The atypical antipsychotics clozapine and olanzapine promote down-regulation and display functional selectivity at human 5-HT7 receptors.

BACKGROUND AND PURPOSE: Classically, ligands of GPCRs have been classified primarily upon their affinity and efficacy to activate a signal transduction pathway. Recent reports indicate that the efficacy of a particular ligand can vary depending on the receptor-mediated response measured (e.g. activating G proteins, other downstream responses, internalization). Previously, we reported that inverse agonists induce both homo- and heterologous desensitization, similar to agonist stimulation, at the Gs -coupled 5-HT7 receptor. The primary objective of this study was to determine whether different inverse agonists at the 5-HT7 receptor also induce internalization and/or degradation of 5-HT7 receptors. EXPERIMENTAL APPROACH: HEK293 cells expressing 5-HT7(a, b or d) receptors were pre-incubated with 5-HT, clozapine, olanzapine, mesulergine or SB269970 and their effects upon receptor density, AC activity, internalization, recruitment of ß-arrestins and lysosomal trafficking were measured. KEY RESULTS: The agonist 5-HT and three out of four inverse agonists tested increased internalization independently of ß-arrestin recruitment. Among these, only the atypical antipsychotics clozapine and olanzapine promoted lysosomal sorting and reduced 5-HT7 receptor density (∼60% reduction within 24 h). Inhibition of lysosomal degradation with chloroquine blocked the clozapine- and olanzapine-induced down-regulation of 5-HT7 receptors. Incubation with SB269970 decreased both 5-HT7(b) constitutive internalization and receptor density but increased 5-HT7(d) receptor density, indicating differential ligand regulation among the 5-HT7 splice variants. CONCLUSIONS AND IMPLICATIONS: Taken together, we found that various ligands differentially activate regulatory processes governing receptor internalization and degradation in addition to signal transduction. Thus, these data extend our understanding of functional selectivity at the 5-HT7 receptor.

Inhibition of phosphodiesterase-3 by levosimendan is sufficient to account for its inotropic effect in failing human heart.

BACKGROUND AND PURPOSE: Levosimendan is known as a calcium sensitizer, although it is also known to inhibit PDE3. We aimed to isolate each component and estimate their contribution to the increased cardiac contractility induced by levosimendan. EXPERIMENTAL APPROACH: Contractile force was measured in electrically stimulated ventricular strips from explanted failing human hearts and left ventricular strips from normal male Wistar rats. PDE activity was measured in a two-step PDE activity assay on failing human ventricle. KEY RESULTS: Levosimendan exerted a positive inotropic effect (PIE) reaching maximum at 10(-5) M in ventricular strips from failing human hearts. In the presence of the selective PDE3 inhibitor cilostamide, the PIE of levosimendan was abolished. During treatment with a PDE4 inhibitor and a supra-threshold concentration of isoprenaline, levosimendan generated an amplified inotropic response. This effect was reversed by ß-adrenoceptor blockade and undetectable in strips pretreated with cilostamide. Levosimendan (10(-6) M) increased the potency of ß-adrenoceptor agonists by 0.5 log units in failing human myocardium, but not in the presence of cilostamide. Every inotropic response to levosimendan was associated with a lusitropic response. Levosimendan did not affect the concentration-response curve to calcium in rat ventricular strips, in contrast to the effects of a known calcium sensitizer, EMD57033 [5-(1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydroquinolin-6-yl)-6-methyl-3,6-dihydro-2H-1,3,4-thiadiazin-2-one]. PDE activity assays confirmed that levosimendan inhibited PDE3 as effectively as cilostamide. CONCLUSIONS AND IMPLICATIONS: Our results indicate that the PDE3-inhibitory property of levosimendan was enough to account for its inotropic effect, leaving a minor, if any, effect to a calcium-sensitizing component.

SERCA2 activity is involved in the CNP-mediated functional responses in failing rat myocardium.

