The use of permeabilized cells to study the ion requirements of receptor-ligand dissociation in endosomes.

The mannose receptor mediates the transport of high-mannose glycoproteins from the cell surface to lysosomes in macrophages. The binding of ligand to the receptor is dependent on both pH and Ca2+. Upon internalization, ligands enter an acidic pre-lysosomal compartment where receptor-ligand dissociation takes place. Acidification is driven by an endosomal proton pump and anion transport is coupled to this acidification step. A permeabilized-cell assay has been designed to characterize the ionic requirements for receptor-ligand dissociation in endosomes. The plasma membrane of macrophages has been permeabilized selectively with digitonin without affecting endosomal membranes. Receptor-ligand dissociation in permeabilized cells required ATP and was blocked by proton ionophores. Di-isothiocyanostilbene-disulphonic acid and N-ethylmaleimide also blocked dissociation, but mitochondrial ATPase inhibitors and vanadate were ineffective. To explore the nature of the anion requirement for acidification, the ability of different anions to compensate for Cl- was tested. For the halide series, Br- was as equally effective as Cl- in supporting receptor-ligand dissociation, but I- was inhibitory. Citrate and gluconate were only partially effective, while SO4(2-), NO3- and PO4(2-) blocked dissociation. Addition of Ca2+ to permeabilized-cell preparations impaired ATP-dependent dissociation without affecting endosome acidification. These results suggest that the endosomal membrane has a Ca2+ conductance that would permit the rapid efflux of Ca2+ from endosomes during acidification, and this would appear to be a necessary step for efficient sorting of Ca2+-dependent receptors from their ligands.     

Activation of decarboxyfactor X by a protein from Russell's viper venom. Purification and partial characterization of activated decarboxyfactor X.

1. Incubation of decarboxyfactor X with the factor X-activating enzyme from Russell's Viper venom revealed the generation of amidase activity towards Bz-Ile-Glu-Gly-Arg-pNA, but not of activity in blood coagulation. 2. The rate of activation of both factor X and decarboxyfactor X depends on the ability of the zymogens to bind Ca2+. The relationship between Ca2+ concentration and velocity of the activation reaction is sigmoid in the case of factor X, but hyperbolic with decarboxyfactor X. 3. Activated decarboxyfactor X was purified by powder column electrophoresis. 4. Identical changes of primary structure accompanied the activation of factor X and decarboxyfactor X. Identical molecular weight and common antigenic determinants were found in factor Xa and decarboxyfactor Xa. The amino acid composition was identical except for 12 glutamic acid residues in decarboxyfactor Xa and gamma-carboxyglutamic acid residues in factor Xa. 5. Unlike factor X, activated factor X has a very low electrophoretic mobility in the presence of Ca2+ at pH 8.6. This is probably due to self association of factor Xa under the influence of Ca2+. The electrophoretic mobility of activated decarboxyfactor X is only slightly decreased compared to decarboxyfactor X in the presence of Ca2+.     

Differential sensitivity of human platelet P2X1 and P2Y1 receptors to disruption of lipid rafts

ATP-stimulated P2X1 and ADP-stimulated P2Y1 receptors play important roles in platelet activation. An increase in intracellular Ca2+ represents a key signalling event coupled to both of these receptors, mediated via direct gating of Ca2+-permeable channels in the case of P2X1 and phospholipase-C-dependent Ca2+ mobilisation for P2Y1. We show that disruption of cholesterol-rich membrane lipid rafts reduces P2X1 receptor-mediated calcium increases by approximately 80%, while P2Y1 receptor-dependent Ca2+ release is unaffected. In contrast to artery, vas deferens, bladder smooth muscle, and recombinant expression in cell lines, where P2X1 receptors show almost exclusive association with lipid rafts, only approximately 20% of platelet P2X1 receptors are co-expressed with the lipid raft marker flotillin-2. We conclude that lipid rafts play a significant role in the regulation of P2X1 but not P2Y1 receptors in human platelets and that a reserve of non-functional P2X1 receptors may exist.     

