I(f)-dependent modulation of pacemaker rate mediated by cAMP in the presence of ryanodine in rabbit sino-atrial node cells

I(f) contributes to generation and autonomic control of spontaneous activity of cardiac pacemaker cells through a cAMP-dependent, Ca(2+)-independent mechanism of rate regulation. However, disruption of Ca(2+) release from sarcoplasmic reticulum (SR) by ryanodine (Ry) has been recently shown to slow spontaneous rate and inhibit beta-adrenergic receptor (betaAR)-induced rate acceleration, leading to the suggestion that the target of betaAR modulation of pacemaking is the intracellular Ca(2+)-regulatory process. We have investigated whether the Ry-induced decrease of betaAR rate modulation alternatively involves disruption of the betaAR-adenylate-cyclase-cAMP-I(f) mechanism. Prolonged exposure to Ry (3 microM, >2 min) slowed spontaneous rate of pacemaker cells by 29.8% via a depolarizing shift of take-off potential (TOP) without significantly changing early diastolic depolarization rate. Ry depressed rate acceleration caused by isoproterenol (Iso) (1 microM, 23.6% in control vs. 8.0%), but did not modify that caused by two membrane-permeable cAMP analogs, CPT-cAMP (300 microM, 17.7% vs. 17.3%) and Rp-cAMPs (50 microM, 18.0% vs. 20.6%). Consistent with the rate effect, exposure to Ry decreased the shift induced by Iso, but not that induced by either cAMP analog on the I(f)-activation curve. We conclude that disruption of Ry receptor function and SR Ca(2+) release depresses betaAR-induced modulation of heart rate, but does not impair cAMP-dependent rate acceleration mediated by I(f). However, abolishment of normal Ca(2+) homeostasis may result in the failure of betaAR agonists to sufficiently elevate cAMP near f-channels. The molecular basis for Ca(2+)-dependent interference in beta-adrenergic signaling remains to be determined.     

Microtubules mobility affects the modulation of L-type I(Ca) by muscarinic and beta-adrenergic agonists in guinea-pig cardiac myocytes

To investigate the interaction of cytoskeleton with the receptor modulation of ionic currents, we studied the effect of muscarinic and beta-adrenergic stimulation in adult guinea-pig ventricular cardiac myocytes treated with paclitaxel and colchicine, two drugs that respectively stabilize or destabilize microtubules. We observed that the stabilization of microtubules with paclitaxel (1 microM for 1-4 h) did not markedly affect either the kinetics of I(Ca), or the stimulatory effect of isoproterenol (Iso, 1 microM); however paclitaxel significantly blunted the response to carbachol (CCh, 1 microM). In agreement with the electrophysiological measurements, Iso induced a similar enhancement of intracellular cAMP levels in both control and paclitaxel-treated cells, while the response to CCh 1 microM was significantly reduced in paclitaxel-treated cells. The reduction of muscarinic response induced by paclitaxel was also evident in atrial cells, in which the stimulation of I(KACh) by CCh 1 microM was reduced to about 10%. Compared to the muscarinic response, paclitaxel did not have significant effect on the purinergic (adenosine 1-10 microM) modulation of I(Ca). In contrast to paclitaxel, in colchicine-treated cells, I(Ca) was not enhanced by beta-adrenergic stimulation, but instead reduced by CCh, even in the absence of previous stimulation. In conclusion, our data suggest that microtubule stabilization significantly affects the muscarinic modulation of I(Ca), by interacting with the receptor or the G-protein rather than on the intracellular signaling cascade.     