BACKGROUND AND PURPOSES: Myocardial C-type natriuretic peptide (CNP) levels are increased in heart failure. CNP can induce negative inotropic (NIR) and positive lusitropic responses (LR) in normal hearts, but its effects in failing hearts are not known. We studied the mechanism of CNP-induced NIR and LR in failing hearts and determined whether sarcoplasmatic reticulum Ca(2+) ATPase2 (SERCA2) activity is essential for these responses. EXPERIMENTAL APPROACH: Contractility, cGMP levels, Ca(2+) transient amplitudes and protein phosphorylation were measured in left ventricular muscle strips or ventricular cardiomyocytes from failing hearts of Wistar rats 6 weeks after myocardial infarction. KEY RESULTS: CNP increased cGMP levels, evoked a NIR and LR in muscle strips, and caused phospholamban (PLB) Ser(16) and troponin I (TnI) Ser(23/24) phosphorylation in cardiomyocytes. Both the NIR and LR induced by CNP were reduced in the presence of a PKG blocker/cGMP analogue (Rp-8-Br-Pet-cGMPS) and the SERCA inhibitor thapsigargin. CNP increased the amplitude of the Ca(2+) transient and increased SERCA2 activity in cardiomyocytes. The CNP-elicited NIR and LR were not affected by the L-type Ca(2+) channel activator BAY-K8644, but were abolished in the presence of isoprenaline (induces maximal activation of cAMP pathway). This suggests that phosphorylation of PLB and TnI by CNP causes both a NIR and LR. The NIR to CNP in mouse heart was abolished 8 weeks after cardiomyocyte-specific inactivation of the SERCA2 gene. CONCLUSIONS AND IMPLICATIONS: We conclude that CNP-induced PLB and TnI phosphorylation by PKG in concert mediate both a predictable LR as well as the less expected NIR in failing hearts.

The functional activity of inhibitory G protein (G(i)) is not increased in failing heart ventricle.

Beta-adrenergic receptor (ßAR) inotropic effects are attenuated and muscarinic receptor-mediated inhibition thereof is enhanced in heart failure. We investigated if increased G(i) activity contributes to attenuated ßAR-inotropic effects and potentiates muscarinic accentuated antagonism in failing rat ventricle. Contractility was measured in ventricular strips and adenylyl cyclase (AC) activity in ventricular membranes from rats with post-infarction heart failure (HF) or Sham-operated controls (Sham). The maximal ßAR-mediated inotropic effect of isoproterenol was reduced by ~70% and basal, ßAR- & forskolin-stimulated AC activity was significantly lower in HF vs. Sham. Carbachol-evoked antagonism of the ßAR-mediated inotropic response was complete only in HF despite a ~40% reduction in the ability of carbachol to inhibit ßAR-stimulated AC. However, neither the relative efficacy (contractility decreased by ~46%) nor the potency of carbachol to inhibit the ßAR inotropic response differed between Sham and HF ventricle. Pertussis toxin (PTX) inactivation of G(i) did not increase the maximal ßAR inotropic effect or the attenuated basal, ßAR- & forskolin-stimulated AC activity in HF, but increased the potency of isoproterenol only in Sham (~0.5 log unit). In HF ventricle pretreated with PTX, simultaneous inhibition of phosphodiesterases 3,4 (PDE3,4) alone produced a larger inotropic response than isoproterenol in ventricle untreated with PTX (84% and 48% above basal respectively). In the absence of PTX, PDE3,4 inhibition evoked negligible inotropic effects in HF. These data are not consistent with the hypothesis that increased G(i) activity contributes to the reduced ßAR-mediated inotropic response and AC activity in failing ventricle. The data, however, support the hypothesis that G(i), through chronic receptor independent inhibition of AC, together with PDE3,4 activity, is necessary to maintain a low basal level of contractility.

Cyclic AMP-dependent inotropic effects are differentially regulated by muscarinic G(i)-dependent constitutive inhibition of adenylyl cyclase in failing rat ventricle.

