Synaptic deficits in layer 5 neurons precede overt structural decay in 5xFAD mice.

Synaptic decay and neurodegeneration are hallmarks of Alzheimer's disease that are thought to precede dementia. Recently, we have reported that the first signs of neuritic dystrophy in a new transgenic mouse model of familial Alzheimer's disease (FAD) called the "5xFAD" are axonal dystrophy followed by loss of spines on basal dendrites. The 5xFAD mouse has profound loss of layer 5 neurons by 12months, and these initial structural insults appear between 4 and 6months of age. Here, we test, for the first time, if synaptic failure of layer 5 neurons in the 5xFAD mouse precedes these structural changes. We used longitudinal, in vivo two-photon fluorescence imaging of bigenic 5xFAD/YFP mice to assess the overall structural stability of layer 5 neurons in young mice (age less than 14weeks). We found these neurons to be structurally and morphologically sound. In parallel, we used in vitro, whole-cell patch clamp electrophysiology of layer 5 pyramidal neurons, from mice aged 8-12weeks, to reveal significant pre- and postsynaptic defects in these cells. Thus our data suggest that layer 5 neurons in the 5xFAD mouse model have synaptic deficits at an early time point, before any overt structural dystrophy, and that such synaptic failure, with co-temporal biochemical changes, may be an early step in neuronal loss.

In vitro and in vivo characterisation of anti-murine IL-13 antibodies recognising distinct functional epitopes.

Interleukin-13 (IL-13) sequentially binds to IL-13Ralpha1 and IL-4Ralpha forming a high affinity signalling complex. This receptor complex is expressed on multiple cell types in the airway and signals through signal transducer and activator of transcription factor-6 (STAT-6) to stimulate the production of chemokines, cytokines and mucus. Antibodies have been generated, using the UCB Selected Lymphocyte Antibody Method (UCB SLAM), that block either binding of murine IL-13 (mIL-13) to mIL-13Ralpha1 and mIL-13Ralpha2, or block recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. Monoclonal antibody (mAb) A was shown to bind to mIL-13 with high affinity (K(D) 11 pM) and prevent binding of mIL-13 to mIL-13Ralpha1. MAb B, that also bound mIL-13 with high affinity (K(D) 8 pM), was shown to prevent recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. In vitro, mAbs A and B similarly neutralised mIL-13-stimulated STAT-6 activation and TF-1 cell proliferation. In vivo, mAbs A and B demonstrated equipotent, dose-dependent inhibition of eotaxin generation in mice stimulated by intraperitoneal administration of recombinant mIL-13. In an allergic lung inflammation model in mice, mAbs A and B equipotently inhibited muc5ac mucin mRNA upregulation in lung tissue measured two days after intranasal allergen challenge. These data support the design of therapeutics for the treatment of allergic airway disease that inhibits assembly of the high affinity IL-13 receptor signalling complex, by blocking the binding of IL-13 to IL-13Ralpha1 and IL-13Ralpha2, or the subsequent recruitment of IL-4Ralpha.

EGFRvIII undergoes activation-dependent downregulation mediated by the Cbl proteins.

The overexpression or mutation of tyrosine kinases (TKs), such as the epidermal growth factor receptor (EGFR), can lead to the development of cancer. The most common mutation of the EGFR in glioblastomas is the deletion of exons 2-7 known as the EGFRvIII. This mutant receptor cannot bind EGF but, instead, is constitutively active. The Cbl family of ubiquitin ligases (Cbl, Cbl-b, and Cbl-c) targets the activated EGFR for degradation. As the EGFRvIII is transforming, we investigated whether it could be downregulated by the Cbl proteins. The overexpression of all three Cbl proteins resulted in the ubiquitination and degradation of the EGFRvIII. As with the wild-type EGFR, the TK-binding domain and the RING finger of Cbl-b are sufficient for the downregulation of the EGFRvIII. Also, we found that Cbl-b is recruited to the EGFRvIII and inhibits the transformation of NIH 3T3 cells by the EGFRvIII. Mutation of the Cbl-binding site (Y1045F) in the EGFRvIII inhibits its ubiquitination and downregulation by Cbl-b and enhances its ability to transform. Furthermore, the EGFR TK inhibitor, AG 1478, prevents the downregulation of the EGFRvIII by the Cbl proteins and antagonizes the ability of an immunotoxin directed against the EGFRvIII to kill cells expressing this receptor. In conclusion, the EGFRvIII does not transform by escaping regulation by Cbl proteins and this activation-induced downregulation of the EGFRvIII has an important role in mediating the toxicity of anti-EGFRvIII immunotoxins.