The heart rate-lowering agent ivabradine inhibits the pacemaker current I(f) in human atrial myocytes

Introduction: It has been speculated that pacemaker current (I _f ) in human atria could play a role in causing ectopic atrial automaticity. Ivabradine is a novel selective and specific I _f inhibitor in the sinus node that reduces heart rate without any negative inotropic effect. The aim of the study was to explore possible effects of ivabradine on I _f in atrial myocytes.
Methods and Results:  Using patch-clamp technique, we studied effects of ivabradine on I _f present in atrial myocytes isolated from human right appendages of patients undergoing cardiac surgery. The identification of HCN isoforms was obtained by means of multiplex single-cell RT-PCR. Ivabradine induced a marked concentration and use-dependent I _f inhibition with an IC₅₀ at steady state of 2.9 μM. Time constant of block development (Tau_on) decreases with the increase in the ivabradine concentration. Use-dependent inhibition induced by ivabradine (3 μM) was not modified in the presence of cAMP (10 μM) in the pipette solution. Multiplex single-cell RT-PCR indicates that the major HCN gene subtype detected in atria was HCN2. HCN4 is detected weakly and HCN1 is not significantly detected.
Conclusions:  Ivabradine inhibits I _f current in the nonpacemaker cell with characteristics similar to those described previously in rabbit sinus node cells, but revealed a lesser sensitivity for I _f recorded in human atrial cell than hHCN4 subunits considered as the major contributors to native f -channels in human sinoatrial node. A potential protection of atrial arrhythmias by ivabradine is discussed.     

Lysophosphatidylcholine enhances I(Ks) currents in cardiac myocytes through activation of G protein, PKC and Rho signaling pathways

Lysophosphatidylcholine (LPC) is a bioactive phospholipid that accumulates rapidly in the ischemic myocardium. In recent years, it has been shown that some of the actions of LPC are mediated through the activation of the membrane G proteins. However, the precise mechanism(s) responsible for the LPC-related intracellular signaling in the regulation of cardiac ion channels are still poorly understood. The present study was undertaken to examine whether LPC regulates the slow component of the delayed rectifier K⁺ current (I_Ks) and, if so, what intracellular signals are important for this process. Isolated guinea pig cardiac myocytes were voltage-clamped using the whole-cell configuration of the patch-clamp method. The bath application of 1-palmitoyl-lysophosphatidylcholine (LPC-16) concentration-dependently (EC₅₀ = 0.7 μM) and reversibly increased I_Ks in atrial cells, but failed to potentiate I_Ks in ventricular myocytes. In contrast, 1-oleoyl-lysophosphatidylcholine (LPC-18:1) only produced a slight I_Ks increase, and 1-caproyl-lysophosphatidylcholine (LPC-6) or the LPC-16 precursor (phosphatidylcholine) had no effect on I_Ks. Pretreatment of atrial cells with an antibody against the N-terminus of the G2A receptor significantly reduced the LPC-16-induced potentiation of I_Ks. The inhibition of heterotrimeric G protein, phospholipase C (PLC) and protein kinase C (PKC) significantly reduced LPC-16-induced enhancement of I_Ks. Moreover, the blockade of Rho and Rho-kinase by specific inhibitors also inhibited the activity of LPC-16. Immunohistochemical studies demonstrated that G2A was densely distributed in the plasma membrane of atrial myocytes. Therefore, the present study suggests that the activation of a G protein (probably Gα_q) by LPC-16 potentiates I_Ks currents through the PLC-PKC and Rho-kinase pathways.     

Modulation of rate by autonomic agonists in SAN cells involves changes in diastolic depolarization and the pacemaker current

Two distinct intracellular mechanisms have been proposed to affect the firing rate of cardiac pacemaker cells: one involves modulation of the I(f) current by the second messenger cAMP, and one relies upon disruption or alteration of SR Ca2+ transients during activity. Although both mechanisms are necessary for proper automaticity and autonomic rate control, the specific contribution of each to pacemaking is still debated. We investigated if the two processes can be separated based on potentially different effects on action potential characteristics during rate modulation. To identify specific I(f)-mediated effects, we used the selective I(f) blocker ivabradine and found that ivabradine (3 microM) slows rate (-16.2%) by selectively reducing (-31.9%) the steepness of early diastolic depolarization (EDD). On the other hand ryanodine (3 microM), used to evaluate the effects of abolishment of SR Ca2+ transients, slowed rate (-31.3%) by depolarizing the take-off potential (TOP, 18.1%) without affecting EDD. We therefore used these two parameters to identify I(f)-based or SR Ca2+ transients-based processes and analyzed the effects on action potential's characteristics of Rp-cAMPs (50 microM), a membrane permeable cAMP analogue directly activating f-channels; we found that Rp-cAMPs accelerates rate by increasing EDD (+42.3%) without modifying TOP. Finally, rate modulation was achieved by muscarinic (ACh 0.01 microM) or beta-adrenergic (Iso 1 microM) stimulation; in both cases, rate changes were associated with modifications of EDD (ACh, -29.3% and Iso, +47.6%) and not of TOP. We conclude that rate-related changes in the EDD induced by autonomic agonists are mediated by I(f) and not by processes involving SR Ca2+ transients.     

