Ly-6C regulates endothelial adhesion and homing of CD8+ T cells by activating integrin-dependent adhesion pathways

Ly-6C belongs to the Ly-6 family of glycosyl phosphatidylinositol-anchored surface glycoproteins and is expressed on a subset of mature CD8⁺ T cells. Ly-6C ligation can mediate T cell activation and causes interleukin 2 secretion in cytolytic T cell clones. We characterize herein a new mAb 1G7.G10 against Ly-6C that recognizes an epitope involved in lymphocyte adhesion and in lymphocyte homing. Pretreatment of lymph node lymphocytes and of purified CD8⁺ T cells (but not of lymphocytes depleted of CD8⁺ T cells) with 1G7.G10 reduced their in vitro binding to lymph node high endothelial venules by 28% and 34%, respectively. This effect was bypassed by cross-linking Ly-6C molecules with 1G7.G10 and a second-step antibody. The in vivo homing of (donor) CD8⁺ T lymphocytes to lymph nodes was reduced by Ly-6C blocking with 1G7.G10 (whole antibody) or with its fragments [F(ab) or F(ab)₂] by 20% or by 32% and 48%, respectively. Cross-linking of Ly-6C in vitro induced very late antigen-4 and lymphocyte function-associated antigen 1-mediated aggregation of CD8⁺ T cells, suggesting that ligand binding to Ly-6C leads to activation of integrins. This activation may facilitate homing of Ly-6C⁺ CD8⁺ T cells in vivo.     

Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase

To determine the mechanisms responsible for the termination of Ca²⁺-activated Cl⁻ currents (I_Cl(Ca)), simultaneous measurements of whole cell currents and intracellular Ca²⁺ concentration ([Ca²⁺]_i) were made in equine tracheal myocytes. In nondialyzed cells, or cells dialyzed with 1 mM ATP, I_Cl(Ca) decayed before the [Ca²⁺]_i decline, whereas the calcium-activated potassium current decayed at the same rate as [Ca²⁺]_i. Substitution of AMP-PNP or ADP for ATP markedly prolonged the decay of I_Cl(Ca), resulting in a rate of current decay similar to that of the fall in [Ca²⁺]_i. In the presence of ATP, dialysis of the calmodulin antagonist W7, the Ca²⁺/calmodulin-dependent kinase II (CaMKII) inhibitor KN93, or a CaMKII-specific peptide inhibitor the rate of I_Cl(Ca) decay was slowed and matched the [Ca²⁺]_i decline, whereas H7, a nonspecific kinase inhibitor with low affinity for CaMKII, was without effect. When a sustained increase in [Ca²⁺]_i was produced in ATP dialyzed cells, the current decayed completely, whereas in cells loaded with 5′-adenylylimidodiphosphate (AMP-PNP), KN93, or the CaMKII inhibitory peptide, I_Cl(Ca) did not decay. Slowly decaying currents were repeatedly evoked in ADP- or AMP-PNP-loaded cells, but dialysis of adenosine 5′-O-(3-thiotriphosphate) or okadaic acid resulted in a smaller initial I_Cl(Ca), and little or no current (despite a normal [Ca²⁺]_i transient) with a second stimulation. These data indicate that CaMKII phosphorylation results in the inactivation of calcium-activated chloride channels, and that transition from the inactivated state to the closed state requires protein dephosphorylation.     

Inositol 1,4,5-tris-phosphate activation of inositol tris-phosphate receptor Ca2+ channel by ligand tuning of Ca2+ inhibition

Inositol 1,4,5-tris-phosphate (IP₃) binding to its receptors (IP₃R) in the endoplasmic reticulum (ER) activates Ca²⁺ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca²⁺ concentration signals including temporal oscillations and propagating waves. IP₃-mediated Ca²⁺ release is also controlled by cytoplasmic Ca²⁺ concentration with both positive and negative feedback. Single-channel properties of the IP₃R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP₃R on cytoplasmic Ca²⁺ and IP₃ concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) response centered at ≈300 nM–1 μM, the open probability remained elevated (≈0.8) in the presence of saturating levels (10 μM) of IP₃, even as [Ca²⁺]_i was raised to high concentrations, displaying two distinct types of functional Ca²⁺ binding sites: activating sites with half-maximal activating [Ca²⁺]_i (K_act) of 210 nM and Hill coefficient (H_act) ≈2; and inhibitory sites with half-maximal inhibitory [Ca²⁺]_i (K_inh) of 54 μM and Hill coefficient (H_inh) ≈4. Lowering IP₃ concentration was without effect on Ca²⁺ activation parameters or H_inh, but decreased K_inh with a functional half-maximal activating IP₃ concentration (K_IP3) of 50 nM and Hill coefficient (H_IP3) of 4 for IP₃. These results demonstrate that Ca²⁺ is a true receptor agonist, whereas the sole function of IP₃ is to relieve Ca²⁺ inhibition of IP₃R. Allosteric tuning of Ca²⁺ inhibition by IP₃ enables the individual IP₃R Ca²⁺ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca²⁺ concentration signaling and quantal Ca²⁺ release.     

Inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ release by cAMP-dependent protein kinase in a living cell

Interaction of intracellular free calcium ([Ca²⁺]_i) and cAMP signaling mechanisms was examined in intact single megakaryocytes by using a combination of single-cell fluorescence microscopy to measure [Ca²⁺]_i and flash photolysis of caged Ca²⁺, inositol 1,4,5-trisphosphate (IP₃), or cAMP to elevate rapidly the concentration of these compounds inside the cell. Photolysis of caged IP₃ stimulated Ca²⁺ release from an IP₃-sensitive store. The cAMP-elevating agent carbacyclin inhibited this IP₃-induced rise in [Ca²⁺]_i but did not affect the rate of Ca²⁺ removal from the cytoplasm after photolysis of caged Ca²⁺. Photolysis of caged cAMP during ADP-induced [Ca²⁺]_i oscillations caused the [Ca²⁺]_i oscillation to transiently cease without affecting the rate of Ca²⁺ uptake and/or extrusion. We conclude that the principal mechanism of cAMP-dependent inhibition of Ca²⁺ mobilization in megakaryocytes appears to be by inhibition of IP₃-induced Ca²⁺ release and not by stimulation of Ca²⁺ removal from the cytoplasm. Two inhibitors of cAMP-dependent protein kinase, a specific peptide inhibitor of the catalytic subunit of cAMP protein kinase and KT5720, blocked the inhibitory effect of carbacyclin, indicating that the inhibition of IP₃-induced Ca²⁺-release by carbacyclin is mediated by cAMP-dependent protein kinase.     

Neuregulins stimulate the functional expression of Ca2+-activated K+ channels in developing chicken parasympathetic neurons

The developmental expression of macroscopic Ca²⁺-activated K⁺ currents (I_K[Ca]) in chicken ciliary ganglion (CG) neurons is dependent in part on trophic factors released from preganglionic nerve terminals. Neuregulins are expressed in the preganglionic neurons that innervate the chicken CG and are therefore plausible candidates for this activity. Application of 1 nM β1-neuregulin peptide for 12 hr evokes a large (7- to 10-fold) increase in I_K[Ca] in embryonic day 9 CG neurons, even in the presence of a translational inhibitor. A similar posttranslational effect is produced by high concentrations (10 nM) of epidermal growth factor and type α transforming growth factor but not by 10 nM α2-neuregulin peptide or by neurotrophins at 40 ngml⁻¹. β1-neuregulin treatment for 12 hr also confers Ca²⁺ sensitivity onto large-conductance (285 pS) K⁺ channels observed in inside–out patches. β-Neuregulins have no effect on voltage-activated Ca²⁺ currents of CG neurons. These data support the hypothesis that β-neuregulins mediate the trophic effects of preganglionic nerve terminals on the electrophysiological differentiation of developing CG neurons.     

Endothelial Ca2+ waves preferentially originate at specific loci in caveolin-rich cell edges