Application of two-photon flash photolysis to reveal intercellular communication and intracellular Ca2+ movements.

Two-photon excitation makes it possible to excite molecules in volumes of much less than 1 fl. In two-photon flash photolysis (TPFP) this property is used to release effector molecules from caged precursors with high three-dimensional resolution. We describe and examine the benefits of using TPFP in model solutions and in a number of cell systems to study their spatial and temporal properties. Using TPFP of caged fluorescein, we determined the free diffusion coefficient of fluorescein (D=4 x 0(-6) cm(2)/s at 20 degrees C, which is in close agreement with published values). TPFP of caged fluorescein in lens tissue in situ revealed spatial nonuniformities in intercellular fiber cell coupling by gap junctions. At the lens periphery, intercellular transport was predominantly directed along rows of cells, but was nearly isotropic further from the periphery. To test an algorithm aiming to reconstruct the Ca(2+) release flux underlying physiological Ca(2+) signals in heart muscle cells, TPFP of DM-Nitrophen was utilized to generate artificial microscopic Ca(2+) signals with known underlying Ca(2+) release flux. In an experiment with mouse oocytes, the recently developed Ca(2+) cage dimethoxynitrophenyl-ethyleneglycol-bis-(beta-aminoethylether)-N,N,N('),N(') tetraacetic acid-4 (DMNPE-4) was released in the oocyte cytosol and inside a nucleolus. Analysis of the resulting fluorescence changes suggested that the effective diffusion coefficient within the nucleolus was half of that in the cytosol. These experiments demonstrate the utility of TPFP as a novel tool for the optical study of biomedical systems.

Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons.

Dendritic spines serve as preferential sites of excitatory synaptic connections and are pleomorphic. To address the structure-function relationship of the dendritic spines, we used two-photon uncaging of glutamate to allow mapping of functional glutamate receptors at the level of the single synapse. Our analyses of the spines of CA1 pyramidal neurons reveal that AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptors are abundant (up to 150/spine) in mushroom spines but sparsely distributed in thin spines and filopodia. The latter may be serving as the structural substrates of the silent synapses that have been proposed to play roles in development and plasticity of synaptic transmission. Our data indicate that distribution of functional AMPA receptors is tightly correlated with spine geometry and that receptor activity is independently regulated at the level of single spines.

Ion selectivities of the Ca(2+) sensors for exocytosis in rat phaeochromocytoma cells.

1. The ion selectivities of the Ca(2+) sensors for the two components of exocytosis in rat phaeochromocytoma (PC12) cells were examined by measurement of membrane capacitance and amperometry. The cytosolic concentrations of metal ions were increased by photolysis of caged-Ca(2+) compounds and measured with low-affinity indicators benzothiazole coumarin (BTC) or 5-nitrobenzothiazole coumarin (BTC-5N). 2. The Ca(2+)-induced increases in membrane capacitance comprised two phases with time constants of 30--100 ms and 5 s. Amperometric events reflecting the exocytosis of large dense-core vesicles occurred selectively in the slow phase, even with increases in the cytosolic Ca(2+) concentration of > 0.1 mM. 3. The slow component of exocytosis was activated by all metal ions investigated, including Cd(2+) (median effective concentration, 18 pM), Mn(2+) (500 nM), Co(2+) (900 nM), Ca(2+) (8 microM), Sr(2+) (180 microM), Ba(2+) (280 microM) and Mg(2+) (> 5 mM). In contrast, the fast component of exocytosis was activated by Cd(2+) (26 pM), Mn(2+) (620 nM), Ca(2+) (24 microM) and Sr(2+) (320 microM), but was only slightly increased by Ba(2+) (> 2 mM) and Co(2+) and not at all by Mg(2+). 4. The fast component, but not the slow component, was competitively blocked by Na(+) (median effective concentration, 44 mM) but not by Li(+), K(+) or Cs(+). Thus, the Ca(2+) sensor for the fast component of exocytosis is more selective than is that for the slow component; moreover, this selectivity appears to be based on ionic radius, with cations with radii of 0.84 to 1.13 A (1 A = 0.1 nm) being effective. 5. These data support a role for synaptotagmin--phospholipid as the Ca(2+) sensor for the exocytosis of large dense-core vesicles and they suggest that an additional Ca(2+)-sensing mechanism operates in the synchronous exocytosis of synaptic-like vesicles.