TGFbeta regulates the expression of G alpha(i2) via an effect on the localization of ras

The negative chronotropic response of the heart to parasympathetic stimulation is mediated via the interaction of M(2) muscarinic receptors, Galpha(i2) and the G-protein coupled inward rectifying K(+) channel, GIRK1. Here TGFbeta(1) is shown to decrease the expression of Galpha(i2) in cultured chick atrial cells in parallel with attenuation of the negative chronotropic response to parasympathetic stimulation. The response to the acetylcholine analogue, carbamylcholine, decreased from a 95+/-2% (+/-SEM, n=8) inhibition of beat rate in control cells to 18+/-2% (+/-SEM,n =8) in TGFbeta(1) treated cells. Data support the conclusion that TGFbeta regulation of Galpha(i2) expression was mediated via an effect on Ras. TGFbeta(1) inhibited Galpha(i2) promoter activity by 56+/-6% (+/-SEM, n=4) compared to control. A dominant activating Ras mutant reversed the effect of TGFbeta on Galpha(i2) expression and stimulated Galpha(i2) promoter activity 1.7 fold above control. A dominant negative Ras mutant mimicked the effect of TGFbeta(1) on Galpha(i2) promoter activity. TGFbeta had no effect on the ratio of GDP/GTP bound Ras, but markedly decreased the level of membrane associated Ras and increased the level of cytoplasmic Ras compared to control. Furthermore, farnesol, a precursor to farnesylpyrophosphate, the substrate for the farnesylation of Ras, not only reversed TGFbeta(1) inhibition of Ras localization to the membrane, but also reversed TGFbeta(1) inhibition of Galpha(i2)promoter activity. FTI-277, a specific inhibitor of the farnesylation of Ras, mimicked the effect of TGFbeta(1) on Ras localization and Galpha(i2) promoter activity. These data suggest a novel relationship between TGFbeta signaling, regulation of Ras function and the autonomic response of the heart.     

Dominant-negative suppression of I(K1) in the mouse heart leads to altered cardiac excitability

The inward rectifier potassium current in the heart, I(K1), has been suggested to play a significant role in cardiac excitability by contributing to the late phase of action potential (AP) repolarization and the stabilization of resting potential. To further assess the role of I(K1) in cardiac excitability we have produced transgenic mice expressing a dominant-negative subunit of the Kir2.1 channel, a major molecular determinant of I(K1) in the heart, and studied the effects of I(K1) suppression on major potassium currents, APs and the overall electrical activity of the heart. Kir2.1 channel subunits with a mutated signature sequence (AAA for GYG substitution) were expressed in the heart under control of the alpha-myosin heavy chain promoter. Two lines of transgenic mice were established, both expressing high levels of Kir2.1-AAA-GFP (GFP, green fluorescent protein) subunits in all major parts of the heart. In ventricular myocytes isolated from transgenic mice, I(K1) was reduced by 95% in both lines, leading to a significant prolongation of APs. Surface ECG recordings from anesthetized transgenic mice revealed significant changes in key parameters of excitability, including prolongation of QRS complexes and QT intervals. This study confirms the significant role of I(K1) in control of AP repolarization and major ECG intervals in the intact heart.     