Stimulus response coupling in the human neutrophil. I. Kinetic analysis of changes in calcium permeability

The relationship between receptor-ligand interaction in human neutrophils and initiation of enhanced Ca permeability ("45Ca uptake") has been correlated with cell function. Different ligands varied in their efficacy in provoking 45Ca permeability changes: chemotactic peptide f-Met-Leu-Phe greater than concanavalin A greater than immune complexes greater than phorbol myristate acetate. Mixtures of stimuli at optimal concentrations elicited no summation of responses, indicating that interaction of f-Met-Leu-Phe, concanavalin A, and phorbol myristate acetate with their respective "receptors" regulates a common site of 45Ca uptake. The onset of Ca uptake was an early event, preceding onset of aggregation, O-2 generation, and degranulation. Enhanced 45Ca permeability during neutrophil activation is dependent on mobilization of intracellular Ca, since 8-(N,N-diethylamino)-octyl:3,4,5-trimethoxybenzoate hydrochloride was a potent inhibitor of the Ca permeability response. In contrast, calmodulin antagonists did not inhibit Ca permeability changes; the requirement for calmodulin in the physiological responses of aggregation, O-2 generation, and degranulation must therefore be subsequent to activation of the "Ca translocator." We propose a role for a "Ca-translocating mechanism" as an amplifying factor in neutrophil activation.     

Receptor regulation of calcium release and calcium permeability in parotid gland cells

The mechanism by which hormones and neurotransmitters regulate fluid secretion in exocrine glands apparently involves the regulation of transmembrane movements of electrolytes, a process for which Ca serves as a second messenger. Analysis of the kinetics of efflux of 86Rb+ (a marker for K+) indicates that the initial phase of the response to secretagogues is mediated through the release of Ca from a cellular pool inaccessible to chelating agents. By investigating the movements of 45Ca under nearly steady-state conditions, we find that this cellular pool can be filled from the extracellular space without a concomitant elevation in ionized intracellular Ca2+. This suggests that the cellular pool is probably associated with the plasma membrane. We have also investigated the possible role of phosphatidic acid in the mechanism by which receptors mobilize Ca2+. Our results suggest that phosphatidic acid, formed on receptor activation, may directly mediate Ca influx into the acinar cell.     

Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.

A rapid rise in the level of cytosolic free calcium ([Ca2+]i) is believed to be one of several early triggering signals in the activation of T lymphocytes by antigen. Although Ca2+ release from intracellular stores and its contribution to Ca2+ signaling in many cell types is well documented, relatively little is known regarding the role and mechanism of Ca2+ entry across the plasma membrane. We have investigated mitogen-triggered Ca2+ signaling in individual cells of the human T-leukemia-derived line, Jurkat, using fura-2 imaging and patch-clamp recording techniques. Phytohemagglutinin (PHA), a mitogenic lectin, induces repetitive [Ca2+]i oscillations in these cells peaking at micromolar levels with a period of 90-120 s. The oscillations depend critically upon Ca2+ influx across the plasma membrane, as they are rapidly terminated by removal of extracellular Ca2+, addition of Ca(2+)-channel blockers such as Ni2+ or Cd2+, or membrane depolarization. Whole-cell and perforated-patch recording methods were combined with fura-2 measurements to identify the mitogen-activated Ca2+ conductance involved in this response. A small, highly selective Ca2+ conductance becomes activated spontaneously in whole-cell recordings and in response to PHA in perforated-patch experiments. This conductance has properties consistent with a role in T-cell activation, including activation by PHA, lack of voltage-dependent gating, inhibition by Ni2+ or Cd2+, and regulation by intracellular Ca2+. Moreover, a tight temporal correlation between oscillations of Ca2+ conductance and [Ca2+]i suggests a role for the membrane Ca2+ conductance in generating [Ca2+]i oscillations in activated T cells.     