BACKGROUND AND PURPOSE: ß-Adrenoceptor (ß-AR)-mediated inotropic effects are attenuated and G(i) proteins are up-regulated in heart failure (HF). Muscarinic receptors constitutively inhibit cAMP formation in normal rat cardiomyocytes. We determined whether constitutive activity of muscarinic receptors to inhibit adenylyl cyclase (AC) increases in HF and if so, whether it modifies the reduced ß-AR- or emergent 5-HT4-mediated cAMP-dependent inotropic effects. EXPERIMENTAL APPROACH: Contractility and AC activity were measured and related to each other in rat ventricle with post-infarction HF and sham-operated (Sham) controls with or without blockade of muscarinic receptors by atropine and inactivation of G(i) protein by pertussis toxin (PTX). KEY RESULTS: Isoprenaline-mediated inotropic effects were attenuated and basal, isoprenaline- and forskolin-stimulated AC activity was reduced in HF compared with Sham. Atropine or PTX pretreatment increased forskolin-stimulated AC activity in HF hearts. ß-AR-stimulated AC and maximal inotropic response were unaffected by atropine in Sham and HF. In HF, the potency of serotonin (5-HT) to evoke an inotropic response was increased in the presence of atropine with no change in the maximal inotropic response. Interestingly, PTX pretreatment reduced the potency of 5-HT to evoke inotropic responses while increasing the maximal inotropic response. CONCLUSIONS AND IMPLICATIONS: Although muscarinic constitutive inhibition of AC is increased in HF, it does not contribute to the reduced ß-AR-mediated inotropic effects in rat ventricle in HF. The data support the hypothesis that there are differences in the functional compartmentation of 5-HT4 and ß-AR AC signalling in myocardium during HF.

Activation of muscarinic receptors elicits inotropic responses in ventricular muscle from rats with heart failure through myosin light chain phosphorylation.

BACKGROUND AND PURPOSE: Muscarinic stimulation increases myofilament Ca(2+) sensitivity with no apparent inotropic response in normal rat myocardium. Increased myofilament Ca(2+) sensitivity is a molecular mechanism promoting increased contractility in failing cardiac tissue. Thus, muscarinic receptor activation could elicit inotropic responses in ventricular myocardium from rats with heart failure, through increasing phosphorylation of myosin light chain (MLC). EXPERIMENTAL APPROACH: Contractile force was measured in left ventricular papillary muscles from male Wistar rats, 6 weeks after left coronary artery ligation or sham surgery. Muscles were also frozen, and MLC-2 phosphorylation level was quantified. KEY RESULTS: Carbachol (10 micromol.L(-1)) evoked a positive inotropic response only in muscles from rats with heart failure approximating 36% of that elicited by 1 micromol.L(-1) isoproterenol (20 +/- 1.5% and 56 +/- 6.1% above basal respectively). Carbachol-evoked inotropic responses did not correlate with infarction size but did correlate with increased left ventricular end diastolic pressure, heart weight/body weight ratio and lung weight, primary indicators of the severity of heart failure. Only muscarinic receptor antagonists selective for M(2) receptors antagonized carbachol-mediated inotropic effects with the expected potency. Carbachol-evoked inotropic responses and increase in phosphorylated MLC-2 were attenuated by MLC kinase (ML-9) and Rho-kinase inhibition (Y-27632), and inotropic responses were abolished by Pertussis toxin pretreatment. CONCLUSION AND IMPLICATIONS: In failing ventricular muscle, muscarinic receptor activation, most likely via M(2) receptors, provides inotropic support by increasing MLC phosphorylation and consequently, myofilament Ca(2+) sensitivity. Enhancement of myofilament Ca(2+) sensitivity, representing a less energy-demanding mechanism of inotropic support may be particularly advantageous in failing hearts.

5-HT4-elicited positive inotropic response is mediated by cAMP and regulated by PDE3 in failing rat and human cardiac ventricles.

BACKGROUND AND PURPOSE: The left ventricle in failing hearts becomes sensitive to 5-HT parallelled by appearance of functional G(s)-coupled 5-HT(4) receptors. Here, we have explored the regulatory functions of phosphodiesterases in the 5-HT(4) receptor-mediated functional effects in ventricular muscle from failing rat and human heart. EXPERIMENTAL APPROACH: Extensive myocardial infarctions were induced by coronary artery ligation in Wistar rats. Contractility was measured in left ventricular papillary muscles of rat, 6 weeks after surgery and in left ventricular trabeculae from explanted human hearts. cAMP was quantified by RIA. KEY RESULTS: In papillary muscles from postinfarction rat hearts, 5-HT(4) stimulation exerted positive inotropic and lusitropic effects and increased cAMP. The inotropic effect was increased by non-selective PDE inhibition (IBMX, 10 microM) and selective inhibition of PDE3 (cilostamide, 1 microM), but not of PDE2 (EHNA, 10 microM) or PDE4 (rolipram, 10 microM). Combined PDE3 and PDE4 inhibition enhanced inotropic responses beyond the effect of PDE3 inhibition alone, increased the sensitivity to 5-HT, and also revealed an inotropic response in control (sham-operated) rat ventricle. Lusitropic effects were increased only during combined PDE inhibition. In failing human ventricle, the 5-HT(4) receptor-mediated positive inotropic response was regulated by PDEs in a manner similar to that in postinfarction rat hearts. CONCLUSIONS AND IMPLICATIONS: 5-HT(4) receptor-mediated positive inotropic responses in failing rat ventricle were cAMP-dependent. PDE3 was the main PDE regulating this response and involvement of PDE4 was disclosed by concomitant inhibition of PDE3 in both postinfarction rat and failing human hearts. 5-HT, PDE3 and PDE4 may have pathophysiological functions in heart failure.