Endometrial response to raloxifene compared with placebo, cyclical hormone replacement therapy, and unopposed estrogen in postmenopausal women.

OBJECTIVE: To determine the endometrial effects of raloxifene 60 mg/day in postmenopausal women as assessed by vaginal bleeding and endometrial thickness. DESIGN: Data from 1157 postmenopausal women were analyzed from a database consisting of four independent, double-blind, randomized, placebo-controlled trials (range = 6-30 months duration), a 24-month open-label randomized, cyclical hormone replacement therapy (HRT)-controlled trial, and a 6-month double-blind, randomized, unopposed estrogen-controlled trial. Vaginal bleeding rate was derived from self-reported adverse events collected at least every 6 months. Endometrial thickness was measured by ultrasonography at regular intervals. RESULTS: Raloxifene 60 mg/day was not significantly different from placebo with regard to the incidence of vaginal bleeding, the baseline-to-endpoint change in endometrial thickness, or the proportion of women experiencing an increase in endometrial thickness above baseline after either 12 or 24 months of therapy. Unexpected bleeding was reported significantly more frequently in the unopposed estrogen groups compared with the raloxifene group (raloxifene 60 mg/day, 0% versus estrogen, 50%; p = 0.002). A significantly greater baseline-to-endpoint increase in endometrial thickness was observed in both the HRT and estrogen groups compared with their respective raloxifene comparison group (raloxifene 60 mg/day, 0.01 +/- 2.0 mm versus HRT, 1.8 +/- 3.2; p < 0.001; raloxifene 60 mg/day, 1.1 +/- 1.7 mm versus estrogen, 7.8 +/- 3.8; p < 0.001). No cases of endometrial hyperplasia or cancer were diagnosed in the placebo or raloxifene 60 mg/day groups. Endometrial hyperplasia was diagnosed in one case in the HRT group and in two cases in the estrogen group. CONCLUSION: Raloxifene 60 mg/day for up to 30 months is not associated with vaginal bleeding or increased endometrial thickness in postmenopausal women.

Adverse events reported by postmenopausal women in controlled trials with raloxifene.

OBJECTIVE: To assess the incidence of adverse events in postmenopausal women treated with raloxifene compared with placebo, hormone replacement therapy (HRT), or unopposed estrogen. METHODS: Common treatment groups were pooled across eight randomized, parallel clinical trials (6-30 months' duration) of raloxifene to create the following three databases: placebo-controlled, HRT-controlled, and estrogen-controlled databases. Incidence and severity of all treatment-emergent adverse events, defined as events that first occurred or worsened during treatment, were compared among groups in each of the databases. RESULTS: Discontinuation rates overall, and those related to adverse events, were not significantly different between treatment groups in any database. There was no significant difference in incidence of vaginal bleeding or breast discomfort between women treated with raloxifene (60 mg/d) or placebo. Both of these events were reported more frequently in women receiving HRT or estrogen. Vaginal bleeding was responsible for significantly more discontinuations from the HRT groups compared with the raloxifene group. Hot flashes was the only event common to all three databases that was significantly increased in the raloxifene group, but this event did not increase the discontinuation rates. The incidence of leg cramps was greater in raloxifene-treated women compared with placebo-treated women in the placebo-controlled database, but did not cause any discontinuations of therapy. Raloxifene had no effect on the incidence of vaginal symptoms or central nervous system events. CONCLUSION: Raloxifene had an adverse event profile distinct from HRT and unopposed estrogen and was well tolerated by postmenopausal women.