P2X7 nucleotide receptor mediation of membrane pore formation and superoxide generation in human promyelocytes and neutrophils

The P2X(7) receptor, which induces cation channel opening imparting significant permeability to Ca2+ and pore formation with changes in the plasma membrane potential, has been known to be rather restrictedly expressed in cells of the macrophage lineage including dendrites, mature macrophages, and microglial cells. However, we show here that the P2X(7) receptor is also expressed in cells of granulocytic lineage such as HL-60 promyelocytes, granulocytic differentiated cells, and neutrophils. Exposure of these cells to 2',3'-O-(4-benzoyl)benzoyl-ATP (BzATP) triggered intracellular Ca2+ rise through the mediation of phospholipase C-independent and suramin-sensitive pathways. BzATP also induced depolarization of the plasma membrane in the absence of extracellular Ca2+, whereas it hyperpolarized the cells in the presence of external Ca2+, probably in part through the activation of Ca2+-activated K(+) channels. However, the hyperpolarization phenomenon was markedly attenuated in differentiated HL-60 cells and neutrophils. RT-PCR and Northern blot analysis revealed the presence of P2X(7) receptors on both HL-60 and neutrophil-like cells. This was further confirmed by pore formation through which the uptake of Lucifer yellow and YO-PRO1 occurred on BzATP treatment. BzATP stimulated in a concentration-dependent manner the production of superoxide in differentiated HL-60 cells via a pathway partially dependent on extracellular Ca2+. Moreover, in human neutrophils, BzATP was a more effective inducer of superoxide generation than PMA. Taken together, this is a first demonstration of the expression of P2X(7) receptors on neutrophils, which shows that the receptor is functionally involved in the defense mechanism by activation of the respiratory burst pathway.     

Simultaneous measurement of Ca2+ release and influx into smooth muscle cells in response to caffeine. A novel approach for calculating the fraction of current carried by calcium

Activation of ryanodine receptors on the sarcoplasmic reticulum of single smooth muscle cells from the stomach muscularis of Bufo marinus by caffeine is accompanied by a rise in cytoplasmic [Ca2+] ([Ca2+]i), and the opening of nonselective cationic plasma membrane channels. To understand how each of these pathways contributes to the rise in [Ca2+]i, one needs to separately monitor Ca2+ entry through them. Such information was obtained from simultaneous measurements of ionic currents and [Ca2+]i by the development of a novel and general method to assess the fraction of current induced by an agonist that is carried by Ca2+. Application of this method to the currents induced in these smooth muscle cells by caffeine revealed that approximately 20% of the current passing through the membrane channels activated following caffeine application is carried by Ca2+. Based on this information we found that while Ca2+ entry through these channels rises slowly, release of Ca2+ from stores, while starting at the same time, is much faster and briefer. Detailed quantitative analysis of the Ca2+ release from stores suggests that it most likely decays due to depletion of Ca2+ in those stores. When caffeine was applied twice to a cell with only a brief (30 s) interval in between, the amount of Ca2+ released from stores was markedly diminished following the second caffeine application whereas the current carried in part by Ca2+ entry across the plasma membrane was not significantly affected. These and other studies described in the preceding paper indicate that activation of the nonselective cation plasma membrane channels in response to caffeine was not caused as a consequence of emptying of internal Ca2+ stores. Rather, it is proposed that caffeine activates these membrane channels either by direct interaction or alternatively by a linkage between ryanodine receptors on the sarcoplasmic reticulum and the nonselective cation channels on the surface membrane.     

Modulation of thymocyte membrane potential by concanavalin A.

Binding of convanavalin A, a potent mitogenic lectin, to thymocyte surface membrane causes depolarization of membrane potential. The effect is suppressed by alpha-methyl-D-glucoside, a haptenic inhibitor of this lectin, or by low temperature. Colchicine and cytochalasin B aslso suppress the change. These data indicate that perturbation of thymocyte surface membrane receptors induced by concanavalin A might be linked to change in the functional state of cellular cytoskeletal systems in turn causing depolarization of thymocyte surface membrane. The initial event generated by receptor-ligand interaction on the outer surface could be translated into cellular interior action only under highly fluid conditions of membrane lipid. Depolarization of thymocyte plasma membrane may be involved in the triggering mechanism of metabolic burst associated with blastoid transformation.     

