, amiloride-insensitive alkalinization of 0.08 {dollar}\pm{dollar} 0.02 pH unit. AngII also increased the rate of acid extrusion by 3.67 {dollar}\pm{dollar} 0.50 fold in a HCO{dollar}\sb3\sp-{dollar}-dependent and Cl{dollar}\sp-{dollar}-independent manner, indicating the activation of the Na{dollar}\sp+{dollar}/HCO{dollar}\sb3\sp-{dollar}-symport. The AngII activation of the symport is mediated through an AT{dollar}\sb2{dollar}-like signalling pathway since the pH{dollar}\sb{lcub}\rm i{rcub}{dollar} response was blocked by the AT{dollar}\sb2{dollar} receptor antagonist, CGP 42112A, and was unaffected by the AT{dollar}\sb1{dollar} inactivator, DTT. Superfusion of the myocytes with 5 {dollar}\mu{dollar}M arachidonic acid (ARA) mimicked the AngII-mediated alkalinization, suggesting further that ARA may mediate the response. Moreover, the AngII- and the ARA-induced responses were blocked with staurosporine, a PKC inhibitor. In summary, AngII activates the Na{dollar}\sp+{dollar}/HCO{dollar}\sb3\sp-{dollar} symport through the AT{dollar}\sb2{dollar} pathway via ARA and possibly through PKC. Although TPA and AngII both alkalinize the cell, they do so through two distinct pathways, perhaps by activating different PKC isoforms.

The homeostatic mechanism for the respiratory system involves the tight control of expression of several gene products. One such gene is a member of the multi-substrate oxidase system called cytochrome P450 (P450). Pulmonary P450s may be involved in the tissue-specific detoxification, and/or bio-activation of chemical agents. Additionally, pulmonary cytochrome P450s, particularly the human iso-form 4B1, could play an important role in the development and homeostasis of the pulmonary tissue by removing androgens that cause deleterious effects on lung maturation. Human cytochrome P450 4B1 (CYP 4B1) is a predominant cytochrome P450 activity in the lung. CYP 4B1 protein is reported to bio-activate some pulmonary toxicants. Therefore it may mediate chemical-induced damage to that organ. However, the high expression levels of CYP 4B1 gene products in the lung (approximately 80% of the total P450 activity) may suggest the involvement of the iso-form in normal physiological functions for pulmonary tissue. On the other hand, the results of this and other laboratories have shown an apparent species-specific difference in the catalytic activity of 4B1 protein in man versus rodents. For instance, the rodent iso-form is potent at activating pro-carcinogens and devoid of androgen hydrolyzing activities towards androgen metabolism. The human 4B1 protein has higher 6-beta-testosterone hydroxylase activity while the rodent iso-form is devoid of this action. Furthermore, the manner in which the 4B1 gene is regulated appears to be species-specific. Therefore, my studies have been directed toward further defining the genetic mechanism of action that regulates the human 4B1 gene expression levels. My research objectives have been divided into three specific aims: to determine the presence or absence of cis-acting elements located in the 3.0 Kbp upstream fragment of the human 4B1 gene; to determine the DNA structure of the 4.5 Kbp fragment that overlaps and lies further upstream to the 3.0 Kbp regulatory region of the human 4B1 gene; and to determine the actions of a contiguous 6.5 Kbp fragment containing the entire putative upstream region of 4B1 gene on transcription of the luciferase reporter gene in human lung cells. The results of my studies show the presence of multiple promoter elements in addition to the TATA-box that are contained in the CYP 4B 13.0 Kbp upstream fragment. Nucleotide sequence analysis of the 4.5 Kbp upstream fragment that contains 1.0 Kbp overlapping fragment with the 3.0 Kbp fragment based on restriction mapping confirms that it is indeed part of the contiguous region of the CYP 4B1 gene. A number of consensus DNA regulatory motifs have been identified. (Abstract shortened by UMI.)

Endothelial cell injury and/or dysfunction contributes to a variety of complications associated with Gram-negative septicemia including systemic vascular collapse, disseminated intravascular coagulation, and vascular leak syndromes. Endotoxin or bacterial lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria, directly provokes endothelial injury in vitro and in vivo. Specifically, endotoxin in the presence of serum induces F-actin depolymerization, opening of the paracellular pathway, and increased endothelial monolayer permeability to macromolecules. We have identified lipid A as the bioactive moiety of bacterial lipopolysaccharide responsible for inducing an array of endothelial cell responses including increased protein tyrosine phosphorylation, actin depolymerization, increased monolayer permeability, and apoptosis. We have also found that the influence of endotoxin on endothelial cell actin organization and barrier function is mediated, in part, through a signaling pathway that is dependent on tyrosine phosphorylation events. Protein tyrosine kinase inhibition, which blocks endotoxin-induced tyrosine phosphorylation of the focal adhesion protein paxillin, protects against downstream endothelial responses including actin depolymerization, intercellular gap formation, and loss of barrier function. Further, we have studied the effect that caspase-mediated cleavage of adherens junction proteins has on mediating endotoxin-induced changes in cell-cell and cell-matrix adhesion. Endotoxin-provoked increments in transendothelial albumin flux and endothelial cell detachment occur at doses and times which are compatible with endotoxin-induced caspase activation and apoptosis. Proteins associated with the zonula adherens and focal adhesions, which mediate cell-cell and cell-matrix adhesion, respectively, are targets of caspase proteolysis. Cleavage of focal adhesion kinase leads to its dissociation from paxillin, a substrate for focal adhesion kinase tyrosine phosphorylation. The time-dependent cleavage of focal adhesion kinase and its dissociation from paxillin parallels a decrease in the phosphotyrosine content of paxillin. Caspase inhibition blocks focal adhesion kinase cleavage, decrements in paxillin phosphotyrosine content, and endothelial cell detachment, but fails to protect against endotoxin-induced endothelial barrier dysfunction. Protein tyrosine kinase inhibition, however, fails to block proteolysis but does protect against increased monolayer permeability. These findings suggest a bifurcation in the pathways through which endotoxin influences cell-cell adhesion and opening of the paracellular pathway versus endothelial cell adhesion to and detachment from the underlying extracellular matrix.

