The localization of various Ca2+ transport and signaling proteins in secretory cells is highly restricted, resulting in polarized agonist-stimulated Ca2+ waves. In the present work, we examined the possible roles of the Sec6/8 complex or the exocyst in polarized Ca2+ signaling in pancreatic acinar cells. Immunolocalization by confocal microscopy showed that the Sec6/8 complex is excluded from tight junctions and secretory granules in these cells. The Sec6/8 complex was found in at least two cellular compartments, part of the complex showed similar, but not identical, localization with the Golgi apparatus and part of the complex associated with Ca2+ signaling proteins next to the plasma membrane at the apical pole. Accordingly, immunoprecipitation (IP) of Sec8 did not coimmunoprecipitate βCOP, Golgi 58K protein, or mannosidase II, all Golgi-resident proteins. By contrast, IP of Sec8 coimmunoprecipitates Sec6, type 3 inositol 1,4,5-trisphosphate receptors (IP3R3), and the Gβγ subunit of G proteins from pancreatic acinar cell extracts. Furthermore, the anti-Sec8 antibodies coimmunoprecipitate actin, Sec6, the plasma membrane Ca2+ pump, the G protein subunits Gαq and Gβγ, the β1 isoform of phospholipase C, and the ER resident IP3R1 from brain microsomal extracts. Antibodies against the various signaling and Ca2+ transport proteins coimmunoprecipitate Sec8 and the other signaling proteins. Dissociation of actin filaments in the immunoprecipitate had no effect on the interaction between Sec6 and Sec8, but released the actin and dissociated the interaction between the Sec6/8 complex and Ca2+ signaling proteins. Hence, the interaction between the Sec6/8 and Ca2+ signaling complexes is likely mediated by the actin cytoskeleton. The anti-Sec6 and anti-Sec8 antibodies inhibited Ca2+ signaling at a step upstream of Ca2+ release by IP3. Disruption of the actin cytoskeleton with latrunculin B in intact cells resulted in partial translocation of Sec6 and Sec8 from membranes to the cytosol and interfered with propagation of agonist-evoked Ca2+ waves. Our results suggest that the Sec6/8 complex has multiple roles in secretory cells including governing the polarized expression of Ca2+ signaling complexes and regulation of their activity.

Shin contributed equally to this workDarier's disease (DD) is a high penetrance, autosomal dominant mutation in the ATP2A2 gene, which encodes the SERCA2 Ca 2+ pump. Here we have used a mouse model of DD, a SERCA2 +/± mouse, to de®ne the adaptation of Ca 2+ signaling and Ca 2+ -dependent exocytosis to a deletion of one copy of the SERCA2 gene. The [Ca 2+ ] i transient evoked by maximal agonist stimulation was shorter in cells from SERCA2 +/± mice, due to an up-regulation of speci®c plasma membrane Ca 2+ pump isoforms. The change in cellular Ca 2+ handling caused~50% reduction in [Ca 2+ ] i oscillation frequency. Nonetheless, agonist-stimulated exocytosis was identical in cells from wild-type and SERCA2 +/± mice. This was due to adaptation in the levels of the Ca 2+ sensors for exocytosis synaptotagmins I and III in cells from SERCA2 +/± mice. Accordingly, exocytosis was~10-fold more sensitive to Ca 2+ in cells from SERCA2 +/± mice. These ®ndings reveal a remarkable plasticity and adaptability of Ca 2+ signaling and Ca 2+ -dependent cellular functions in vivo, and can explain the normal function of most physiological systems in DD patients.

Endothelin 1 (ET-1), a potent vasoconstrictor peptide expressed by endothelium, is also produced in the heart in response to a variety of stresses. It induces hypertrophy in cultured cardiac myocytes but only at concentrations far greater than those found in plasma. We tested whether ET-1 generated by cardiac myocytes in vivo is a local signal for cardiac hypertrophy. To avoid the perinatal lethality seen in systemic ET-1-null mice, we used the Cre͞loxP system to generate mice with cardiac myocyte-specific disruption of the ET-1 gene. We used the ␣-myosin heavy chain promoter to drive expression of Cre and were able to obtain 75% reduction in ET-1 mRNA in cardiac myocytes isolated from these mice at baseline and after stimulation, in vivo, for 24 h with tri-iodothyronine (T3). Necropsy measurements of cardiac mass indexed for body weight showed a 57% reduction in cardiac hypertrophy in response to 16 days of exogenous T3 in mice homozygous for the disrupted ET-1 allele compared to siblings with an intact ET-1 gene. Moreover, in vivo MRI showed only a 3% increase in left ventricular mass indexed for body weight in mice with the disrupted allele after 3 weeks of T3 treatment versus a 27% increase in mice with an intact ET-1 gene. A reduced hypertrophic response was confirmed by planimetry of cardiac myocytes. We conclude that ET-1, produced locally by cardiac myocytes, and acting in a paracrine͞autocrine manner, is an important signal for myocardial hypertrophy that facilitates the response to thyroid hormone. C ardiac hypertrophy is characterized by an increase in myocardiocyte size and is accompanied by qualitative and quantitative changes in gene expression, protein synthesis, and physiological performance (1). Cardiac hypertrophy helps to maintain cardiac output in the presence of increased demand or afterload, yet it is one of the most important predisposing risk factors for sudden death and the development of heart failure in human populations. A greater understanding of hypertrophy, and the capacity to regulate it in human disease, could have profound clinical implications.Many ligand͞receptor systems, with their specific and interwoven downstream signaling pathways, have been implicated in cardiomyocyte hypertrophy in both cell culture and intact animals (reviewed in ref.2). These include the peptide endothelin 1 (ET-1) and its receptors ET A and ET B on the cell surface of cardiomyocytes. Several lines of evidence suggest that ET-1 functions in a paracrine͞autocrine manner in cardiac hypertrophy. In cell cultures of cardiomyocytes, ET-1 induces hypertrophy as assessed by cell size, increased myofibrillogenesis, and transcriptional changes associated with cardiac hypertrophy such as expression of atrial natriuretic factor (3, 4). Of note, in these studies the concentrations of ET-1 required to produce hypertrophy were 10 Ϫ7 to 10 Ϫ9 M, far greater than those encountered in plasma (10 Ϫ12 to 10 Ϫ13 M) (5). Although cardiac synthesis of ET-1 is negligible under normal circumstances, conditions that stimulate...