Stimulation of endothelial cells (ECs) with ATP evoked an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i). In a single bovine aortic EC, the [Ca²⁺]_i rise started at a specific peripheral locus and propagated throughout the entire cell as a Ca²⁺ wave. The initiation locus was constant upon repeated stimulation with ATP or other agonists (bradykinin and thrombin). The Ca²⁺ wave was unaffected by the removal of extracellular Ca²⁺, demonstrating its dependence on intracellular Ca²⁺ release. Microinjection of heparin into the cell inhibited the ATP-induced Ca²⁺ responses, indicating that the Ca²⁺ wave is at least partly mediated by the inositol 1,4,5-trisphosphate receptor. Immunofluorescence staining revealed that caveolin, a marker protein for caveolae, is distributed heterogeneously in the cell and that Ca²⁺ waves preferentially originate at caveolin-rich cell edges. In contrast to caveolin, internalized transferrin and subunits of the clathrin-associated adaptor complexes such as adaptor protein-1 and -2 were diffusely distributed. Disruption of microtubules by Colcemid led to redistribution of caveolin away from the edges into the perinuclear center of the cell, and the ATP-induced [Ca²⁺]_i increase was initiated on the rim of the centralized caveolin. Thus, caveolae may be involved in the initiation of ATP-induced Ca²⁺ waves in ECs.     

Strained DNA is kinked by low concentrations of Zn2+

A novel atomic force microscope with a magnetically oscillated tip has provided unprecedented resolution of small DNA fragments spontaneously adsorbed to mica and imaged in situ in the presence of divalent ions. Kinks (localized bends of average angle 78°) were observed in axially strained minicircles consisting of tandemly repeated d(A)₅ and d(GGGCC[C]) sequences. The frequency of kinks in identical minicircles increased 4-fold in the presence of 1 mM Zn²⁺ compared with 1 mM Mg²⁺. Kinking persisted in mixed Mg²⁺/Zn²⁺ electrolytes until the Zn²⁺ concentration dropped below 100 μM, indicating that this type of kinking may occur under physiological conditions. Kinking appears to replace intrinsic bending, and statistical analysis shows that kinks are not localized within any single sequence element. A surprisingly small free energy is associated with kink formation.     

Ca2+-independent inhibition of inositol trisphosphate receptors by calmodulin: Redistribution of calmodulin as a possible means of regulating Ca2+ mobilization

The interactions between calmodulin, inositol 1,4,5-trisphosphate (InsP₃), and pure cerebellar InsP₃ receptors were characterized by using a scintillation proximity assay. In the absence of Ca²⁺, ¹²⁵I-labeled calmodulin reversibly bound to multiple sites on InsP₃ receptors and Ca²⁺ increased the binding by 190% ± 10%; the half-maximal effect occurred when the Ca²⁺ concentration was 184 ± 14 nM. In the absence of Ca²⁺, calmodulin caused a reversible, concentration-dependent (IC₅₀ = 3.1 ± 0.2 μM) inhibition of [³H]InsP₃ binding by decreasing the affinity of the receptor for InsP₃. This effect was similar at all Ca²⁺ concentrations, indicating that the site through which calmodulin inhibits InsP₃ binding has similar affinities for calmodulin and Ca²⁺-calmodulin. Calmodulin (10 μM) inhibited the Ca²⁺ release from cerebellar microsomes evoked by submaximal, but not by maximal, concentrations of InsP₃. Tonic inhibition of InsP₃ receptors by the high concentrations of calmodulin within cerebellar Purkinje cells may account for their relative insensitivity to InsP₃ and limit spontaneous activation of InsP₃ receptors in the dendritic spines. Inhibition of InsP₃ receptors by calmodulin at all cytosolic Ca²⁺ concentrations, together with the known redistribution of neuronal calmodulin evoked by protein kinases and Ca²⁺, suggests that calmodulin may also allow both feedback control of InsP₃ receptors and integration of inputs from other signaling pathways.     

A single peripheral CD8+ T cell can give rise to progeny expressing type 1 and/or type 2 cytokine genes and can retain its multipotentiality through many cell divisions