Two-photon and UV-laser flash photolysis of the Ca2+ cage, dimethoxynitrophenyl-EGTA-4.

We report efficient two-photon and UV-laser flash photolysis of dimethoxynitrophenyl-EGTA-4 (DMNPE-4), a newly-developed photolabile Ca(2+)-specific chelator. This compound exhibits good two-photon absorption at 705 nm, has a low Mg2+ affinity (approximately 7 mM), a Kd for Ca2+ of 19 nM, a quantum yield of 0.20 and changes its Ca2+ affinity by 21,000-fold upon photolysis. Two-photon excitation photolysis (TPP) experiments were performed with a Ti:Sapphire laser in solutions containing DMNPE-4 with either 0 or 10 mM Mg2+ and compared to that of the widely used Ca2+ cage, DM-nitrophen (Kd for Ca2+ 5 nM, Kd for Mg2+ 2.5 microM, quantum yield 0.18, affinity change 600,000-fold). The resulting Ca2+ signals were recorded with the fluorescent Ca2+ indicator fluo-3 and a laser-scanning confocal microscope in the line-scan mode. In vitro, photolysis of DMNPE-4:Ca2+ produced Ca(2+)-release signals that had comparable amplitudes and time courses in the presence and absence of Mg2+. However, photorelease of Ca2+ from DM-nitrophen was obviated by the presence of Mg2+. In patch-clamped isolated cardiac myocytes, equivalent TPP results were obtained in analogous experiments. Single-photon excitation of DMNPE-4 by Nd:YAG laser flashes produced Na-Ca exchange currents of comparable amplitude in the absence and presence of Mg2+. However, only very small currents were observed in DM-nitrophen solution containing 10 mM Mg2+. In conclusion, both DMNPE-4 and DM-nitrophen undergo TPP, however, only DMNPE-4 exhibits efficient release of Ca2+ in the presence of Mg2+.

Post-priming actions of ATP on Ca2+-dependent exocytosis in pancreatic beta cells.

The role of cytosolic ATP in exocytosis was investigated by using amperometric measurement of insulin exocytosis in pancreatic beta cells, which were stimulated with photolysis of caged Ca2+ compounds. Insulin exocytosis occurred with two rates. We found that ATP hastened and augmented the exocytosis via selective enhancement of the exocytosis with the faster rate. A nonhydrolysable analog of ATP, adenosine 5'-O-(3-thiotriphosphate), which blocks ATPase, was even more effective than ATP, indicating that the phosphorylation event occurred downstream of ATP-dependent vesicle transportation and priming. The action of ATP was eliminated by a competitive antagonist of cAMP, and by an inhibitor of adenylate cyclase. These data characterize an ATP sensing mechanism for the Ca2+-dependent exocytosis involving adenylate-cyclase, cAMP-dependent protein kinase, and, possibly, the fusion machinery itself. Thus, the fast exocytotic machinery requires both phosphorylation and Ca2+ for the final triggering and likely constitutes a distal ATP sensor for insulin exocytosis that acts in concert with ATP-sensitive K+ channels.

Supralinear Ca2+ signaling by cooperative and mobile Ca2+ buffering in Purkinje neurons.

Endogenous high-affinity Ca2+ buffering and its roles were investigated in mouse cerebellar Purkinje cells with the use of a low-affinity Ca2+ indicator and a high-affinity caged Ca2+ compound. Increases in the cytosolic Ca2+ concentration ([Ca2+]i) were markedly facilitated during repetitive depolarization, resulting in the generation of steep micromolar Ca2+ gradients along dendrites. Such supralinear Ca2+ responses were attributed to the saturation of a large concentration (0.36 mM) of a mobile, high-affinity (dissociation constant, 0.37 microM) Ca2+ buffer with cooperative Ca2+ binding sites, resembling calbindin-D28K, and to an immobile, low-affinity Ca2+ buffer. These data suggest that the high-affinity Ca2+ buffer operates as the neuronal computational element that enables efficient coincidence detection of the Ca2+ signal and that facilitates spatiotemporal integration of the Ca2+ signal at submicromolar [Ca2+]i.

