Cdk5-dependent regulation of glucose-stimulated insulin secretion

Tight glycemic control in individuals with diabetes mellitus is essential to prevent or delay its complications. Present treatments to reduce hyperglycemia mainly target the ATP-sensitive K(+) (K(ATP)) channel of pancreatic beta cells to increase insulin secretion. These current approaches are often associated with the side effect of hypoglycemia. Here we show that inhibition of the activity of cyclin-dependent kinase 5 (Cdk5) enhanced insulin secretion under conditions of stimulation by high glucose but not low glucose in MIN6 cells and pancreatic islets. The role of Cdk5 in regulation of insulin secretion was confirmed in pancreatic beta cells deficient in p35, an activator of Cdk5. p35-knockout mice also showed enhanced insulin secretion in response to a glucose challenge. Cdk5 kinase inhibition enhanced the inward whole-cell Ca(2+) channel current and increased Ca(2+) influx across the L-type voltage-dependent Ca(2+) channel (L-VDCC) upon stimulation with high glucose in beta cells, but had no effect on Ca(2+) influx without glucose stimulation. The inhibitory regulation by Cdk5 on the L-VDCC was attributed to the phosphorylation of loop II-III of the alpha(1C) subunit of L-VDCC at Ser783, which prevented the binding to SNARE proteins and subsequently resulted in a decrease of the activity of L-VDCC. These results suggest that Cdk5/p35 may be a drug target for the regulation of glucose-stimulated insulin secretion.     

Loss of plasma membrane phospholipid asymmetry requires raft integrity. Role of transient receptor potential channels and ERK pathway

Cholesterol-rich membrane microdomains, also termed lipid rafts, are implicated in the recruitment of essential proteins for intracellular signal transduction. In nonstimulated cells, phosphatidylserine, an anionic aminophospholipid essential for the hemostatic response, is mostly sequestered in the inner leaflet of the plasma membrane. Cell stimulation by Ca(2+)-mobilizing or apoptogenic agents induces the migration of phosphatidylserine to the exoplasmic leaflet, allowing the assembly and activation of several key enzyme complexes of the coagulation cascade and phagocyte recognition of stimulated or senescent cells. We have recently proposed that store-operated Ca(2+) entry regulates externalization of phosphatidylserine at the cell surface (Kunzelmann-Marche, C., Freyssinet, J.-M., and Martinez, M. C. (2001) J. Biol. Chem. 276, 5134-5139). Here, we show that store-operated Ca(2+) entry and phosphatidylserine exposure are dramatically reduced after raft disruption by methyl-beta-cyclodextrin. In addition, transient receptor potential channel 1-specific antibody was able to significantly decrease Ca(2+)-induced redistribution of phosphatidylserine. Furthermore, store-operated Ca(2+) entry and phosphatidylserine exposure were dependent in part on the extracellular signal-regulated kinase pathway associated with rafts. Hence, raft integrity and store-operated Ca(2+) entry involving transient receptor potential channel 1 channels are essential for completion of the phosphatidylserine transmembrane redistribution process.     

Electrical activity modulates growth cone guidance by diffusible factors

Brief periods of electrical stimulation of cultured Xenopus spinal neurons resulted in a marked alteration in the turning responses of the growth cone induced by gradients of attractive or repulsive guidance cues. Netrin-1-induced attraction was enhanced, and the repulsion induced by myelin-associated glycoprotein (MAG) or myelin membrane fragments was converted to attraction. The effect required the presence of extracellular Ca(2+) during electrical stimulation and appeared to be mediated by an elevation of both cytoplasmic Ca(2+) and cAMP. Thus, electrical activity may influence the axonal path finding of developing neurons, and intermittent electrical stimulation may be effective in promoting nerve regeneration after injury.     

Separation of vesicles of cardiac sarcolemma from vesicles of cardiac sarcoplasmic reticulum. Comparative biochemical analysis of component activities.