Essential role for Ca2+ in regulation of IL-1beta secretion by P2X7 nucleotide receptor in monocytes,macrophages, and HEK-293 cells

Interleukin (IL)-1beta is a proinflammatory cytokine that elicits the majority of its biological activity extracellularly, but the lack of a secretory signal sequence prevents its export via classic secretory pathways. Efficient externalization of IL-1beta in macrophages and monocytes can occur via stimulation of P2X7 nucleotide receptors with extracellular ATP. However, the exact mechanisms by which the activation of these nonselective cation channels facilitates secretion of IL-1beta remain unclear. Here we demonstrate a pivotal role for a sustained increase in cytosolic Ca2+ to potentiate secretion of IL-1beta via the P2X7 receptors. Using HEK-293 cells engineered to coexpress P2X7 receptors with mature IL-1beta (mIL-1beta), we show that activation of P2X7 receptors results in a rapid secretion of mIL-1beta by a process(es) that is dependent on influx of extracellular Ca2+ and a sustained rise in cytosolic Ca2+. Moreover, reduction in extracellular Ca2+ attenuates approximately 90% of P2X7 receptor-mediated IL-1beta secretion but has no effect on enzymatic processing of precursor IL-1beta (proIL-1beta) to mIL-1beta by caspase-1. Similar experiments with THP-1 human monocytes and Bac1.2F5 murine macrophages confirm the unique role of Ca2+ in P2X7 receptor-mediated secretion of IL-1beta. In addition, we report that cell surface expression of P2X7 receptors in the absence of external stimulation also results in enhanced release of IL-1beta and that this can be repressed by inhibitors of P2X7 receptors. We clarify an essential role for Ca2+ in ATP-induced IL-1beta secretion and indicate an additional role of P2X7 receptors as enhancers of the secretory apparatus by which IL-1beta is released.     

Non-genomic immunosuppressive actions of progesterone inhibits PHA-induced alkalinization and activation in T cells

Progesterone is an endogenous immunomodulator, and can suppress T-cell activation during pregnancy. When analyzed under a genome time scale, the classic steroid receptor pathway does not have any effect on ion fluxes. Therefore, the aim of this study was to investigate whether the non-genomic effects on ion fluxes by progesterone could immunosuppress phytohemagglutinin (PHA)-induced human peripheral T-cell activation. The new findings indicated that, first, only progesterone stimulated both [Ca2+]i elevation and pHi decrease; in contrast, estradiol or testosterone stimulated [Ca2+]i elevation and hydrocortisone or dexamethasone stimulated pHi decrease. Secondly, the [Ca2+]i increase by progesterone was dependent on Ca2+ influx, and the acidification was blocked by Na+/H+ exchange (NHE) inhibitor, 3-methylsulphonyl-4-piperidinobenzoyl, guanidine hydrochloride (HOE-694) but not by 5-(N,N-dimethyl)-amiloride (DMA). Thirdly, progesterone blocked phorbol 12-myristate 13-acetate (PMA) or PHA-induced alkalinization, but PHA did not prevent progesterone-induced acidification. Fourthly, progesterone did not induce T-cell proliferation; however, co-stimulation progesterone with PHA was able to suppress PHA-induced IL-2 or IL-4 secretion and proliferation. When progesterone was applied 72 h after PHA stimulation, progesterone could suppress PHA-induced T-cell proliferation. Finally, immobilization of progesterone by conjugation to a large carrier molecule (BSA) also stimulated a rapid [Ca2+]i elevation, pHi decrease, and suppressed PHA-induced proliferation. These results suggested that the non-genomic effects of progesterone, especially acidification, are exerted via plasma membrane sites and suppress the genomic responses to PHA. Progesterone might act directly through membrane specific nonclassical steroid receptors to cause immunomodulation and suppression of T-cell activation during pregnancy.     