Photolabile amiloride derivatives as cation site probes of the Na,K-ATPase.

Treatment of purified canine renal Na,K-ATPase with a range of photoactivatable amiloride derivatives results in inhibition of ATPase activity prior to illumination. Inhibition by amiloride derivatives substituted on a guanidium N could not be prevented by the presence of either K or Na; however, these cations could protect the enzyme against inhibition by derivatives substituted on the 5-position of the pyrazine ring. In the case of 5-(N-ethyl-[2'-methoxy-4'-nitrobenzyl])amiloride (NENMBA), the presence of monovalent cations (Na, K, and Rb) protected the enzyme effectively against inhibition, with concentrations in the millimolar range. ATP did not prevent inhibition; furthermore, native and NENMBA-treated enzyme exhibited normal levels of high affinity [3H]ADP (and hence ATP) binding. The rate of inhibition increased with increasing concentrations of NENMBA. Extensive washing of NENMBA-inhibited enzyme did not restore ATPase activity, showing that NENMBA has an extremely slow off-rate for dissociation from its inhibitory site. Partially inhibited enzyme could be rapidly pelleted and resuspended in NENMBA-free buffer and inhibition was observed to continue, albeit at a somewhat diminished rate, suggesting that NENMBA gains access to its inhibitory site after partitioning into the lipid phase rather than directly from the aqueous solution. Photolysis of NENMBA-inhibited enzyme resulted in covalent incorporation of the reagent into the alpha-subunit of the Na,K-ATPase, as observed by separation of labeled protein on a Laemmli gel and Western analysis using a polyclonal amiloride antibody. Almost all of the covalent labeling could be prevented by the presence of Rb in the incubation and labeling medium. These results suggest that NENMBA inhibits the Na, K-ATPase by disruption of the cation transport domain rather than the catalytic domain of the enzyme and that it promises to be a useful tool for cation site localization.

Millisecond studies of calcium-dependent exocytosis in pituitary melanotrophs: comparison of the photolabile calcium chelators nitrophenyl-EGTA and DM-nitrophen.

DM-nitrophen (DMN) is a photolabile calcium chelator that has been used extensively to study calcium-triggered exocytosis. Nitrophenyl-EGTA (NPE) is a recently synthesized photolabile calcium chelator that, unlike DMN, selectively binds calcium over magnesium. Here, we compare NPE and DMN for their effectiveness in raising cytosolic calcium ([Ca]i) to trigger exocytosis. The whole cell patch clamp technique was used to monitor membrane capacitance (Cm) and to load both calcium indicator dye and photolabile chelators into rat pituitary melanotrophs prior to flash photolysis. In cells dialysed with DMN, a transient increase in [Ca]i was observed immediately after continuity between the patch pipette and the cell cytosol was achieved. This 'loading transient' reflects the release of calcium from DMN during the binding of intracellular magnesium. No such transient was seen with NPE, consistent with the negligible binding of magnesium to this chelator. Following flash photolysis of DMN or NPE, [Ca]i increased, triggering both a rapid exocytic burst and slower sustained phases of exocytosis. When flashes of the same intensity were compared, the photolysis of NPE resulted in smaller increases in [Ca]i and slower exocytic bursts than that of DMN. These findings are in accordance with the properties of the two compounds [Ellis-Davies G.C.R., Kaplan J.H. Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis. Proc Natl Acad Sci USA 1994; 91: 187-191] and the calcium dependency of the exocytic burst [Thomas P., Wong J.G., Lee A.K., Almers W. A low affinity Ca2+ receptor controls the final steps in peptide secretion from pituitary melanotrophs. Neuron 1993; 11: 93-104]. Although NPE is somewhat less effective than DMN in raising [Ca]i, this chelator promises to be a useful and interesting tool for the time-resolved study of calcium-dependent exocytosis in the presence of physiological concentrations of magnesium.