Dual Effect of Nitric Oxide on the Hyperpolarization-activated Inward Current (If) in Sino-atrial Node Cells of the Rabbit

Using the whole-cell voltage-clamp technique, we have investigated the effect of nitric oxide (NO) donor (sodium nitroprusside, SNP) on hyperpolarization-activated inward current,I_f, in isolated rabbit sinoatrial node (SAN) cells.I_fin the basal state increased when NO was applied but decreased whenI_fwas pre-stimulated by isoproterenol (ISO) or by adding cAMP to the pipette solution. Both the stimulatory and the inhibitory effects of NO were abolished by guanylyl cyclase inhibitor, methylene blue (MB), suggesting that the effect of NO is mediated by cGMP. The inhibitory effect of NO was abolished whenI_fwas pre-stimulated by 3-isobutyl-1-methylxanthine (IBMX), which is a phosphodiesterase (PDE) inhibitor, or by adding 8Br-cAMP (which is resistant to PDE) to the pipette solution. An analogue of cGMP, 8Br-cGMP, which is a potent stimulator of cGMP-dependent protein kinase (PKG) but has little effect on PDE, did not inhibitI_fwhenI_fwas pre-stimulated by ISO. In its basal state,I_fwas still increased by 8Br-cGMP, and this effect was not prevented by the pretreatment with H-7, PKG inhibitor. The effect of acetylcholine (ACh) was not identical to that of NOI_fdecreased when pre-stimulated not only by ISO, but also by IBMX. The above results suggest that via cGMP, NO exerts a dual effect onI_f: the inhibitory effect is mediated by cGMP-stimulated PDE, and the stimulatory effect may be attributable to direct binding of cGMP toI_fchannels.     

Effect of 5-HT4 receptor stimulation on the pacemaker current I(f) in human isolated atrial myocytes

OBJECTIVE: 5-HT4 receptors are present in human atrial cells and their stimulation has been implicated in the genesis of atrial arrhythmias including atrial fibrillation. An I(f)-like current has been recorded in human atrial myocytes, where it is modulated by beta-adrenergic stimulation. In the present study, we investigated the effect of serotonin (5-hydroxytryptamine, 5-HT) on I(f) electrophysiological properties, in order to get an insight into the possible contribution of I(f) to the arrhythmogenic action of 5-HT in human atria. METHODS: Human atrial myocytes were isolated by enzymatic digestion from samples of atrial appendage of patients undergoing coeffective cardiac surgery. Patch-clamped cells were superfused with a modified Tyrode's solution in order to amplify I(f) and reduce overlapping currents. RESULTS AND CONCLUSIONS: A time-dependent, cesium-sensitive increasing inward current, that we had previously described having the electrophysiological properties of the pacemaker current I(f), was elicited by negative steps (-60 to -130 mV) from a holding potential of -40 mV. Boltzmann fit of control activation curves gave a midpoint (V1/2) of -88.9 +/- 2.6 mV (n = 14). 5-HT (1 microM) consistently caused a positive shift of V1/2 of 11.0 +/- 2.0 mV (n = 8, p < 0.001) of the activation curve toward less negative potentials, thus increasing the amount of current activated by clamp steps near the physiological maximum diastolic potential of these cells. The effect was dose-dependent, the EC50 being 0.14 microM. Maximum current amplitude was not changed by 5-HT. 5-HT did not increase I(f) amplitude when the current was maximally activated by cAMP perfused into the cell. The selective 5-HT4 antagonists, DAU 6285 (10 microM) and GR 125487 (1 microM), completely prevented the effect of 5-HT on I(f). The shift of V1/2 caused by 1 microM 5-HT in the presence of DAU 6285 or GR 125487 was 0.3 +/- 1 mV (n = 6) and 1.0 +/- 0.6 mV (n = 5), respectively (p < 0.01 versus 5-HT alone). The effect of 5-HT4 receptor blockade was specific, since neither DAU 6285 nor GR 125487 prevented the effect of 1 microM isoprenaline on I(f). Thus, 5-HT4 stimulation increases I(f) in human atrial myocytes; this effect may contribute to the arrhythmogenic action of 5-HT in human atrium.     