Novel aspects of G-protein-coupled receptor signalling--different ways to achieve specificity.

Our understanding of signal transduction via G-protein-coupled receptors (GPCR) has developed dramatically during the last decades. The initial idea of linear signalling pathways transferring information from the cell membrane to the nucleus has evolved into a complicated network of signalling pathways offering the possibility of crosstalk, fine tuning and specific regulation at multiple levels. During the recent meeting on GPCRs at the Karolinska Institutet, Stockholm novel aspects of GPCR signalling were presented and discussed. Here, we will discuss several possibilities for GPCRs to achieve specificity in signal transduction, such as the phenomenon of biased agonism, receptor multimerization, the role of co-receptors, the regulation of heterotrimeric G proteins as well as multiple G(s)-dependent pathways to extracellular single-regulated protein kinases.

Effects of treatment with a 5-HT4 receptor antagonist in heart failure.

BACKGROUND AND PURPOSE: Positive inotropic responses (PIR) to 5-hydroxytryptamine (5-HT) are induced in the left ventricle (LV) in rats with congestive heart failure (CHF); this is associated with upregulation of the G(s)-coupled 5-HT(4) receptor. We investigated whether chronic 5-HT(4) receptor blockade improved cardiac function in CHF rats. EXPERIMENTAL APPROACH: Rats were given either the 5-HT(4) antagonist SB207266 (0.5 mg kg(-1) 24h(-1); MI(int)) or placebo (MI(pl)) through mini-osmotic pumps for 6 weeks subsequent to induction of post-infarction CHF. In vivo cardiac function and ex vivo responses to isoprenaline or 5-HT were evaluated using echocardiography and isolated LV papillary muscles, respectively. mRNA levels were investigated using real-time quantitative RT-PCR. KEY RESULTS: LV diastolic function improved, with 4.6% lower LV diastolic diameter and 24.2% lower mitral flow deceleration in MI(int) compared to MI(pl). SB207266 reduced LV systolic diameter by 6.1%, heart weight by 10.2% and lung weight by 13.1%. The changes in posterior wall thickening and shortening velocity, cardiac output, LV systolic pressure and (dP/dt)(max), parameters of LV systolic function, did not reach statistical significance. The PIR to isoprenaline (10 microM) increased by 36% and the response to 5-HT (10 microM) decreased by 57% in MI(int) compared to MI(pl). mRNA levels for ANP, 5-HT(4(b)) and 5-HT(2A) receptors, MHCbeta, and the MHCbeta/MHCalpha -ratio were not significantly changed in MI(int) compared to MI(pl). CONCLUSIONS AND IMPLICATIONS: Treatment with SB207266 to some extent improved in vivo cardiac function and ex vivo myocardial function, suggesting a possible beneficial effect of treatment with a 5-HT(4) receptor antagonist in CHF.

The cloned human 5-HT7 receptor splice variants: a comparative characterization of their pharmacology, function and distribution.