Laser photolysis of caged calcium: rates of calcium release by nitrophenyl-EGTA and DM-nitrophen.

Nitrophenyl-EGTA and DM-nitrophen are Ca2+ cages that release Ca2+ when cleaved upon illumination with near-ultraviolet light. Laser photolysis of nitrophenyl-EGTA produced transient intermediates that decayed biexponentially with rates of 500,000 s-1 and 100,000 s-1 in the presence of saturating Ca2+ and 290,000 s-1 and 68,000 s-1 in the absence of Ca2+ at pH 7.2 and 25 degrees C. Laser photolysis of nitrophenyl-EGTA in the presence of Ca2+ and the Ca2+ indicator Ca-orange-5N produced a monotonic increase in the indicator fluorescence, which had a rate of 68,000 s-1 at pH 7.2 and 25 degrees C. Irradiation of DM-nitrophen produced similar results with somewhat slower kinetics. The transient intermediates decayed with rates of 80,000 s-1 and 11,000 s-1 in the presence of Ca2+ and 59,000 s-1 and 3,600 s-1 in the absence of Ca2+ at pH 7.2 and 25 degrees C. The rate of increase in Ca(2+)-indicator fluorescence produced upon photolysis of the DM-nitrophen: Ca2+ complex was 38,000 s-1 at pH 7.2 and 25 degrees C. In contrast, pulses in Ca2+ concentration were generated when the chelator concentrations were more than the total Ca2+ concentration. Photoreleased Ca2+ concentration stabilized under these circumstances to a steady state within 1-2 ms.

Kinetics of prephosphorylation reactions and myosin light chain phosphorylation in smooth muscle. Flash photolysis studies with caged calcium and caged ATP.

The pre-myosin light chain (MLC20) phosphorylation components of the lag phase (td) of contractile activation were determined in permeabilized smooth muscles activated by photolytic release of ATP from caged ATP and/or Ca2+ from 4-(2-nitrophenyl)-EGTA (NP-EGTA). Calmodulin (CaM) shortened the td (470 ms at 0 added CaM) that followed Ca2+ release, but its effect (td = approximately 200 ms) saturated at 40 microM. Photolysis of caged ATP following preequilibration with identical [Ca4CaM] shortened td to 41 ms. The rate of phosphorylation was very fast (3.5 s-1 at 22 degrees C in the presence of 5 microM exogenous CaM) following photolysis of caged ATP, and, following Ca2+ release, phosphorylation was accelerated by CaM. Simultaneous photolysis of caged ATP and NP-EGTA was followed by a td of 194 ms at 5 microM CaM and a rate of MLC20 phosphorylation intermediate between these parameters following photolysis of, respectively, NP-EGTA and caged ATP. In the presence of the normal, total endogenous CaM content (37 +/- 4 microM) of protal vein smooth muscles td was 565 ms. Steady state maximum force at pCa 5.5 was increased by much lower (100 nM) exogenous [CaM] than was required (> 2.5 microM) to shorten the td. We estimate the endogenous CaM available under steady state conditions in vivo to be approximately 0.25 microM and probably less during a rapid Ca2+ transient. We conclude that the [CaM] dependence of the kinetics of MLC20 phosphorylation and force development (t1/2 and td) initiated by Ca2+ reflects the recruitment of a slowly diffusible component of total CaM. The relatively long duration of td (197 ms) at saturating [CaM] suggests the contribution to td of an additional component, possibly a prephosphorylation activation/isomerization of the Ca4CaM myosin light chain kinase complex (Török, K., and Trentham, D. R. (1994) Biochemistry 33, 12807-12820). The relatively short delay (108 ms in the presence of 40 microM CaM) following simultaneous photolysis of NP-EGTA and caged ATP suggests that preincubation with ATP (prior to photolysis of NP-EGTA) may inhibit the formation of a preactive Ca2CaM myosin light chain kinase complex.