The lineage relationships between murine CD8⁺ T cells with different cytokine profiles were investigated by paired-daughter analysis in the presence and absence of the type 2 cytokine-inducing stimulus, interleukin 4 (IL-4). Single CD8⁺ CD44^low lymph node T cells were activated to divide at high frequency with IL-2 and immobilized antibodies to CD3, CD8, and LFA-1. When these parent cells were subcloned by transferring their daughter or granddaughter cells into secondary cultures with or without IL-4, the subclones expressed diverse combinations of the mRNAs for the type 1 cytokines, interferon γ (IFN-γ), and IL-2, and the type 2 cytokines, IL-4, IL-5, IL-6, and IL-10. Frequencies of subclones that expressed IL-4, IL-6, and, to a lesser extent, IL-2, IL-5, and IL-10 were higher among those grown with IL-4, but a significant proportion of those grown without exogenous IL-4 also expressed one or more type 2 cytokines. Subclones within 89% of families displayed different cytokine profiles, indicating that their parent cells were multipotential for this function. Because 98% of parent cells yielded subclones that produced type 1 cytokines and 77% yielded type 2 cytokine producers, we conclude that type 1 and type 2 cytokine-producing CD8⁺ T cells can be derived from a common precursor. Similar analyses performed by subcloning after ≥7 or ≥13 cell divisions without IL-4 showed that many CD8⁺ T cells retained the potential to shift toward a type 2 cytokine profile in response to IL-4, even after prolonged expansion under conditions that favored type 1 cytokine expression. CD8⁺ T cells that express type 1 and/or type 2 cytokines therefore are derived from the same peripheral T cell lineage whose multipotentiality can persist through many cell divisions.     

Killing K Channels with TEA+

Tetraethylammonium (TEA⁺) is widely used for reversible blockade of K channels in many preparations. We noticed that intracellular perfusion of voltage-clamped squid giant axons with a solution containing K⁺ and TEA⁺ irreversibly decreased the potassium current when there was no K⁺ outside. Five minutes of perfusion with 20 mM TEA⁺, followed by removal of TEA⁺, reduced potassium current to <5% of its initial value. The irreversible disappearance of K channels with TEA⁺ could be prevented by addition of ≥ 10 mM K⁺ to the extracellular solution. The rate of disappearance of K channels followed first-order kinetics and was slowed by reducing the concentration of TEA⁺. Killing is much less evident when an axon is held at −110 mV to tightly close all of the channels. The longer-chain TEA⁺ derivative decyltriethylammonium (C10⁺) had irreversible effects similar to TEA⁺. External K⁺ also protected K channels against the irreversible action of C10⁺. It has been reported that removal of all K⁺ internally and externally (dekalification) can result in the disappearance of K channels, suggesting that binding of K⁺ within the pore is required to maintain function. Our evidence further suggests that the crucial location for K⁺ binding is external to the (internal) TEA⁺ site and that TEA⁺ prevents refilling of this location by intracellular K⁺. Thus in the absence of extracellular K⁺, application of TEA⁺ (or C10⁺) has effects resembling dekalification and kills the K channels.     

T cell functions in granulocyte/macrophage colony-stimulating factor deficient mice

Immunological functions were analyzed in mice lacking granulocyte/macrophage colony-stimulating factor (GM-CSF). The response of splenic T cells to allo-antigens, anti-mouse CD3 mAb, interleukin 2 (IL-2), or concanavalin A was comparable in GM-CSF +/+ and GM-CSF −/− mice. To investigate the responses of CD8⁺ and CD4⁺ T cells against exogenous antigens, mice were immunized with ovalbumin peptide or with keyhole limpet hemocyanin (KLH). Cytotoxic CD8⁺ T cells with specificity for ovalbumin peptide could not be induced in GM-CSF −/− mice. After immunization with KLH, there was a delay in IgG generation, particularly IgG2a, in GM-CSF −/− mice. Purified CD4⁺ T cells from GM-CSF −/− mice immunized with KLH showed impaired proliferative responses and produced low amounts of interferon-γ (IFN-γ) and IL-4 when KLH-pulsed B cells or spleen cells were used as antigen presenting cells (APC). When enriched dendritic cells (DC) were used as APC, CD4⁺ T cells from GM-CSF −/− mice proliferated as well as those from GM-CSF +/+ mice and produced high amounts of IFN-γ and IL-4. To analyze the rescue effect of DC on CD4⁺ T cells, supernatants from (i) CD4⁺ T cells cultured with KLH-pulsed DC or (ii) DC cultured with recombinant GM-CSF were transferred to cultures of CD4⁺ T cells and KLH-pulsed spleen cells from GM-CSF −/− mice. Supernatants from both DC sources contained a factor or factors that restored proliferative responses and IFN-γ production of CD4⁺ T cells from GM-CSF −/− mice.     