Sarcolemmal and sarcoplasmic reticulum membrane vesicle fractions were isolated from cardiac microsomes. Separation of sarcolemmal and sarcoplasmic reticulum membrane markers was documented by a combination of correlative assay and centrifugation techniques. To facilitate the separation, the crude microsomes were incubated in the presence of ATP, Ca2+, and oxalate to increase the density of the sarcoplasmic reticulum vesicles. After sucrose gradient centrifugation, the densest subfraction (sarcoplasmic reticulum) contained the highest (K+,Ca2+)-ATPase activity and virtually no (Na2+,K+)-ATPase activity, even when latent (Na+,K+)-ATPase activity was unmasked. In addition, the sarcoplasmic reticulum fraction contained no significant sialic acid, beta receptor binding activity, or adenylate cyclase activity. Sarcolemmal membrane fractions were of low buoyant density. Preparations most enriched in sarcolemmal vesicles contained the highest level of all the other parameters and only about 10% of the (K+,Ca2+)-ATPase activity of the sarcoplasmic reticulum fraction. The results suggest that (Na+,K+)-ATPase, sialic acid, beta-adrenergic receptors, and adenylate cyclase can be entirely accounted for by the sarcolemmal content of cardiac microsomes. Gel electrophoresis of the sarcolemmal and sarcoplasmic reticulum membrane fractions showed distinct bands. Membrane proteins exclusive to each of the fractions were also demonstrated by phosphorylation. Cyclic AMP stimulated phosphorylation by [gamma-32P]ATP of two proteins of apparent Mr = 20,000 and 7,000 that were concentrated in sarcoplasmic reticulum, but the stimulation was markedly dependent on the presence of added soluble cyclic AMP-dependent protein kinase. Cyclic AMP also stimulated phosphorylation of membrane proteins in sarcolemma, but this phosphorylation was mediated by an endogenous protein kinase activity. The apparent molecular weights of these phosphorylated proteins were 165,000, 90,000, 56,000, 24,000, and 11,000. The results suggest that sarcolemma may contain an integral enzyme complex, not present in sarcoplasmic reticulum, that contains beta-adrenergic receptors, adenylate cyclase, cyclic AMP-dependent protein kinase, and several substrates of the protein kinase.     

CRHSP-28 regulates Ca(2+)-stimulated secretion in permeabilized acinar cells

CRHSP-28 is a Ca(2+)-regulated heat-stable phosphoprotein, abundant in the apical cytoplasm of epithelial cells that are specialized in exocrine protein secretion. To define a functional role for the protein in pancreatic secretion, recombinant CRHSP-28 (rCRHSP-28) was introduced into streptolysin-O-permeabilized acinar cells, and amylase secretion in response to elevated Ca(2+) was determined. Secretion was enhanced markedly by rCRHSP-28 over a time course that closely corresponded with the loss of the native protein from the intracellular compartment. No effects of rCRHSP-28 were detected until approximately 50% of the native protein was lost from the cytosol. Secretion was enhanced by rCRHSP-28 over a physiological range of Ca(2+) concentrations with 2-3-fold increases in amylase release occurring in response to low micromolar levels of free Ca(2+). Further, rCRHSP-28 augmented secretion in a concentration-dependent manner with minimal and maximal effects occurring at 1 and 25 microg/ml, respectively. Covalent cross-linking experiments demonstrated that native CRHSP-28 was present in a 60-kDa complex in cytosolic fractions and in a high molecular mass complex in particulate fractions, consistent with the slow leak rate of the protein from streptolysin-O-permeabilized cells. Probing acinar lysates with rCRHSP-28 in a gel-overlay assay identified two CRHSP-28-binding proteins of 35 (pp35) and 70 kDa (pp70). Interestingly, preparation of lysates in the presence of 1 mm Ca(2+) resulted in a marked redistribution of both proteins from a cytosolic to a Triton X-100-insoluble fraction, suggesting a Ca(2+)-sensitive interaction of these proteins with the acinar cell cytoskeleton. In agreement with our previous study immunohistochemically localizing CRHSP-28 around secretory granules in acinar cells, gel-overlay analysis revealed pp70 copurified with acinar cell secretory granule membranes. These findings demonstrate an important cell physiological function for CRHSP-28 in the Ca(2+)-regulated secretory pathway of acinar cells.     