Electrical currents generated by a partially purified Na/Ca exchanger from lobster muscle reconstituted into liposomes and adsorbed on black lipid membranes: activation by photolysis of Ca2+.

The Na/Ca exchanger from lobster muscle crossreacts specifically with antibodies raised against the dog heart Na/Ca exchanger. Immunoblots of the lobster muscle and mammalian heart exchangers, following SDS-PAGE, indicate that the invertebrate and mammalian exchangers have similar molecular weights: about 120 kDa. The exchanger from lobster muscle was partially purified and functionally reconstituted into asolectin vesicles which were loaded with 160 mM NaCl. 45Ca uptake by these proteoliposomes was promoted by replacing 160 mM NaCl in the external medium with 160 mM KCl to produce an outwardly-directed Na+ concentration gradient. When the proteoliposomes were adsorbed onto black lipid membranes (BLM), and DM-Nitrophen-Ca2+ ("caged Ca2+") was added to the KCl medium, photolytically-evoked Ca2+ concentration jumps elicited transient electric currents. These currents corresponded to positive charge exiting from the proteoliposomes, and were consistent with the Na/Ca exchanger-mediated exit of 3 Na+ in exchange for 1 entering Ca2+. The current was dependent upon the Ca2+ concentration jump, the protein integrity, and the outwardly directed Na+ gradient. KCl-loaded proteoliposomes did not produce any current. Low external Na+ concentrations augmented the current, whereas Na+ concentrations > 25 mM reduced the current. The dependence of the current on free Ca2+ was Michaelis-Menten-like, with half-maximal activation (KM(Ca)) at < 10 microM Ca2+. Caged Sr2+ and Ba2+, but not Mg2+, also supported photolysis-evoked outward current, as did Ni2+, but not Mn2+. However, Mg2+ and Mn2+ augmented the Ca-dependent current, perhaps by facilitating the adsorption of proteoliposomes to the BLM. The Ca-dependent current was irreversibly blocked by La3+ (added as 200 microM DMN-La3+). The results indicate that the properties of the Na/Ca exchanger can be studied with these electro-physiological methods.

Rapid adaptation of cardiac ryanodine receptors: modulation by Mg2+ and phosphorylation.

Channel adaptation is a fundamental feature of sarcoplasmic reticulum calcium release channels (called ryanodine receptors, RyRs). It permits successive increases in the intracellular concentration of calcium (Ca2+) to repeatedly but transiently activate channels. Adaptation of RyRs in the absence of magnesium (Mg2+) and adenosine triphosphate is an extremely slow process (taking seconds). Photorelease of Ca2+ from nitrophenyl-EGTA, a photolabile Ca2+ chelator, demonstrated that RyR adaptation is rapid (milliseconds) in canine heart muscle when physiological Mg2+ concentrations are present. Phosphorylation of the RyR by protein kinase A increased the responsiveness of the channel to Ca2+ and accelerated the kinetics of adaptation. These properties of the RyR from heart may also be relevant to other cells in which multiple agonist-dependent triggering events regulate cellular functions.

Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis.

The synthesis and properties of a caged calcium are described. The compound is an ortho-nitrophenyl derivative of EGTA. It is synthesized in 10 steps and with 24% overall yield. The photosensitive chelator, nitrophenyl-EGTA, has a Kd value for Ca2+ of 80 nM and for Mg2+ of 9 mM. Upon exposure to UV radiation (approximately 350 nm), the chelator is cleaved, yielding iminodiacetic acid photoproducts with low Ca affinity (Kd = 1 mM). The quantum yield of photolysis of nitrophenyl-EGTA in the presence of Ca2+ is 0.23 and in the absence of Ca2+ is 0.20. In experiments with chemically skinned skeletal muscle fibers, a fully relaxed fiber equilibrated with nitrophenyl-EGTA-Ca2+ complex, in the presence of 1 mM free Mg2+, maximally contracted after a single flash from a frequency-doubled ruby laser (347 nm). Half-maximal tension was achieved in 18 ms at 15 degrees C. Nitrophenyl-EGTA provides a tool for the investigation of the mechanism of Ca(2+)-dependent physiological processes, since under conditions of normal intracellular Ca2+ and Mg2+ concentrations, only Ca2+ is bound by the photolabile chelator and on illumination released rapidly and in high photochemical yield.