Overexpression of the SUP45 gene encoding a Sup35p-binding protein inhibits the induction of the de novo appearance of the [PSI+] prion

[PSI⁺], a non-Mendelian element found in some strains of Saccharomyces cerevisiae, is presumed to be the manifestation of a self-propagating prion conformation of eRF3 (Sup35p). Translation termination factor eRF3 enhances the activity of release factor eRF1 (Sup45p). As predicted by the prion model, overproduction of Sup35p induces the de novo appearance of [PSI⁺]. However, another non-Mendelian determinant, [PIN⁺], is required for this induction. We now show that SUP45 overexpression inhibits the induction of [PSI⁺] by Sup35p overproduction in [PIN⁺] strains, but has no effect on the propagation of [PSI⁺] or on the [PIN] status of the cells. We also show that SUP45 overexpression counteracts the growth inhibition usually associated with overexpression of SUP35 in [PSI⁺] strains. We argue that excess Sup45p inhibits [PSI⁺] seed formation. Because Sup45p complexes with Sup35p, we hypothesize that excess Sup45p may sequester Sup35p, thereby reducing the opportunity for Sup35p conformational flips and/or self-interactions leading to prion formation. This in vivo yeast result is reminiscent of the in vitro finding by investigators of Alzheimer disease that apolipoprotein E inhibits amyloid nucleation, but does not reduce seeded growth of amyloid.     

Targeted inactivation of αi2 or αi3 disrupts activation of the cardiac muscarinic K+ channel, IK+Ach, in intact cells

Cardiac muscarinic receptors activate an inwardly rectifying K⁺ channel, I_K^+Ach, via pertussis toxin (PT)-sensitive heterotrimeric G proteins (in heart G_i2, G_i3, or G_o). We have used embryonic stem cell (ES cell)-derived cardiocytes with targeted inactivations of specific PT-sensitive α subunits to determine which G proteins are required for receptor-mediated regulation of I_K^+Ach in intact cells. The muscarinic agonist carbachol increased I_K^+Ach activity in ES cell-derived cardiocytes from wild-type cells, in cells lacking α_o, and in cells lacking the PT-insensitive G protein α_q. In cells with targeted inactivation of α_i2 or α_i3, channel activation by both carbachol and adenosine was blocked. Carbachol-induced channel activation was restored in the α_i2- and α_i3-null cells by reexpressing the previously targeted gene and guanosine 5′-[γ-thio] triphosphate was able to fully activate I_K^+Ach in excised membranes patches from these mutants. In contrast, negative chronotropic responses to both carbachol and adenosine were preserved in cells lacking α_i2 or α_i3. Our results show that expression of two specific PT-sensitive α subunits (α_i2 and α_i3 but not α_o) is required for normal agonist-dependent activation of I_K^+Ach and suggest that both α_i2- and α_i3-containing heterotrimeric G proteins may be involved in the signaling process. Also the generation of negative chronotropic responses to muscarinic or adenosine receptor agonists do not require activation of I_K^+Ach or the expression of α_i2 or α_i3.     

Low density lipoprotein receptor-negative mice expressing human apolipoprotein B-100 develop complex atherosclerotic lesions on a chow diet: No accentuation by apolipoprotein(a)

We have generated mice with markedly elevated plasma levels of human low density lipoprotein (LDL) and reduced plasma levels of high density lipoprotein. These mice have no functional LDL receptors [LDLR^−/−] and express a human apolipoprotein B-100 (apoB) transgene [Tg(apoB^+/+)] with or without an apo(a) transgene [Tg(apoa^+/−)]. Twenty animals (10 males and 10 females) of each of the following four genotypes were maintained on a chow diet: (i) LDLR^−/−, (ii) LDLR^−/−;Tg(apoa^+/−), (iii) LDLR^−/−;Tg(apoB^+/+), and (iv)LDLR^−/−;Tg(apoB^+/+);Tg(apo^+/−). The mice were killed at 6 mo, and the percent area of the aortic intimal surface that stained positive for neutral lipid was quantified. Mean percent areas of lipid staining were not significantly different between the LDLR^−/− and LDLR^−/−;Tg(apoa^+/−) mice (1.0 ± 0.2% vs. 1.4 ± 0.3%). However, the LDLR^−/−;Tg(apoB^+/+) mice had ≈15-fold greater mean lesion area than the LDLR^−/− mice. No significant difference was found in percent lesion area in the LDLR^−/−;Tg(apoB^+/+) mice whether or not they expressed apo(a) [18.5 ± 2.5%, without lipoprotein(a), Lp(a), vs. 16.0 ± 1.7%, with Lp(a)]. Histochemical analyses of the sections from the proximal aorta of LDLR^−/−;Tg(apoB^+/+) mice revealed large, complex, lipid-laden atherosclerotic lesions that stained intensely with human apoB-100 antibodies. In mice expressing Lp(a), large amounts of apo(a) protein colocalized with apoB-100 in the lesions. We conclude that LDLR^−/−; Tg(apoB^+/+) mice exhibit accelerated atherosclerosis on a chow diet and thus provide an excellent animal model in which to study atherosclerosis. We found no evidence that apo(a) increased atherosclerosis in this animal model.     

