CASK interacts with PMCA4b and JAM-A on the mouse sperm flagellum to regulate Ca2+ homeostasis and motility.

Deletion of the highly conserved gene for the major Ca(2+) efflux pump, Plasma membrane calcium/calmodulin-dependent ATPase 4b (Pmca4b), in the mouse leads to loss of progressive and hyperactivated sperm motility and infertility. Here we first demonstrate that compared to wild-type (WT), Junctional adhesion molecule-A (Jam-A) null sperm, previously shown to have motility defects and an abnormal mitochondrial phenotype reminiscent of that seen in Pmca4b nulls, exhibit reduced (P < 0.001) ATP levels, significantly (P < 0.001) greater cytosolic Ca(2+) concentration ([Ca(2+) ](c)) and ∼10-fold higher mitochondrial sequestration, indicating Ca(2+) overload. Investigating the mechanism involved, we used co-immunoprecipitation studies to show that CASK (Ca(2+) /calmodulin-dependent serine kinase), identified for the first time on the sperm flagellum where it co-localizes with both PMCA4b and JAM-A on the proximal principal piece, acts as a common interacting partner of both. Importantly, CASK binds alternatively and non-synergistically with each of these molecules via its single PDZ (PDS-95/Dlg/ZO-1) domain to either inhibit or promote efflux. In the absence of CASK-JAM-A interaction in Jam-A null sperm, CASK-PMCA4b interaction is increased, resulting in inhibition of PMCA4b's enzymatic activity, consequent Ca(2+) accumulation, and a ∼6-fold over-expression of constitutively ATP-utilizing CASK, compared to WT. Thus, CASK negatively regulates PMCA4b by directly binding to it and JAM-A positively regulates it indirectly through CASK. The decreased motility is likely due to the collateral net deficit in ATP observed in nulls. Our data indicate that Ca(2+) homeostasis in sperm is maintained by the relative ratios of CASK-PMCA4b and CASK-JAM-A interactions.

Effect of magnesium sulfate on contractile force and intracellular calcium concentration in pregnant human myometrium.

OBJECTIVE: This study was undertaken to evaluate the effects of magnesium sulfate (MgSO4) on contractile force and increases in free intracellular calcium concentration ([Ca2+]i) in human myometrial strips from pregnant women. STUDY DESIGN: Simultaneous measurements of isometric tension and [Ca2+]i were measured in myometrial strips obtained at the time of cesarean delivery from pregnant nonlaboring women at term with the use of a fluorescence spectrometer equipped with a displacement force transducer. Changes in [Ca2+]i were measured with fura-2, a Ca(2+)-sensitive fluorescent probe. Myometrial strips were exposed to MgSO4 (5 or 10 mmol/L) for 5, 10, 20, and 30 minutes and observed for spontaneous contractions or stimulated with either oxytocin (OT; 0.1 micromol/L) or potassium chloride (KCl; 90 mmol/L). RESULTS: MgSO4 reduced spontaneous, OT, and KCl-evoked contractions and increases in [Ca2+]i in a time and concentration-dependent manner. After 20 minutes exposure to 5 mmol/L MgSO4, the OT-elicited changes in contractile response and [Ca2+]i were significantly decreased. MgSO4 did not change [Ca2+]i/force relationship of the responses to OT or KCl, or during spontaneous activity. CONCLUSION: At a pharmacologic concentration (5 mmol/L), MgSO4 inhibits contractile response and [Ca2+]i in pregnant human myometrial strips by a pattern that is consistent with both extra- and intracellular mechanisms. At a suprapharmacologic concentration (10 mmol/L), the more immediate effect of MgSO4 is consistent with an extracellular mechanism. MgSO4 does not appear to interfere at the level of the calcium-calmodulin interface, since the [Ca2+]i/force relationship was not changed.

Role of protein kinase Calpha in regulation of [Ca2+](I) and force in human myometrium.