Characteristics of sarcoplasmic reticulum from slowly glycolysing and from rapidly glycolysing pig skeletal muscle post mortem

The composition and function of fragmented sarcoplasmic reticulum from pig skeletal muscle was examined in the period immediately post mortem. Muscle was defined as being either slowly glycolysing or rapidly glycolysing on the basis of colour, pH and concentrations of glycogen and lactate. The microsomal fraction was separated on a discontinuous gradient of 35, 40 and 45% (w/v) sucrose into heavy and intermediate fractions which sedimented to the interfaces, and a light fraction which remained on the surface of the 35%-sucrose layer. The sarcoplasmic reticulum from rapidly glycolysing muscle had a lower buoyant density than had that from slowly glycolysing muscle. This was reflected in the consistent lack of material in the heavy fraction and a greater proportion in the light fraction. The latter material had significantly lower ratios (w/w) of protein to phospholipid (2.3:1 versus 3.8:1) and of protein to cholesterol (10.4:1 versus 15.6:1). There were no gross differences in phospholipid content or in fatty acid composition of individual phospholipid classes in the membranes from the two types of muscle. Analysis of membrane proteins by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed that ATPase (adenosine triphosphatase) was a major component of each fraction and that its contribution to the total protein content of the membrane was greater in rapidly glycolysing muscle, suggesting a loss of non-ATPase proteins. The two fractions of sarcoplasmic reticulum prepared from rapidly glycolysing muscle had approximately one-third the normal activities of Ca(2+) binding and Ca(2+) uptake in the presence of ATP and one-half the passive Ca(2+)-binding capacity in the absence of ATP of the fractions from slowly glycolysing muscle. However, the (Ca(2+)+Mg(2+))-stimulated ATPase activities were similar. Efflux from actively loaded vesicles, after the addition of EDTA, consisted of a rapid and a slow phase. Vesicles from rapidly glycolysing muscle lost 60% of associated Ca(2+) (approx. 0.10mumol of Ca(2+)/mg of protein) during the rapid phase, compared with 30% (approx. 0.17mumol of Ca(2+)/mg of protein) in those from slowly glycolysing muscle. The efflux rate during the slower phase was comparable in both types of vesicles. Analysis of the temperature-dependence of (Ca(2+)+Mg(2+))-stimulated ATPase activity revealed that a high-activation-energy process operating in the temperature range 31-45 degrees C in the intermediate and light fractions from slowly glycolysing muscle was not apparent in vesicles from rapidly glycolysing muscle. Conditions that result in the prolonged activation of glycogenolysis in pig muscle post mortem primarily affect the protein components of the sarcoplasmic-reticular membrane, giving rise to a loss of loosely associated proteins. The function of the membranes observed under these conditions does not appear to be due to enhanced permeability of the membrane to Ca(2+) and may be the result of a defect in the transport of Ca(2+) into the vesicles.     

Atrial natriuretic peptide attenuates elevations in Ca2+ and protects hepatocytes by stimulating net plasma membrane Ca2+ efflux

Elevations in intracellular Ca(2+) concentration and calpain activity are common early events in cellular injury, including that of hepatocytes. Atrial natriuretic peptide is a circulating hormone that has been shown to be hepatoprotective. The aim of this study was to examine the effects of atrial natriuretic peptide on potentially harmful elevations in cytosolic free Ca(2+) and calpain activity induced by extracellular ATP in rat hepatocytes. We show that atrial natriuretic peptide, through protein kinase G, attenuated both the amplitude and duration of ATP-induced cytosolic Ca(2+) rises in single hepatocytes. Atrial natriuretic peptide also prevented stimulation of calpain activity by ATP, taurolithocholate, or Ca(2+) mobilization by thapsigargin and ionomycin. We therefore investigated the cellular Ca(2+) handling mechanisms through which ANP attenuates this sustained elevation in cytosolic Ca(2+). We show that atrial natriuretic peptide does not modulate the release from or re-uptake of Ca(2+) into intracellular stores but, through protein kinase G, both stimulates plasma membrane Ca(2+) efflux from and inhibits ATP-stimulated Ca(2+) influx into hepatocytes. These findings suggest that stimulation of net plasma membrane Ca(2+) efflux (to which both Ca(2+) efflux stimulation and Ca(2+) influx inhibition contribute) is the key process through which atrial natriuretic peptide attenuates elevations in cytosolic Ca(2+) and calpain activity. Moreover we propose that plasma membrane Ca(2+) efflux is a valuable, previously undiscovered, mechanism through which atrial natriuretic peptide protects rat hepatocytes, and perhaps other cell types, against Ca(2+)-dependent injury.     

Plasma membrane Ca(2+)-pump ATPase is not a substrate for cGMP-dependent protein kinase.