Th(IV) Adsorption onto Oxidized Multi-Walled Carbon Nanotubes in the Presence of Hydroxylated Fullerene and Carboxylated Fullerene

The adsorption of Th(IV) onto the surface of oxidized multi-walled carbon nanotubes (oMWCNTs) in the absence and presence of hydroxylated fullerene (C₆₀(OH)_n) and carboxylated fullerene (C₆₀(C(COOH)₂)_n) has been investigated. C₆₀(OH)_n, C₆₀(C(COOH)₂)_n and oMWCNTs have been chosen as model phases because of their representative in carbon nano-materials family. Adsorption experiments were performed by batch procedure as a function of contact time, pH, ionic strength, and temperature. The results demonstrated that the adsorption of Th(IV) was rapidly reached equilibrium and the kinetic process could be described by a pseudo-second-order rate model very well. Th(IV) adsorption on oMWCNTs was dependent on pH but independent on ionic strength. Adsorption isotherms were correlated better with the Langmuir model than with the Freundlich model. The thermodynamic parameters calculated from temperature-dependent adsorption isotherms suggested that Th(IV) adsorption on oMWCNTs was spontaneous and endothermic. Compared with the adsorption of Th(IV) on the same oMWCNTs free of C₆₀(OH)_n or C₆₀(C(COOH)₂)_n, the study of a ternary system showed the inhibition effect of C₆₀(OH)_n at high concentration on the adsorption of Th(IV) in a pH range from neutral to slightly alkaline; whereas the promotion effect of C₆₀(C(COOH)₂)_n, even at its low concentration, on Th(IV) adsorption was observed in acid medium.     

Theoretical investigation of action potential duration dependence on extracellular Ca in human cardiomyocytes

Reduction in [Ca²⁺]_o prolongs the AP in ventricular cardiomyocytes and the QT_c interval in patients. Although this phenomenon is relevant to arrhythmogenesis in the clinical setting, its mechanisms are counterintuitive and incompletely understood. To evaluate in silico the mechanisms of APD modulation by [Ca²⁺]_o in human cardiomyocytes. We implemented the Ten Tusscher-Noble-Noble-Panfilov model of the human ventricular myocyte and modified the formulations of the rapidly and slowly activating delayed rectifier K⁺ currents (I_Kr and I_Ks) and L-type Ca²⁺ current (I_CaL) to incorporate their known sensitivity to intra- or extracellular Ca²⁺. Simulations were run with the original and modified models at variable [Ca²⁺]_o in the clinically relevant 1 to 3 mM range. The original model responds with APD shortening to decrease in [Ca²⁺]_o, i.e. opposite to the experimental observations. Incorporation of Ca²⁺ dependency of K⁺ currents cannot reproduce the inverse relation between APD and [Ca²⁺]_o. Only when I_CaL inactivation process was modified, by enhancing its dependency on Ca²⁺, simulations predict APD prolongation at lower [Ca²⁺]_o. Although Ca²⁺-dependent I_CaL inactivation is the primary mechanism, secondary changes in electrogenic Ca²⁺ transport (by Na⁺/Ca²⁺ exchanger and plasmalemmal Ca²⁺-ATPase) contribute to the reversal of APD dependency on [Ca²⁺]_o. This theoretical investigation points to Ca²⁺-dependent inactivation of I_CaL as a mechanism primarily responsible for the dependency of APD on [Ca²⁺]_o. The modifications implemented here make the model more suitable to analyze repolarization mechanisms when Ca²⁺ levels are altered.     

Ionic basis of ischemia-induced bradycardia in the rabbit sinoatrial node

To investigate the basis of ischemia-induced bradycardia (<60 beats/min), we isolated pacemaker cells from the rabbit sinoatrial node and exposed them to ischemic-like conditions, including omission of glucose, pH 6.6, and either 5.4 or 10 mM KCl to evaluate the role of increased serum [K]. A perforated-patch technique was employed to test the hypothesis that the arrhythmia is caused by attenuation of inward currents that contribute to the diastolic depolarization. After exposure to "ischemic" Tyrode containing 5.4 mM KCl, the pacemaker cells exhibited 13% slower beat rates and action potentials with 6-mV greater overshoots and 44% longer durations. In contrast, after exposure to "ischemic" Tyrode containing 10 mM KCl, the pacemaker cells exhibited a 7-mV depolarization of the maximum diastolic potential but no significant change in the overshoot. Beat rates were slowed by 43%, and the action potentials were prolonged by 46%. "Ischemic" Tyrode containing 5.4 mM KCl increased L-type Ca current, decreased T-type Ca current and reduced Ni-sensitive inward current tails (presumably Na-Ca exchange current), even after treatment with 40 muM ryanodine to block Ca release from the sarcoplasmic reticulum. "Ischemic" Tyrode containing 10 mM KCl increased hyperpolarization-activated inward current at diastolic potentials and reduced the slowly activating component, but not the rapidly activating component, of delayed rectifier K current. Our results suggest that reductions of inward Na-Ca exchange current and T-type Ca current contribute to "ischemia"-induced "bradycardia" in sinoatrial node pacemaker cells.     