Direct visualization of antigen-specific T cells: HTLV-1 Tax11–19- specific CD8+ T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients

Human T lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropic spastic paraparesis is a demyelinating inflammatory neurologic disease associated with HTLV-1 infection. HTLV-1 Tax11–19-specific cytotoxic T cells have been isolated from HLA-A2-positive patients. We have used a peptide-loaded soluble HLA-A2–Ig complex to directly visualize HTLV-1 Tax11–19-specific T cells from peripheral blood and cerebrospinal fluid without in vitro stimulation. Five of six HTLV-1-associated myelopathy/tropic spastic paraparesis patients carried a significant number (up to 13.87%) of CD8⁺ lymphocytes specific for the HTLV-1 Tax11–19 peptide in their peripheral blood, which were not found in healthy controls. Simultaneous comparison of peripheral blood and cerebrospinal fluid from one patient revealed 2.5-fold more Tax11–19-specific T cells in the cerebrospinal fluid (23.7% vs. 9.4% in peripheral blood lymphocyte). Tax11–19-specific T cells were seen consistently over a 9-yr time course in one patient as far as 19 yrs after the onset of clinical symptoms. Further analysis of HTLV-1 Tax11–19-specific CD8⁺ T lymphocytes in HAM/TSP patients showed different expression patterns of activation markers, intracellular TNF-α and γ-interferon depending on the severity of the disease. Thus, visualization of antigen-specific T cells demonstrates that HTLV-1 Tax11–19-specific CD8⁺ T cells are activated, persist during the chronic phase of the disease, and accumulate in cerebrospinal fluid, showing their pivotal role in the pathogenesis of this neurologic disease.     

Depletion of Ca2+ from the sarcoplasmic reticulum of cardiac muscle prompts phosphorylation of phospholamban to stimulate store refilling

Nonmuscle cells have almost ubiquitously evolved a mechanism to detect and prevent Ca²⁺ store depletion—store operated calcium entry. No such mechanism has, as yet, been reported in cardiac myocytes. However, it is conceivable that such a mechanism may play an important role in cardiac Ca²⁺ homeostasis to ensure the availability of sufficient stored Ca²⁺ to maintain normal excitation contraction coupling. We present data that confirms the presence of a mechanism that is able to monitor the Ca²⁺ load of the SR and initiate a signaling process to accelerate Ca²⁺ uptake by the SR when store depletion is detected. Depletion of SR Ca²⁺ activates a protein kinase, the principal SR substrate of which is phospholamban. Phosphorylation of this SR protein promotes Ca²⁺ pump activity and therefore store refilling. Furthermore, a protein kinase activity associated with the SR that is inhibited by Ca²⁺ ions has been identified. We have measured lumenal [Ca²⁺] by using a fluorescent Ca²⁺ indicator and found that by initiating Ca²⁺ uptake and increasing Ca²⁺ load, we can inhibit the protein kinase activity associated with the SR. This confirms that a protein kinase, that is regulated by lumenal [Ca²⁺], has been identified and represents part of a previously unidentified signalling cascade. This local feedback mechanism would allow the myocyte to detect and prevent SR Ca²⁺ load depletion.     