Annexin A6 is an organizer of membrane microdomains to regulate receptor localization and signalling

Annexin A6 (AnxA6) belongs to the conserved annexin protein family--a group of Ca(2+) -dependent membrane binding proteins. It is the largest of all annexin proteins and upon activation, binds to negatively charged phospholipids in the plasma membrane and endosomes. In addition, AnxA6 associates with cholesterol-rich membrane microdomains termed lipid rafts. Membrane cholesterol triggers Ca(2+) -independent translocation of AnxA6 to membranes and AnxA6 levels determine the number of caveolae, a form of specialized rafts at the cell surface. AnxA6 also has an F-actin binding domain and interacts with cytoskeleton components. Taken together, this suggests that AnxA6 has a scaffold function to link membrane microdomains with the organization of the cytoskeleton. Such a link facilitates AnxA6 to participate in plasma membrane repair and it would also impact on receptor signalling at the cell surface, growth factor, and lipoprotein receptor trafficking, Ca(2+) -channel activity and T cell activation. Hence, the regulation of cell surface receptors by AnxA6 may be facilitated by its unique structure that allows recruitment of interaction partners and simultaneously bridging specialized membrane domains with cortical actin surrounding activated receptors.     

Interaction of CD2 with its ligand, LFA-3, in human T cell proliferation

Recently, it has been demonstrated that lymphocyte function-associated Ag (LFA-3) is a natural ligand for CD2 and that this receptor-ligand interaction functions in cell-cell adhesion. In this report, we demonstrate that LFA-3 plays a role in T cell activation. L cells were transfected with human genomic DNA and sorted for expression of LFA-3. We demonstrate that LFA-3+ L cells, together with anti-CD3 mAb or with suboptimal doses of PHA, stimulate proliferation of human peripheral blood T cells. Furthermore, thymocyte proliferation was induced by LFA-3+ L cells and suboptimal doses of PHA. Proliferation was inhibited by mAb directed against either CD2 or LFA-3. Stimulation of thymocytes by the combination of PHA and LFA-3+ L cells resulted in the increased expression of the IL-2R, as well as of the surface Ag 4F2, transferrin receptor, and HLA-DR. These data support the conclusion that LFA-3 plays a role in CD2-dependent T cell activation. LFA-3 is widely distributed and is expressed on all APC and target cells. Thus, the ability of the CD2/LFA-3 interaction to costimulate with an anti-CD3 mAb suggests that the CD2/LFA-3 interaction may be involved not only in an Ag-independent alternate pathway of T cell activation but also in Ag-specific T cell activation.     

Adhesion frequency assay for in situ kinetics analysis of cross-junctional molecular interactions at the cell-cell interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics to the binding curve returns the 2D affinity and off-rate. The assay has been validated using interactions of Fcγ receptors with IgG Fc, selectins with glycoconjugate ligands, integrins with ligands, homotypical cadherin binding, T cell receptor and coreceptor with peptide-major histocompatibility complexes. The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology, membrane anchor, molecular orientation and length, carrier stiffness, curvature, and impingement force, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules. The method has also been used to study the concurrent binding of dual receptor-ligand species, and trimolecular interactions using a modified model. The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods. To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin α(L)β(2) on neutrophils with dimeric E-selectin in the solution to activate α(L)β(2).     

Fibrinogen binding to human platelet plasma membranes. Identification of two steps requiring divalent cations

Fibrinogen binding to platelet plasma membranes, which is a prerequisite for platelet aggregation, was determined by incubating 125I-labeled fibrinogen with isolated membranes and measuring the amount of radioactivity sedimenting with the membranes through 15% sucrose. Fibrinogen binding was optimal at 10(-3) M Ca2+. Scatchard analyses of the fibrinogen binding showed that the membrane capacity for fibrinogen was 1.6 X 10(-12) mol/mg of membrane protein, with a dissociation constant (Kd) = 1.2 X 10(-8) M. When Ca2+ levels were manipulated by the addition of varying amounts of EGTA at a fixed Mg2+ concentration of 3 X 10(-3) M, specific binding of fibrinogen to platelet membranes occurred only at Ca2+ concentrations greater than or equal to 10(-6) M. Membranes isolated from platelets of an individual with Glanzmann's thrombasthenia bound only 12% as much fibrinogen as control platelets. The data in the present study suggest that there are two divalent cation binding sites that must be occupied for fibrinogen to bind: one site is specific for calcium and is saturated at 10(-6) M Ca2+; the other site is less specific and is saturated at a 10(-3) M concentration of either Ca2+ or Mg2+. Fibrinogen binding to intact platelets and, consequently, platelet aggregation only required 10(-3) M extracellular divalent cation and was not specific for Ca2+. These data indicate that the cytoplasm is a potential source for the requirement of 10(-6) M Ca2+, and that changes in the intracellular concentration of Ca2+ may cause the expression of fibrinogen receptors during ADP-induced platelet activation.     