Serotonin (5-hydroxytryptamine, 5-HT) receptor pre-mRNA is alternatively spliced in human tissue to produce three splice variants, h5-HT7(a), h5-HT7(b) and h5-HT7(d), which differ only in their carboxyl terminal tails. Using membranes from transiently and stably transfected HEK293 cells expressing the three recombinant h5-HT7 splice variants we compared their pharmacological profiles and ability to activate adenylyl cyclase. Using PCR on cDNA derived from various human tissues, the 5-HT7(a) and 5-HT7(b) splice variants were detected in every tissue examined. The h5-HT7(d) splice variant was detected in 13 of 16 tissues examined, with predominant expression in the heart, small intestine, colon, ovary and testis. All three h5-HT7 splice variants displayed high affinity binding for [3H]5-HT (pKd=8.8-8.9) in the presence and absence of 100 microM GTP and had similar binding affinities for all 17 ligands evaluated. In HEK293 cells expressing similar, high levels of receptor (approximately 10,000 fmol/mg protein), 5-CT (5-carboxamidotryptamine), 5-MeOT (5-methoxytryptamine) and 5-HT were full agonists while 8-OH-DPAT ((2R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin) was a partial agonist with relative efficacy of approximately 0.8. Even at this high receptor level, EC50 values for stimulation of adenylyl cyclase were 10- to 50-fold higher than the Kd values, indicating a lack of spare receptors. No significant differences in coupling to adenylyl cyclase were observed between the three splice variants over a wide range of receptor expression levels. For antagonists, binding affinities determined by displacement of [3H]5-HT binding and by competitive inhibition of 5-HT-stimulated adenylyl cyclase activity were essentially identical amongst the splice variants. These studies indicate that the three human splice variants are pharmacologically indistinguishable and that modifications of the carboxyl tail do not influence coupling to adenylyl cyclase.

5HT4(a) and 5-HT4(b) receptors have nearly identical pharmacology and are both expressed in human atrium and ventricle.

5-Hydroxytryptamine (5-HT) increases human heart rate and atrial contractile force and hastens atrial relaxation through 5-HT4 receptors. Moreover, 5-HT may be arrhythmogenic and give rise to atrial fibrillation. It is not clear which splice variant(s) of the 5-HT4 receptor is expressed and mediates these effects of 5-HT in the human heart. Previous studies have indicated different pharmacological properties of 5-HT4 receptors in human heart and mouse colliculi neurones, possibly due to expression of different splice variants. We therefore cloned the human 5-HT4(b) receptor and compared its pharmacological properties with those of the cloned human 5-HT4(a) receptor and searched for the corresponding mRNA in human tissues. The primary structures of the two human 5-HT4 receptor splice variants are identical except for divergent C-terminal tails of 28 and 29 amino acids in the 5-HT4(a) and 5-HT4(b) receptors, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that both variants were coexpressed in various tissues, including cardiac atrium and ventricle. Additional bands suggested the presence of more than two human 5-HT4 receptor splice variants. With cloned receptors stably expressed in HEK293 cells or transiently expressed in COS-7 cells, [3H]GR 113808 consistently showed slightly higher binding affinity to h5-HT4(b) than to h5-HT4(a) receptor (pKd 0.1-0.2 log units higher). Competition of agonists, partial agonists and antagonists for [3H]GR113808 binding revealed no significant differences between the two receptors. For 5-HT4 receptor agonists and antagonists, their potencies in stimulating or inhibiting, respectively, 5-HT-stimulated adenylyl cyclase activity correlated well with their binding affinities. Tropisetron and SB207710 showed partial agonist activity at high receptor expression levels for both isoforms. Cisapride and renzapride were both partial agonists at moderate receptor levels and full agonists at high receptor levels. Cisapride was more potent than renzapride while the converse was the case in human atrium, for which cisapride had lower affinity and agonist potency than at the recombinant receptors. The binding affinities and agonist potencies of ligands for both 5-HT4(a) and 5-HT4(b) receptors correlated with the corresponding affinities and potencies in human atrium. The agonist potency of SB207710 was around 10 times lower than its binding and blocking affinity for both splice variants, suggesting that activation of adenylyl cyclase and blockade of 5-HT responses are mediated through different conformational states. The similar pharmacological properties of the two human 5-HT4 receptor splice variants together with their expression in human atrium would be consistent with mediation of the cardiostimulant effects of 5-HT through both variants. However, the effects of cisapride appear either mediated through non-a and non-b splice variants of the 5-HT4 receptor or 5-HT4(a) and 5-HT4(b) receptor expression in human atrial cells alters somewhat their pharmacological profile through still unknown mechanisms.