Modification of lysine 501 in Na,K-ATPase reveals coupling between cation occupancy and changes in the ATP binding domain.

Treatment of the canine renal Na,K-ATPase with N-(2-nitro-4-isothiocyanophenyl)-imidazole (NIPI) results in irreversible loss of enzymatic activity. The reactivity of the protein toward the probe is about 10-fold greater in the E1.Na or sodium-bound form than when it is in the E2.K or potassium-bound form. Fully inactivated enzyme does not bind ATP but binds sodium and potassium and undergoes the major enzyme conformational transitions (Ellis-Davies, G. C. R., and Kaplan, J. H. (1990) J. Biol. Chem. 265, 20570-20576). Labeling of the sodium pump protein with [3H]NIPI in either the E1.Na or E2.K conformations results in the covalent incorporation of one molecule of probe per alpha-subunit of ATPase. Trypsin treatment of purified alpha-subunit, and separation of the digest using reverse-phase high performance liquid chromatography, yields five major radioactive fragments in each case. Amino acid sequencing indicates that only one residue, lysine 501, is labeled by NIPI. This suggests that the integrity of the domain containing Lys501 is essential for both high and low affinity binding of ATP by the Na,K-ATPase. Furthermore, the spatial organization of the protein close to lysine 501 is changed in the E1.Na and E2.K form of the enzyme. This change is reflected in the greater reactivity of lysine 501 in the E1.Na conformation and indicates that the binding of either sodium or potassium ions to their sites on the sodium pump has differential effects on the nucleotide binding domain containing lysine 501.

Release characteristics, ovarian activity and menstrual bleeding pattern with a single contraceptive implant releasing 3-ketodesogestrel.

The properties of a single contraceptive subdermal implant releasing 3-ketodesogestrel were assessed in fifteen women over twelve months. Serum levels of 3-ketodesogestrel were monitored regularly following insertion and after removal. The mean serum level of 3-ketodesogestrel was 245 pg/ml after 72 h (steady state) and 176 pg/ml after twelve months. All volunteers demonstrated ovulation inhibition throughout the study. Transient oestradiol peaks occurred during the study. No luteal activity was noted. The cervical mucus was rapidly rendered hostile to sperm migration. Two women withdrew from the study during the first six months for medical reasons. Both volunteers cited bleeding irregularity as the main cause, one complaining of oligomenorrhoea, the other of prolonged bleeding/spotting episodes. A small but significant increase in weight was noted during the study period.

PIP: 15 sterilized women participated in a clinical trial of the implant Implanon (Organon), a single ethylene vinyl acetate rod containing 60 mg 3-ketodesogestrel (3-KDG), the metabolite of desogestrel. The rod is 40 mm long, 2 mm in diameter and is packaged in its inserter. In this trial the implants were treated to simulate the 2nd year of use. The study subjects underwent intensive hormone and ultrasound monitoring for 72 hours after insertion, twice weekly for 6 weeks and at 6-month intervals. 13 women completed 6 months, 7 completed 12 months, and 5 continued the trial 24 months. There were no complications related to insertion or removal. 3-KDG levels rose to a steady state of 245 pg/ml by 72 hours, then fell to a mean of 17 pg/ml at 12 months. 90 pg/ml of 3-KDG is the critical serum level for anovulation. After removal, 3-KDG declined to 54 pg/ml in 3 days. Follicle development tended toward small follicles or those larger than 10 mm. There was no luteal activity, and LH, FSH and progesterone remained in the follicular phase range. Estradiol levels were not low enough to risk osteoporosis. There was no significant change in serum sex hormone binding globulin. Systolic blood pressure decreased significantly at 12 months; mean weight gain was 3.7 kg (range from loss of 4 kg to gain of 22 kg); a variety of bleeding irregularities were recorded by individual women.