Recent findings implicate protein kinase C in regulation of contraction of uterine muscle (myometrium). However, the role of protein kinase C isoforms in myometrial contraction remains uncertain. Therefore, this study examined protein kinase Calpha's role in regulation of contraction and intracellular calcium concentration ([Ca2+](I)) of myometrium from term pregnant women. The authors demonstrated that protein kinase Calpha inhibitor Go6976 decreased the amplitude of potassium chloride-induced myometrial contractions in a time-dependent manner. The treatment of the myometrial strips with protein kinase Calpha-specific antisense oligodeoxynucleotides decreased the potassium chloride-induced contraction and [Ca2+](I) response to 39.3% + 6.8% and 50.0% + 3.3%, respectively, compared to control. The sense oligonucleotides treatment did not significantly change the potassium chloride responses (89.8% + 6.8% and 93.9% + 4.5% of the control for the contraction and [Ca2+](I), respectively). These data, coupled with the observation that protein kinase Calpha levels are elevated in the pregnant myometrium, suggest the involvement of protein kinase Calpha in regulation of human uterine contraction.

Dynamic interactions between L-type voltage-sensitive calcium channel Cav1.2 subunits and ahnak in osteoblastic cells.

Voltage-sensitive Ca(2+) channels (VSCCs) mediate Ca(2+) permeability in osteoblasts. Association between VSCC alpha(1)- and beta-subunits targets channel complexes to the plasma membrane and modulates function. In mechanosensitive tissues, a 700-kDa ahnak protein anchors VSCCs to the actin cytoskeleton via the beta(2)-subunit of the L-type Ca(v)1.2 (alpha(1C)) VSCC complex. Ca(v)1.2 is the major alpha(1)-subunit in osteoblasts, but the cytoskeletal complex and subunit composition are unknown. Among the four beta-subtypes, the beta(2)-subunit and, to a lesser extent, the beta(3)-subunit coimmunoprecipitated with the Ca(v)1.2 subunit in MC3T3-E1 preosteoblasts. Fluorescence resonance energy transfer revealed a complex between Ca(v)1.2 and beta(2)-subunits and demonstrated their association in the plasma membrane and secretory pathway. Western blot and immunohistochemistry showed ahnak association with the channel complex in the plasma membrane via the beta(2)-subunit. Cytochalasin D exposure disrupted the actin cytoskeleton but did not disassemble or disrupt the function of the complex of L-type VSCC Ca(v)1.2 and beta(2)-subunits and ahnak. Similarly, small interfering RNA knockdown of ahnak did not disrupt the actin cytoskeleton but significantly impaired Ca(2+) influx. Collectively, we showed that Ca(v)1.2 and beta(2)-subunits and ahnak form a stable complex in osteoblastic cells that permits Ca(2+) signaling independently of association with the actin cytoskeleton.

Voltage-sensitive ion channels and cancer.

Plasma membrane voltage-sensitive ion channels classically have been associated with a variety of inherited diseases or "channelopathies" that range in the severity of symptoms from mild to lethal. Ion channels are found throughout the body and are responsible for facilitated diffusion of ions down the electrochemical gradient across cells membranes in various tissues. Voltage-sensitive ion channels open in response to changes in the membrane potential and are primarily found in excitable cells and tissues. Potassium, calcium, and sodium channels play critical roles in the development of major diseases, such as hyperkalemia, epilepsy, congenital myotonia and several cardiac arrythmias. Recently, cancer studies have begun to define the role of voltage-sensitive ion channels in the progression of cancer to a more malignant phenotype. In cancer, the increased expression or increased kinetics of voltage-sensitive ion channels is associated with an increasing malignant potential as evinced by their role in cell proliferation, migration and survival; as such, these channels are becoming the targets of significant drug development efforts to block or reduce voltage-sensitive ion channel activity in order to prevent or combat malignant disease.

Magnesium sulfate induces translocation of protein kinase C isoenzymes alpha and delta in myometrial cells from pregnant women.

OBJECTIVES: The purpose of this study was to determine the effect of magnesium sulfate on protein kinase C (PKC) translocation in myometrial cells from pregnant women. STUDY DESIGN: Myometrium was obtained at the time of cesarean delivery from women at term before labor. Cultured myometrial cells were treated with magnesium sulfate (3, 5, and 10 mmol/L), oxytocin (0.1 micromol/L), or 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.1 micromol/L). The translocation of PKC isozymes alpha (calcium dependent) and delta (calcium independent) from cytosol to membrane fractions was assessed with use of Western blot analysis. RESULTS: In unexposed control cells, the majority of PKC alpha and delta was located in the cytosol fraction. Exposure to magnesium sulfate for 60 minutes induced translocation of both PKC alpha and delta at concentrations as low as 5 and 3 mmol/L, respectively. The magnitude of magnesium sulfate induced translocation for PKC delta is similar to that seen after oxytocin or TPA exposure but less for PKC alpha. Exposure to oxytocin for 30 minutes and 60 minutes induced translocation of PKC alpha and delta, respectively. Exposure to TPA for 5 and 30 minutes induced translocation of PKC alpha and PKC delta, respectively. In calcium-free media, only TPA induced translocation of these two isoenzymes. CONCLUSION: Magnesium sulfate stimulates PKC translocation in cultured myometrial cells from pregnant women. Magnesium sulfate and oxytocin require extracellular calcium to induce translocation of both PKC alpha and delta.