A Xenopus oocyte β subunit: Evidence for a role in the assembly/expression of voltage-gated calcium channels that is separate from its role as a regulatory subunit

Two closely related β subunit mRNAs (xo28 and xo32) were identified in Xenopus oocytes by molecular cloning. One or both appear to be expressed as active proteins, because: (i) injection of Xenopus β antisense oligonucleotides, but not of sense or unrelated oligonucleotides, significantly reduced endogenous oocyte voltage-gated Ca²⁺ channel (VGCC) currents and obliterated VGCC currents that arise after injection of mammalian α₁ cRNAs (α_1C and α_1E); (ii) coinjection of a Xenopus β antisense oligonucleotide and excess rat β cRNA rescued expression of α₁ Ca²⁺ channel currents; and (iii) coinjection of mammalian α₁ cRNA with cRNA encoding either of the two Xenopus β subunits facilitated both activation and inactivation of Ca²⁺ channel currents by voltage, as happens with most mammalian β subunits. The Xenopus β subunit cDNAs (β3xo cDNAs) predict proteins of 484 aa that differ in only 22 aa and resemble most closely the sequence of the mammalian type 3 β subunit. We propose that “α₁ alone” channels are in fact tightly associated α₁β3xo channels, and that effects of exogenous β subunits are due to formation of higher-order [α₁β]β_n complexes with an unknown contribution of β3xo. It is thus possible that functional mammalian VGCCs, rather than having subunit composition α₁β, are [α₁β]β_n complexes that associate with α₂δ and, as appropriate, other tissue-specific accessory proteins. In support of this hypothesis, we discovered that the last 277-aa of α_1E have a β subunit binding domain. This β binding domain is distinct from the previously known interaction domain located between repeats I and II of calcium channel α₁ subunits.     

CD8+ T-cell-derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppress HIV-1 replication in monocyte/macrophages

It has been demonstrated that CD8⁺ T cells produce a soluble factor(s) that suppresses human immunodeficiency virus (HIV) replication in CD4⁺ T cells. The role of soluble factors in the suppression of HIV replication in monocyte/macrophages (M/M) has not been fully delineated. To investigate whether a CD8⁺ T-cell-derived soluble factor(s) can also suppress HIV infection in the M/M system, primary macrophages were infected with the macrophage tropic HIV-1 strain Ba-L. CD8⁺ T-cell-depleted peripheral blood mononuclear cells were also infected with HIV-1 IIIB or Ba-L. HIV expression from the chronically infected macrophage cell line U1 was also determined in the presence of CD8⁺ T-cell supernatants or β-chemokines. We demonstrate that: (i) CD8⁺ T-cell supernatants did, but β-chemokines did not, suppress HIV replication in the M/M system; (ii) antibodies to regulated on activation normal T-cell expressed and Secreted (RANTES), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β did not, whereas antibodies to interleukin 10, interleukin 13, interferon α, or interferon γ modestly reduced anti-HIV activity of the CD8⁺ T-cell supernatants; and (iii) the CD8⁺ T-cell supernatants did, but β-chemokines did not, suppress HIV-1 IIIB replication in peripheral blood mononuclear cells as well as HIV expression in U1 cells. These results suggest that HIV-suppressor activity of CD8⁺ T cells is a multifactorial phenomenon, and that RANTES, MIP-1α, and MIP-1β do not account for the entire scope of CD8⁺ T-cell-derived HIV-suppressor factors.     

Bradykinin inhibits M current via phospholipase C and Ca2+ release from IP3-sensitive Ca2+ stores in rat sympathetic neurons

A variety of intracellular signaling pathways can modulate the properties of voltage-gated ion channels. Some of them are well characterized. However, the diffusible second messenger mediating suppression of M current via G protein-coupled receptors has not been identified. In superior cervical ganglion neurons, we find that the signaling pathways underlying M current inhibition by B₂ bradykinin and M₁ muscarinic receptors respond very differently to inhibitors. The bradykinin pathway was suppressed by the phospholipase C inhibitor U-73122, by blocking the IP₃ receptor with pentosan polysulfate or heparin, and by buffering intracellular calcium, and it was occluded by allowing IP₃ to diffuse into the cytoplasm via a patch pipette. By contrast, the muscarinic pathway was not disrupted by any of these treatments. The addition of bradykinin was accompanied by a [Ca²⁺]_i rise with a similar onset and time to peak as the inhibition of M current. The M current inhibition and the rise of [Ca²⁺]_i were blocked by depletion of Ca²⁺ internal stores by thapsigargin. We conclude that bradykinin receptors inhibit M current of sympathetic neurons by activating phospholipase C and releasing Ca²⁺ from IP₃-sensitive Ca²⁺ stores, whereas muscarinic receptors do not use the phospholipase C pathway to inhibit M current channels.