Receptors for neuropeptide Y, gamma-aminobutyric acid and dopamine differentially regulate Ca2+ currents in Xenopus melanotrope cells via the G(i) protein beta/gamma-subunit

Secretion of alpha-melanophore-stimulating hormone (alphaMSH) from pituitary melanotrope cells of the amphibian Xenopus laevis is under inhibitory synaptic control by three neurotransmitters produced by the suprachiasmatic nucleus: gamma-aminobutyric acid (GABA), neuropeptide Y (NPY) and dopamine (DA). These inhibitory effects occur through G(i)-protein-coupled receptors (G(i)PCR), and differ in strength: GABA(B)-receptor-induced inhibition is the weakest, whereas DA (via a D2-receptor) and NPY (via a Y1-receptor) strongly inhibit, with NPY having a long-lasting effect. Previously it was shown that DA inhibits two (R- and N-type channel) of the four voltage-operated Ca2+ channels in the melanotrope, and that only part of this inhibition is mediated by beta/gamma-subunits of the G(i) protein. We here demonstrate that also the Y1- and GABA(B)-receptor inhibit only part of the total Ca2+ current (I(Ca)), with fast activation and inactivation kinetics. However, GABA(B)-mediated inhibition is weaker than the inhibitions induced via Y1- and D2-receptors (-21 versus -27% and -30%, respectively). Using a depolarizing pre-pulse protocol it was demonstrated that GABA(B)-induced inhibition of I(Ca) most likely depends on Gbeta/gamma-subunit activation whereas Y1- and D2- induced inhibitions are only partially mediated by Gbeta/gamma-subunits. No differences were found between the Y1- and D2-induced inhibitions. These results imply that activation of different G(i)PCR inhibits the I(Ca) through different mechanisms, a phenomenon that may underlie the different potencies of the suprachiasmatic neurotransmitters to inhibit alphaMSH secretion.     

Ins(1,4,5)P(3) regulates phospholipase Cbeta1 expression in cardiomyocytes

The functional significance of the Ca²⁺-releasing second messenger inositol(1,4,5)trisphosphate (Ins(1,4,5)P₃, IP₃) in the heart has been controversial. Ins(1,4,5)P₃ is generated from the precursor lipid phosphatidylinositol(4,5)bisphosphate (PIP₂) along with sn-1,2-diacylglycerol, and both of these are important cardiac effectors. Therefore, to evaluate the functional importance of Ins(1,4,5)P₃ in cardiomyocytes (NRVM), we overexpressed IP₃ 5-phosphatase to increase degradation. Overexpression of IP₃ 5-phosphatase reduced Ins(1,4,5)P₃ responses to α₁-adrenergic receptor agonists acutely, but with longer stimulation, caused an overall increase in phospholipase C (PLC) activity, associated with a selective increase in expression of PLCβ1, that served to normalise Ins(1,4,5)P₃ content. Similar increases in PLC activity and PLCβ1 expression were observed when Ins(1,4,5)P₃ was sequestered onto the PH domain of PLCδ1, a high affinity selective Ins(1,4,5)P₃-binding motif. These findings suggested that the available level of Ins(1,4,5)P₃ selectively regulates the expression of PLCβ1. Cardiac responses to Ins(1,4,5)P₃ are mediated by type 2 IP₃-receptors. Hearts from IP₃-receptor (type 2) knock-out mice showed heightened PLCβ1 expression. We conclude that Ins(1,4,5)P₃ and IP₃-receptor (type 2) regulate PLCβ1 and thereby maintain levels of Ins(1,4,5)P₃. This implies some functional significance for Ins(1,4,5)P₃ in the heart.     