Activation of the COOH-terminal Src kinase (Csk) by cAMP-dependent protein kinase inhibits signaling through the T cell receptor.

In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor-CD3 complex (TCR/CD3) and inhibits T cell function via a previously unknown proximal target. Here we examine the mechanism for this PKA-mediated immunomodulation. cAMP treatment of Jurkat and normal T cells reduces Lck-mediated tyrosine phosphorylation of the TCR/CD3 zeta chain after T cell activation, and decreases Lck activity. Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on zeta chain phosphorylation. PKA phosphorylates Csk at S364 in vitro and in vivo leading to a two- to fourfold increase in Csk activity that is necessary for cAMP-mediated inhibition of TCR-induced interleukin 2 secretion. Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin. We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation.

Identification of the adenylyl cyclase-activating 5-hydroxytryptamine receptor subtypes expressed in the rat submandibular gland.

1. Serotonin (5-hydroxytryptamine, 5-HT) has been shown to increase cyclic AMP production in dispersed cell aggregates from the major salivary glands of the rat. The goal of the present study was to identify the 5-HT receptor subtypes that mediate these effects in rat submandibular glands (SMG). 2. Among the 5-HT receptor subtypes identified in the rat, 5-HT(4(a,b)), 5-HT(6) and 5-HT(7(a,b,c)) activate adenylyl cyclase (AC). We used subtype specific primers to screen rat SMG by reverse transcription-PCR. Results indicate the presence of mRNA for 5-HT(4(b)) and 5-HT(7(a)) but not for 5-HT(4(a)), 5-HT(6) and 5-HT(7(b,c)). 3. In dispersed SMG cells, 5-carboxyamidotryptamine (5-CT), a 5-HT(7) receptor agonist, stimulated cyclic AMP synthesis with higher potency (EC(50)=27+/-5 nM) but lower efficacy than 5-HT, suggesting a 5-HT(7) component and an additional component in the response to 5-HT. The 5-HT(7) contribution was further supported by antagonism of the 5-CT effect by metergoline, a 5-HT(7) antagonist, which exhibited an affinity (K(i)=50 nM) similar to that obtained at the cloned 5-HT(7) receptor. 4. In the presence of a maximally effective concentration of 5-CT, 5-HT produced an additional increase in cyclic AMP production that was inhibited by the 5-HT(4) receptor antagonist, GR113808, suggesting that the second component of cyclic AMP production is mediated by 5-HT(4) receptors. 5. These findings indicate the presence in rat SMG of both 5-HT(4(b)) and 5-HT(7(a)) receptors positively coupled to AC.

Ephrin-B2 is a candidate ligand for the Eph receptor, EphB6.

No ligand has hitherto been designated for the Eph receptor tyrosine kinase family member, EphB6. Here, expression of an EphB6 ligand in the pro-B leukemic cell line, Reh, is demonstrated by binding of soluble EphB6-Fc fusion protein to the Reh cells. The ligand belongs to the subgroup of membrane spanning ligands, as suggested by the fact that phosphatidylinositol-specific phospholipase C treatment did not abrogate binding of EphB6-Fc. Two transmembrane Eph receptor ligands, ephrin-B1 and ephrin-B2, were identified in Reh cells. Analysis of EphB6-Fc fusion protein binding to ephrin-B1 or ephrin-B2 transfected COS cells revealed a high-affinity saturable binding between EphB6-Fc and ephrin-B2, but not with ephrin-B1. In mice, EphB6 has previously been shown to be expressed in thymus. Here, we show expression of EphB6 in human thymus, as well as the expression of ephrin-B2 in both human and mouse thymus. We conclude that ephrin-B2 may be a physiological ligand for the EphB6 receptor.

Activation of the cAMP signaling pathway increases apoptosis in human B-precursor cells and is associated with downregulation of Mcl-1 expression.