A plasma membrane Ca(2+)-pump ATPase preparation purified from porcine aorta was incubated with cGMP-dependent protein kinase (G-kinase) under the conditions under which dose-dependent stimulation of the enzyme by G-kinase was observed. Several proteins were phosphorylated, but two isoforms of plasma membrane Ca(2+)-pump ATPase with molecular masses of 135- and 145-kDa were not phosphorylated. The protein that was phosphorylated by G-kinase and identified in our previous study as the 135-kDa isoform of Ca(2+)-pump ATPase, on the basis of its almost identical mobility on SDS-PAGE, was found to be another protein with a molecular mass of 138 kDa. Fractionation of the enzyme preparation after incubation with G-kinase by a newly developed calmodulin affinity chromatographic method resulted in the separation of all the G-kinase substrates from the two isoforms of plasma membrane Ca(2+)-pump ATPase. These results suggest that the direct phosphorylation of the Ca(2+)-pump ATPase does not occur in association with the stimulation of the plasma membrane Ca(2+)-pump ATPase by G-kinase.     

Cellular distribution of three mammalian Ca2+-binding proteins related to Torpedo calelectrin.

Addition of Ca2+ to post-microsomal fractions of bovine adrenal or liver produced a sedimentable complex of membrane vesicles and cytoplasmic proteins. Proteins with apparent mol. wts. 70 000, 36 000 and 32 500 were solubilized from this complex by Ca2+ chelation. The 36 000 mol. wt. protein (p36) was immunoprecipitated by an antiserum specific for pp36, a major substrate for Rous sarcoma virus src-gene tyrosine kinase. This protein was present in many mesenchymal cells and associated with membrane cytoskeleton of bovine fibroblasts in a Ca2+-dependent manner. The 70 000 and 32 500 mol. wt. proteins were widely distributed in established cell lines, but were not clearly associated with cell organelles in tissue sections, nor retained in cytoskeleton preparations. On immunoblots p36 reacted strongly with antibodies produced against the electric fish protein Torpedo calelectrin and the similar Ca2+-binding properties and subunit mol. wts. of these proteins suggests that they might be functionally related. Since Torpedo calelectrin, p70, p36 and p32.5 were bound by lipid vesicles or microsomal membranes at micromolar free Ca2+ concentrations, regulated association with intrinsic membrane components may be involved in the functions of these widespread proteins.     

Intra- and intercellular Ca(2+)-transient propagation in normal and high glucose solutions in ROS cells during mechanical stimulation

Experiments using confocal laser microscopy on the rat osteosarcoma cell line (ROS 17/2.8) indicate that mechanical stimulation elicits pronounced [Ca2+](i)transients in the MS (mechanically stimulated) cell, which then propagate to the NB (neighbouring) cells. Experiments with Ca(2+)-free solutions or gadolinium suggest that Ca(2+)-influx through stretch-sensitive channels is required. When intracellular stores are depleted with thapsigargin, mechanical stimulation was able to evoke a Ca(2+)transient of reduced amplitude that disappeared entirely after subsequent blocking of Ca(2+)-influx. Heptanol inhibited intercellular propagation of the Ca(2+)transient, demonstrating the involvement of gap junctions in the propagation of the Ca(2+)transient in ROS cells. PKC activation has only a small inhibitory effect, while inhibition of PKC or tyrosine kinase was ineffective. PKA activation reduced the amplitude of the [Ca2+](i)-rise in NB cells, and decreased the percentage of responsive cells. Cells grown in 50mM glucose for 72h presented only a very limited decrease of the Ca(2+)-rise during mechanical stimulation in the MS and NB cells compared to control conditions. PKC downregulation in high glucose did not modulate this effect.The results of our experiments indicate that PKC or sustained high glucose concentrations do not affect gap junctional communication in ROS cells, while activation of PKA has an inhibitory effect. This might indicate that osteoblastic dysfunction in diabetes could be directly related to the high glucose concentrations and not to inhibition of the intercellular communication.     

Neuronal Ca(2+) sensor 1. Characterization of the myristoylated protein, its cellular effects in permeabilized adrenal chromaffin cells, Ca(2+)-independent membrane association, and interaction with binding proteins, suggesting a role in rapid Ca(2+) signal transduction.