Effects of ryanodine in skinned cardiac cells

Ryanodine (1 X 10(-5) M) did not affect the Ca2+ sensitivity of the myofilaments of skinned (sarcolemma removed by microdissection) cardiac cells from the rat ventricle. Ryanodine (1 X 10(-5) M) inhibited three types of Ca2+ release from the sarcoplasmic reticulum (SR), which have different mechanisms: 1) Ca2+-induced release of Ca2+ triggered by a rapid and transient increase of [free Ca2+] at the outer surface of the SR; 2) caffeine-induced release of Ca2+; 3) spontaneous cyclic release of Ca2+ occurring in the continuous presence of a [free Ca2+] sufficient to overload the SR. These results suggest that the three types of Ca2+ release are through the same channel across the SR membrane, although the gating mechanisms are different for the three types. Ryanodine also diminished the rate of Ca2+ accumulation into the SR. Even in the presence of 1 X 10(-5) M ryanodine the SR accumulated Ca2+ that could be released when the SR was sufficiently overloaded with Ca2+. Thus, ryanodine pretreatment did not permit the direct activation of the myofilaments by externally applied Ca2+. The approximately 1000-fold difference in the effective concentrations of ryanodine in intact vs. skinned cardiac cells suggests that low concentrations of ryanodine act in the intact cardiac tissues through processes or on structures that are destroyed by the skinning procedure. No significant differences were observed in the effects of ryanodine in skinned cardiac cells from different adult mammalian species.     

Multistep regulation of Leydig cell function

LH controls Leydig cell steroidogenesis by interaction with specific membrane receptors initiating membrane coupling events. Stimulation of the androgen pathways occurs mainly through cAMP mediated mechanism including LH induced guanyl nucleotide binding, membrane phosphorylation and adenylate cyclase activation. cAMP dependent kinase activation presumably causes phosphorylation of key proteins of the steroidogenic pathway and consequent increase in testosterone production. The hormone also appears to facilitate the androgen stimulus by a cyclic AMP independent mechanism located at the plasma membrane or intracellular sites. The stimulatory event can be negatively influenced by the action of certain peptide hormones (i.e. angiotensin II) through the guanyl nucleotide inhibitory subunit of adenylate cyclase (Gi). In recent studies we have presented evidence for a Ca2+ sensitive kinase system present in purified cell membranes. Gpp(NH)p, GTP, and phospholipid in presence of nanomolar Ca2+ induce phosphate incorporation into Mr 44,500 substrate with marked inhibition at microM Ca2+. Similarly a biphasic pattern of activation was observed with adenylate cyclase activity. Membrane phosphorylation may be a modifier of LH-stimulated adenylate cyclase activity and possibly other LH induced actions in the activated Leydig cell membrane. Furthermore we have defined the stimulatory effects of forskolin on all Leydig cell cyclic AMP pools and have provided additional evidence of functional compartmentalization and/or cAMP independent facilitory stimulus of steroidogenesis by the trophic hormone. The demonstration of a novel high affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation mediated by the Gi subunit of adenylate cyclase has provided a new approach for direct evaluation of functional inhibitory influence of Gi subunit in the Leydig cell. The cultured fetal Leydig cell system has provided a useful model to elucidate mechanisms involved in the development of gonadotropin induced estradiol mediated desensitization of steroidogenesis. We have isolated from the fetal testis a small population (2-5% of total) of transitional cells with morphological characteristics of cells found in 15 day postnatal testis but functional capabilities of the adult cell. We have also demonstrated after appropriate treatment (i.e. estrogen, and frequent or a high gonadotropin dose) the emergence of a functional adult-like cell type from the fetal Leydig cell population.     

Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease

A Ca2+-dependent protease I), which hydrolyzes casein at Ca2+ concentrations lower than the 10(-5) M range, is purified roughly 4000-fold from the soluble fraction of rat brain. This protease is able to activate Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) by limited proteolysis analogously to the previously known Ca2+-dependent analogously to the previously known Ca2+-dependent protease (Ca2+ protease II) which is active at the millimolar range of Ca2+ (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616). The protein kinase fragment thus produced shows a molecular weight of about 5.1 X 10(4), and is significantly smaller than native protein kinase C (Mr = 7.7 X 10(4). Although protein kinase C may be normally activated in a reversible manner by the simultaneous presence of phospholipid and diacylglycerol at Ca2+ concentrations less than 10(-6) M, this enzyme fragment is fully active without any lipid fractions and independent of Ca2+. The limited proteolysis of protein kinase C is markedly enhanced in the velocity by the addition of phospholipid and diacylglycerol, which are both required for the reversible activation of the enzyme. However, casein hydrolysis by this protease is not affected by phospholipid and diacylglycerol. Available evidence suggests that, at lower concentrations of this divalent cation, Ca2+ protease I reacts preferentially with the active form of protein kinase C which is associated with membrane, and converts it to the permanently active form. In contrast, the inactive form of protein kinase C, which is free of membrane phospholipid, does not appear to be very susceptible to the proteolytic attack. It remains unknown, however, whether this mechanism of irreversible activation of protein kinase C does operate in physiological processes. It is noted that Ca2+ protease II, which is active at higher concentrations of Ca2+, proteolytically activates protein kinase C irrespective of the presence and absence of phospholipid and diacylglycerol.     

Characterization of volume-sensitive, calcium-permeating pathways in the osteosarcoma cell line UMR-106-01

Measurements of cell volume changes, free cytosolic Ca2+ concentration [( Ca2+]i) with Fura 2 and cell membrane potential with 3,3'-dipropylthiodicarbocyanine iodide were used to study the effect of cell volume change on Ca2+ influx and the membrane potential of the osteoblastic osteosarcoma cell line, UMR-106-01. Swelling the cells by hypo-osmotic stress was followed by reduction in cell volume which was markedly impaired by removal of medium Ca2+. Accordingly, cell swelling resulted in [Ca2+]i increase only in the presence of medium Ca2+. The cell swelling-activated Ca2+ entry pathway was active at resting membrane potentials, and Ca2+ influx through this pathway markedly increased upon cell hyperpolarization. A linear relationship between Ca2+ entry and the potential across the plasma membrane was observed. Thus, the volume-activated Ca2+ permeating pathway in UMR-106-01 cells has conductive properties. These pathways do not spontaneously inactivate with time when the cells are not allowed to volume regulate. The pathway can be blocked by micromolar concentrations of nicardipine and La3+ but display very low sensitivity to diltiazem and verapamil. Activation of the volume-sensitive, Ca2+ permeating pathway was not dependent on an increase in [Ca2+]i. Likewise, activation of the pathway was independent of a change in membrane potential between -85 and -3 mV. The increase in [Ca2+]i resulted in hyperpolarization of the cells, probably due to activation of Ca2+-activated K+ channels. The volume-sensitive pathways were partially active under isotonic conditions. Their activity was inhibited by cell shrinkage and increased by cell swelling. The pathways were sensitive to small changes in cell volume, particularly around a medium osmolarity of 310 mosM.     

Toll-like receptors as molecular switches

Members of the Toll family of single-pass transmembrane receptors are key mediators of innate immunity in both vertebrates and invertebrates. They respond to various pathogen-associated stimuli and transduce the complex signalling responses that are required for inflammation and for the subsequent development of adaptive immunity. Here, we propose a molecular mechanism for signalling by the Toll and Toll-like receptors that involves a series of protein conformational changes initiated by dimerization of their extracellular domains. The initial dimerization event, which is triggered by the interaction of the receptor with its ligand, might disrupt a pre-formed but non-functional dimer. Formation of a stable receptor-ligand complex then relieves constitutive autoinhibition, enabling receptor-receptor association of the extracellular juxtamembrane regions and cytoplasmic signalling domains. This activation process constitutes a tightly regulated, unidirectional molecular switch.