Role of Ca2+ in diperoxovanadate-induced cytoskeletal remodeling and endothelial cell barrier function.

Diperoxovanadate (DPV), a potent inhibitor of protein tyrosine phosphatases and activator of tyrosine kinases, alters endothelial barrier function via signaling pathways that are incompletely understood. One potential pathway is Src kinase-mediated tyrosine phosphorylation of proteins such as cortactin that regulate endothelial cell (EC) cytoskeleton assembly. As DPV modulates endothelial cell signaling via protein tyrosine phosphorylation, we determined the role of DPV-induced intracellular free calcium concentration ([Ca2+]i) in activation of Src kinase, cytoskeletal remodeling, and barrier function in bovine pulmonary artery endothelial cells (BPAECs). DPV in a dose- and time-dependent fashion increased [Ca2+]i, which was partially blocked by the calcium channel blockers nifedipine and Gd3+. Treatment of cells with thapsigargin released Ca2+ from the endoplasmic reticulum, and subsequent addition of DPV caused no further change in [Ca2+]i. These data suggest that DPV-induced [Ca2+]i includes Ca release from the endoplasmic reticulum and Ca influx through store-operated calcium entry. Furthermore, DPV induced an increase in protein tyrosine phosphorylation, phosphorylation of Src and cortactin, actin remodeling, and altered transendothelial electrical resistance in BPAECs. These DPV-mediated effects were significantly attenuated by BAPTA (25 microM), a chelator of [Ca2+]i. Immunofluorescence studies reveal that the DPV-mediated colocalization of cortactin with peripheral actin was also prevented by BAPTA. Chelation of extracellular Ca2+ by EGTA had marginal effects on DPV-induced phosphorylation of Src and cortactin; actin stress fibers formation, however, affected EC barrier function. These data suggest that DPV-induced changes in [Ca2+]i regulate endothelial barrier function using signaling pathways that involve Src and cytoskeleton remodeling.

Magnesium sulfate inhibits the oxytocin-induced production of inositol 1,4,5-trisphosphate in cultured human myometrial cells.

OBJECTIVE: The purpose of this study was to determine the effects of magnesium sulfate on inositol trisphosphate production and the mechanism of these effects. STUDY DESIGN: Myometrium was obtained at the time of cesarean delivery from women before labor at term. Inositol trisphosphate was measured in the primary myometrial cell cultures after stimulation with oxytocin, sodium fluoride, or Bay K 8644 with or without preincubation with magnesium sulfate or nifedipine. Experiments were performed in either calcium-containing or calcium-free medium that contained egtazic acid and after preincubation with the intracellular calcium chelator BAPTA-acetoxymethylester. Inositol trisphosphate production was measured by radioreceptor assay. In separate experiments, changes in intracellular calcium concentrations ([Ca(2+)](i)) were measured with the use of Fura-2 and spectrophotofluorometry. RESULTS: Oxytocin, sodium fluoride, and Bay K 8644 increased inositol trisphosphate production 2- to 4-fold. Preincubation with magnesium sulfate (3 x 10(-3) mol/L) for > or = 5 minutes decreased oxytocin-, sodium fluoride-, and Bay K 8644-induced inositol trisphosphate production in either calcium-containing or calcium-free media. Preincubation with BAPTA-acetoxymethylester decreased oxytocin-stimulated inositol trisphosphate production by 78% in calcium-containing media and completely prevented the oxytocin response in calcium-free media. Magnesium sulfate decreased inositol trisphosphate production in calcium-containing media but had no additional effect in calcium-free media. Oxytocin and Bay K 8644 increased [Ca(2+)](i) in either calcium-containing or calcium-free media, and magnesium sulfate reduced this in both cases. CONCLUSION: Magnesium sulfate appears to inhibit phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activity and subsequent calcium release in cultured myometrial cells by a direct effect on phospholipase C.