Overexpression of frequenin and its orthologue neuronal Ca(2+) sensor 1 (NCS-1) has been shown to increase evoked exocytosis in neurons and neuroendocrine cells. The site of action of NCS-1 and its biochemical targets that affect exocytosis are unknown. To allow further investigation of NCS-1 function, we have demonstrated that NCS-1 is a substrate for N-myristoyltransferase and generated recombinant myristoylated NCS-1. The bacterially expressed NCS-1 shows Ca(2+)-induced conformational changes. The possibility that NCS-1 directly interacts with the exocytotic machinery to enhance exocytosis was tested using digitonin-permeabilized chromaffin cells. Exogenous NCS-1 was retained in permeabilized cells but had no effect on Ca(2+)-dependent release of catecholamine. In addition, exogenous NCS-1 did not regulate cyclic nucleotide levels in this system. These data suggest that the effects of NCS-1 seen in intact cells are likely to be due to an action on the early steps of stimulus-secretion coupling or on Ca(2+) homeostasis. Myristoylated NCS-1 bound to membranes in the absence of Ca(2+) and endogenous NCS-1 was tightly membrane-associated. Using biotinylated NCS-1, a series of specific binding proteins were detected in cytosol, chromaffin granule membrane, and microsome fractions of adrenal medulla. These included proteins distinct from those detected by biotinylated calmodulin, demonstrating the presence of multiple specific Ca(2+)-independent and Ca(2+)-dependent binding proteins as putative targets for NCS-1 action. A model for NCS-1 function, from these data, indicates a constitutive membrane association independent of Ca(2+). This differs from the Ca(2+) myristoyl switch model for the closely related recoverin and suggests a possible action in rapid Ca(2+) signal transduction in response to local Ca(2+) signals.     

Sperm protein (sp50) binds to acromosome and plasma membranes in a Ca2+-dependent manner: Possible role in acrosome reaction

Annexins are a family of Ca²⁺-binding proteins involved in the exocytotic process. The presence and the role of annexins in mammalian spermatozoa have not been well established. Two annexin-like proteins were obtained from guinea pig testis, a doublet of Mr 31–33 kD (p31/33) and a protein of Mr 50 kD (p50). Both proteins were able to bind to erythrocyte ghosts in a Ca²⁺-dependent fashion. Polyclonal antibodies against p31/33 reacted with two major proteins, Mrs 50 kD (sp50) and 42 kD (sp42), from mature and immature guinea pig spermatozoa. p50 and sp50 are likely the native proteins from testis and spermatozoa, respectively, and they are seemingly related. By immunofluorescence, sp50 was only found in the apical acrosome region of immature and capacitated and noncapacitated spermatozoa, and its location was intracellular. In spermatozoa undergoing acrosome reaction, sp50 was detected in the whole acrosome, while in spermatozoa that had undergone acrosome reaction sp50 was not detected. However, in the protein pattern of acrosome reaction vesicles, anti-p31/33 antibody revealed diffuse bands of Mr 35–38 kD. sp50 was able to bind to plasma membrane fragments and acrosome outer membrane from demembranated sperm in a Ca²⁺-dependent fashion. The presence of sp50 in the acrosome region, its distribution throughout the acrosome membrane just before the acrosome reaction, and its ability to bind both plasma and outer acrosome membranes in a Ca²⁺-dependent manner suggest that sp50 may participate in the acrosome reaction mechanism in guinea pig spermatozoa. © 1996 Wiley-Liss, Inc.     

Membrane-bound annexin V isoforms (CaBP33 and CaBP37) and annexin VI in bovine tissues behave like integral membrane proteins.

The distribution of annexin V isoforms (CaBP33 and CaBP37) and of annexin VI in bovine lung, heart, and brain subfractions was investigated with special reference to the fractions of these proteins which are membrane-bound. In addition to EGTA-extractable pools of the above proteins, membranes from lung, heart, and brain contain EGTA-resistant annexins V and VI which can be solubilized with detergents (Triton X-100 or Triton X-114). A strong base like Na2CO3, which is usually effective in extracting membrane proteins, only partially solubilizes the membrane-bound, EGTA-resistant annexins analyzed here. Also, only 50-60% of the Triton X-114-soluble annexins partition in the aqueous phase, the remaining fractions being recovered in the detergent-rich phase. Altogether, these findings suggest that, by an as yet unknown mechanism, following Ca(2+)-dependent association of annexin V isoforms and annexin VI with membranes, substantial fractions of these proteins remain bound to membranes in a Ca(2+)-independent way and behave like integral membrane proteins. These results further support the possibility that the above annexins might play a role in membrane trafficking and/or in the regulation of the structural organization of membranes.     