SPARC (Secreted Protein Acidic and Rich in Cysteine) and thrombospondin-1 (TSP) are secreted glycoproteins that influence multiple endothelial cell (EC) functions including cell attachment to and spreading on substrates, cell motility and angiogenesis. These two counteradhesive proteins have been grouped together solely on a functional basis. By definition, both SPARC and TSP influence EC-extracellular matrix interactions and promote focal adhesion disassembly. We studied whether SPARC and TSP influence homophilic EC-EC adhesion and vascular endothelial barrier function. Actin-dependent homophilic EC-EC adhesion is vital to the formation and maintenance of an endothelial barrier. Although the signal transduction pathways responsible for regulating endothelial barrier function and opening of the paracellular pathway are unclear, tyrosine phosphorylation of actin-dependent cell-cell adherens junction proteins diminishes homophilic cell-cell adhesion. We have now demonstrated that SPARC and TSP regulate movement of a 14C-bovine serum albumin (BSA) tracer molecule across postconfluent EC monolayers in a tyrosine phosphorylation-dependent manner, in vitro. Pretreatment of EC monolayers with the protein tyrosine kinase (PTK) inhibitors, herbimycin A or genistein, protected against both SPARC- and TSP-induced barrier dysfunction. In contrast, pretreatment of EC monolayers with the protein tyrosine phosphatase (PTP) inhibitors, sodium orthovanadate (vanadate) or phenylarsine oxide enhanced the SPARC- and TSP-induced increments in 14C-BSA flux. SPARC- and TSP-exposed EC monolayers also exhibited actin reorganization and intercellular gap formation using F-actin epifluorescence microscopy. Further, PTK inhibition protected against intercellular gap formation. In the presence of PTP inhibition, both SPARC- and TSP-exposed EC exhibited dose-and-time dependent increases in protein tyrosine phosphorylation by immunoblotting. Several tyrosine phosphorylated proteins were identified as cell-cell adherens junction proteins. Further, these increases in protein tyrosine phosphorylation were localized to the EC-EC boundaries using epifluorescence microscopy. These findings indicate that SPARC and TSP regulate an endothelial paracellular pathway through protein tyrosine phosphorylation. In addition, our studies with the PTP inhibitor, vanadate, corroborate our findings with SPARC and TSP, and suggest that vascular endothelial barrier function is regulated through tyrosine phosphorylation of cell-cell adherens junction proteins.

Adult frog phasic skeletal muscle cells have slow inward calcium currents (ICa) with no obvious role in excitation-contraction coupling (ECC). We have found that embryonic skeletal myocytes are capable of contracting in the early stages of development, at the time when the T-system and the SR may not be fully developed. Contractions could then be triggered at this stage by Ca2+ flowing into the cells through ICa. The present thesis was undertaken to test the hypothesis that ICa may have a role in ECC during early stages of skeletal muscle cell development. This study was carried out in embryonic skeletal myocytes from Xenopus laevis cultured for 1-15 days. We recorded ICa with the patch-clamp technique and, Ba2+ as the carrier. Cell capacitance and current density indicated that ICa and total membrane area increase with time, reaching a plateau at around days 12-15. Cytosolic Ca2+ changes were observed from fluorescence images. While [Ca2+]i increased continuously during 700 ms depolarizing pulses in young cells, older cells showed an early [Ca2+]i rise followed by a steady lower [Ca2+]i. In young cells [Ca2+]i did not increase when the cells were field stimulated in the presence of 10-7 M [Ca2+]0, while 1-week-old cells showed an increase in [Ca2+]i. We studied the SR functional development by measuring contracture thresholds and [Ca2+]i in response to caffeine. Young cells responded to higher caffeine concentrations than older cells. Development of the T-tubular and SR membranes was investigated with fluorescence confocal microscopy. One-day-cultured myocytes are devoid of T-tubular membrane, except for short and sparse invaginations. Within 3 days, a T-tubular system forms and continues to grow until day 12 in culture. The SR is present in the periphery of the cell from day one. At this stage the SR is scanty but more abundant than the T-tubules and does not form a particular pattern. The longitudinal and transverse pattern of the SR forms within two weeks as a complex and highly dense structure. Our results indicate that (1) a different process of ECC is taking place during the early stages of embryonic skeletal muscle development and (2) the role of ICa is to provide an influx of calcium that may enable the cell to contract, probably by a mechanism similar to calcium-induced calcium release.