Functional inhibitory human leucocyte antigen class I receptors on natural killer (NK) cells in patients with chronic NK lymphocytosis

Chronic natural killer (NK) lymphocytosis is a rare disorder characterized by an indolent clinical course. Despite high NK cell numbers, many patients present with only mild clinical symptoms, and are often asymptomatic. NK cells are equipped with a range of receptors that bind human leucocyte antigen (HLA)-class I molecules. The killer immunoglobulin-like receptors (KIR, CD158) bind groups of HLA alleles, the CD94/NKG2 receptors bind HLA-E, and the CD85j (ILT2, LIR-1) receptor binds to the relatively non-polymorphic alpha3 domain of HLA molecules. Inhibitory HLA class I receptors silence NK cells against cells expressing normal levels of HLA class I. Analysis of NK cells in six patients with chronic NK lymphocytosis revealed a high level of the inhibitory CD94/NKG2A receptor on all NK cells. In four patients, KIR were absent, in one patient a single KIR was expressed in the absence of self-ligand, and in one patient CD85j and multiple KIR were expressed. Cytotoxicity assays demonstrated that all HLA class I receptors were functional. The ability of monoclonal antibodies to block the receptors and allow killing of autologous target cells established that both receptor and ligand expression were adequate for inhibitory function. We propose that the silent behaviour of NK cells in patients with chronic NK lymphocytosis is due to effective inhibitory HLA class I receptors.     

Immunohistochemical detection of pregnancy and placental proteins in human and cynomolgus monkey (macaca fascicularis) peripheral blood cells

Summary With use of an enzyme-bridge immunoperoxidase method, the localization of pregnancy-specific ₁-glycoprotein (SP₁), pregnancy-associated proteins (SP₂, SP₃) and placental tissue proteins (FSF, PP₁, PP₂ and PP₄ to PP₁₂) was investigated in human and cynomolgus peripheral blood cells.Blood cells of cynomolgus monkeys revealed the same staining results as in man: SP₁, SP₂, SP₃, PP₂, PP₄, PP₆, PP₇, PP₈, PP₁₀ and PP₁₂ were found to be positive in the polymorphonuclear neutrophils. Monocyte macrophages stained positively only for PP₂ and PP₁₀. Lymphocytes and erythrocytes did not stain positively for any of the proteins examined.A much stronger positive staining for SP₂ and SP₃ was found in the polymorphonuclear neutrophils from a human female taking contraceptive pills and pregnant cynomolgus monkeys. Otherwise there was no difference in staining results irrespective of whether the specimens were from males, or pregnant or non-pregnant females.The results reported here indicate that the so-called placenta-specific proteins SP₁, PP₁₀ and PP₁₂ are apparently also synthesized by normal blood cells and appear to be not specific to the placenta. Only PP₅ and PP₁₁ seem to be highly specific to the trophoblast.     

Fluorescent protein complementation assays: new tools to study G protein-coupled receptor oligomerization and GPCR-mediated signaling

G protein-coupled receptor (GPCR) signaling is mediated by protein–protein interactions at multiple levels. The characterization of the corresponding protein complexes is therefore paramount to the basic understanding of GPCR-mediated signal transduction. The number of documented interactions involving GPCRs is rapidly growing, and appreciating the functional significance of these complexes is clearly the next challenge. New experimental approaches including protein complementation assays (PCAs) have recently been used to examine the composition, plasma membrane targeting, and desensitization of protein complexes involved in GPCR signaling. These methods also hold promise for better understanding of drug-induced effects on GPCR interactions. This review focuses on the application of fluorescent PCAs for the study of GPCR signaling. Potential applications of PCAs in high-content screens are also presented. Non-fluorescent PCA techniques as well as combined assays for the detection of ternary and quaternary protein complexes are briefly discussed.