Mechanism of intracellular Ca(2+)-wave propagation elicited by mechanical stimulation in cultured endothelial CPAE cells

Intra- and intercellular Ca(2+)-signaling during mechanical stimulation in calf pulmonary artery endothelial cells (CPAE) was investigated with digital fluorescence microscopy. Mechanical stimulation of a CPAE cell in a Ca(2+)-containing solution revealed a rise of the free intracellular Ca(2+)-concentration ([Ca(2+)](i)) in the mechanically stimulated cell (MS) proceeding to the neighboring (NB) cells as an intercellular Ca(2+)-wave. Experiments in Ca(2+)-free solution, containing 2mM EGTA, demonstrated that a detectable [Ca(2+)](i)-transient in the MS cell is not always a requisite for intercellular communication (IC). The Ca(2+)-wave propagation was not affected by changes in membrane potential and was not mediated by voltage-dependent Ca(2+)-channels. Ca(2+)-influx through the Ni(2+)-sensitive Ca(2+)-pathway occurred in the MS as could be assessed by Mn(2+)-quenching experiments. The intra- and intercellular Ca(2+)-wave was triggered by the release of thapsigargin-sensitive intracellular Ca(2+)-stores. Phospholipase C (PLC) inhibition by U73122 reduced the Ca(2+)-amplitude of the MS cell and almost completely inhibited the IC, indicating that the Ca(2+)-release in the MS and NB cells is PLC/inositol 1,4,5-trisphosphate (IP(3)) mediated.     

Isolation and characterization of recoverin-like Ca(2+)-binding protein from rat brain.

Rat brain was found, by immunoblot analysis, to have a protein of Mr 23,000 (P23k) that was clearly different from recoverin and was labeled with an antiserum raised against the NH2-terminus of recoverin. P23k could not be detected by an antiserum raised against the COOH-terminus of recoverin. Blots with 45Ca demonstrated that P23k bound Ca2+. This calciprotein was further purified by Ca(2+)-dependent hydrophobic interaction and ion-exchange chromatography. In SDS polyacrylamide gel electrophoresis, P23k had an apparent Mr of 21,000 in the presence of 10 microM Ca2+ and 23,000 in the absence of Ca2+ (0.1 mM EGTA). The isoelectric point of P23k was 5.6. Ca(2+)-binding analysis indicated that P23k bound 2 moles of Ca2+ per mole of protein and had two binding sites with dissociation constants of 13 microM and 0.2 microM. Purified P23k bound to the crude membrane fractions from the cerebellum, cerebrum and retina in a Ca(2+)-dependent manner. Partial amino acid sequence analysis of proteolytic fragments of P23k revealed the sequence homology between P23k and recoverin. These results suggested that P23k may act as a Ca(2+)-sensitive regulator by forming a complex with its target on the membrane.     

Development and dissipation of Ca(2+) gradients in adrenal chromaffin cells

We used pulsed laser imaging to measure the development and dissipation of Ca(2+) gradients evoked by the activation of voltage-sensitive Ca(2+) channels in adrenal chromaffin cells. Ca(2+) gradients appeared rapidly (<5 ms) upon membrane depolarization and dissipated over several hundred milliseconds after membrane repolarization. Dissipation occurred with an initial fast phase, as the steep gradient near the membrane collapsed, and a slower phase as the remaining shallow gradient dispersed. Inhibition of active Ca(2+) uptake by the endoplasmic reticulum (thapsigargin) and mitochondria (carbonylcyanide p-trifluoro-methoxyphenylhydrazone/oligomycin) had no effect on the size of Ca(2+) changes or the rate of gradient dissipation, suggesting that passive endogenous Ca(2+) buffers are responsible for the slow Ca(2+) redistribution. We used a radial diffusion model incorporating Ca(2+) diffusion and binding to intracellular Ca(2+) buffers to simulate Ca(2+) gradients. We included a 3D optical sectioning model, simulating the effects of out-of-focus light, to allow comparison with the measured gradients. Introduction of a high-capacity immobile Ca(2+) buffer, with a buffer capacity on the order of 1000 and appropriate affinity and kinetics, approximated the size of the Ca(2+) increases and rate of dissipation of the measured gradients. Finally, simulations without exogenous buffer suggest that the Ca(2+) signal due to Ca(2+) channel activation is restricted by the endogenous buffer to a space less than 1 microm from the cell membrane.     

Effects of PMCA and SERCA pump overexpression on the kinetics of cell Ca(2+) signalling

The dynamic interactions of the main pathways for active Ca(2+) transport have been analysed in living cells by altering the expression of their components. The plasma membrane (PMCA) and the endoplasmic reticulum (ER) (SERCA) Ca(2+) pumps were transiently overexpressed in CHO cells, and the Ca(2+) homeostasis in the subcellular compartments was investigated using specifically targeted chimaeras of the Ca(2+)- sensitive photoprotein aequorin. In resting cells, overexpression of the PMCA and SERCA pumps caused a reduction and an increase in ER [Ca(2+)] levels, respectively, while no significant differences were detected in cytosolic and mitochondrial [Ca(2+)]. Upon stimulation with an inositol 1,4, 5-trisphosphate (IP(3))-generating agonist, the amplitude of the mitochondrial and cytosolic Ca(2+) rises correlated with the ER [Ca(2+)] only up to a threshold value, above which the feedback inhibition of the IP(3) channel by Ca(2+) appeared to be limiting.     

The calmodulin-stimulated (Ca2+ + Mg2+)-ATPase in hemoglobin S erythrocyte membranes: effects of sickling and oxidative agents

A decrease in the reactivity of erythrocyte membrane (Ca2+ + Mg2+)-ATPase to calmodulin stimulation has been observed in aging red cells and in various types of hemolytic anemias, particularly in sickle red cell membranes. Unlike the aging process, the defect in the (Ca2+ + Mg2+)-ATPase from SS red blood cells is not secondary to a decrease in calmodulin activity and is already present in the least dense SS red blood cells separated on a discontinuous density gradient. Deoxygenated AS red cells were forced to sickle by lowering the pH, raising the osmolarity of the buffer (sickling pulse). Under these conditions an inhibition of the calmodulin-stimulated enzyme was observed only if several cycles of oxygenation/deoxygenation were applied. No alteration of the enzyme could be detected after submitting AS red blood cells to other conditions or in AA red blood cells submitted to the same treatments. This suggests that oxidative processes are involved in the alterations of the (Ca2+ + Mg2+)-ATPase activity. Treatment of membranes from AA erythrocytes by thiol group reagents and malondialdehyde, a by-product of auto-oxidation of membrane unsaturated lipids and a cross-linking agent of cytoskeletal proteins, led to a partial inhibition of the calmodulin-stimulated (Ca2+ + Mg2+)-ATPase. We postulate that the hyperproduction of free radicals described in the SS red blood cells and involved in the destabilization of the membrane may be also responsible for the (Ca2+ + Mg2+)-ATPase failure.     

Effect of Zinc on Calmodulin-Stimulated Protein Kinase II and Protein Phosphorylation in Rat Cerebral Cortex

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.     

Mechanism regulating Ca2+-dependent mechanosensory behaviour in sea urchin spermatozoa

Flagellar movement of the sea urchin sperm is regulated by intracellular Ca(2+). Flagellasialin, a polysialic acid-containing glycoprotein, as well as other membrane proteins seems responsible for the Ca(2+) control. To elucidate the mechanism of Ca(2+) dynamics underlying flagellar movement, we analysed the sperm's mechanosensory behavioural responses by using microtechniques. In sea water containing 10 mM Ca(2+), the sperm swim in circular paths. When a mechanical stimulus was applied to the sperm head with a glass microstylus, the sperm showed a series of flagellar responses, consisting of a stoppage of beating (quiescence) and a recovery of swimming in a straight path, followed by swimming in a circular path again; as the result the sperm avoided the obstacle. Ca(2+)-imaging with Fluo-4 showed that the intracellular Ca(2+) was high in the quiescence and gradually decreased after that. The effects of blockers and antibodies against candidate components revealed that the Ca(2+) influx was induced by Ca(2+) channels and the Ca(2+) efflux was induced by a flagellasialin-related Ca(2+)-efflux system, plasma membrane Ca(2+)-ATPases and the K(+)-dependent Na(+)/Ca(2+) exchanger. The results show that the Ca(2+)-dependent mechanosensory behaviour of the sea urchin sperm is regulated by organized functioning of the membrane environment including the plasma membrane proteins and flagellasialin.