During B- and T-cell ontogeny, extensive apoptosis occurs at distinct stages of development. Agents that increase intracellular levels of cAMP induce apoptosis in thymocytes and mature B cells, prompting us to investigate the role of cAMP signaling in human CD10+ B-precursor cells. We show for the first time that forskolin (which increases intracellular levels of cAMP) increases apoptosis in the CD10- cells in a dose-dependent manner (19%-94% with 0-1,000 microM forskolin after 48 hours incubation, IC50 = 150 microM). High levels of apoptosis were also obtained by exposing the cells to the cAMP analogue 8-chlorophenylthio-cAMP (8-CPT-cAMP). Specific involvement of cAMP-dependent protein kinase (PKA) was demonstrated by the ability of a cAMP antagonist, Rp-isomer of 8-bromo-adenosine- 3', 5'- monophosphorothioate (Rp-8-Br-cAMPS), to reverse the apoptosis increasing effect of the complementary cAMP agonist, Sp-8-Br-cAMPS. Furthermore, we investigated the expression of Bcl-2 family proteins. We found that treatment of the cells with forskolin or 8-CPT-cAMP for 48 hours resulted in a fourfold decline in the expression of Mcl-1 (n = 6, P = 0.002) compared to control cells. The expression of Bcl-2, Bcl-xL, or Bax was largely unaffected. Mature peripheral blood B cells showed a smaller increase in the percentage of apoptotic cells in response to 8-CPT-cAMP (1.3-fold, n = 6, P = 0.045) compared to B-precursor cells, and a smaller decrease in Mcl-1 levels (1.5-fold, n = 4, P = 0.014). Taken together, these findings show that cAMP is important in the regulation of apoptosis in B-progenitor and mature B cells and suggest that cAMP-increased apoptosis could be mediated, at least in part, by a decrease in Mcl-1 levels.

Isozymes of cyclic AMP-dependent protein kinases (PKA) in human lymphoid cell lines: levels of endogenous cAMP influence levels of PKA subunits and growth in lymphoid cell lines.

Activation of the cAMP signaling pathway in lymphoid cells is known to inhibit cell proliferation of T and B cells as well as cytotoxicity of natural killer (NK) cells. In order to find suitable model systems to study cAMP-mediated processes, we have examined the expression of cAMP-dependent protein kinase (PKA), endogenous levels of cAMP, and cell proliferation in eight cell lines of B lineage origin, four cell lines of T lineage origin, and normal human B and T cells. We demonstrated that the expression of mRNA and protein for one of the regulatory (R) subunits of PKA (RIalpha) was present in all the cells investigated, in contrast to the other R subunits (RIbeta, RIIalpha, and RIIbeta). Furthermore, three T cell lines and one B cell line expressed only RIalpha and C, implying these cells to contain solely PKA type I. Moreover, for the RI subunit, we observed an apparent reciprocal relationship between levels of mRNA and protein. Generally, RIalpha protein was low in cell lines where mRNA was elevated and vice versa. This was not the case for the RII subunits, where high levels of mRNA were associated with elevated levels of protein. Interestingly, we demonstrated an inverse correlation between levels of endogenous cAMP and cell growth as determined by [3H]-thymidine incorporation and cell-doubling rate (P < 0.05). Taken together, our results demonstrate great differences in PKA isozyme composition, which should be taken into consideration when using lymphoid cell lines as model system for cAMP/PKA effects in normal lymphocytes.

Kinetic properties of the C-terminal Src kinase, p50csk.

Csk is an important regulator of tyrosine kinases of the Src family. In this paper, we have characterised the kinetics and catalytic properties of a highly active and stable enzyme obtained in milligram amounts by expressing the enzyme as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli. Using the synthetic polyamino acid poly(Glu, Tyr) as substrate, phosphotransferase activity was linear for 7-8 min with Mg2+ and 5 min with Mn2+. With Mg2+ and Mn2+, respectively, K(m) (ATP) was 56.9 +/- 6.2 and 5.4 +/- 0.6 microM and Vmax was 293 +/- 52 and 217 +/- 38 pmol phosphate transferred (microgram Csk)-1 min-1. Optimal concentrations of Mg2+ and Mn2+ were 4-10 mM and 2-3 mM, respectively, and higher concentrations of both cations were inhibitory. The Csk activity was highly sensitive to monovalent (Na+, K+) and divalent (Ca2+) cations, the sensitivity being 2-5-fold higher with Mg2+ than Mn2+. Physiological concentrations of Ca2+ (less than 10 microM) were without effect. Autophosphorylation of Csk was demonstrated in vitro, but did not influence the catalytic activity. Addition of inorganic phosphate above 100 microM strongly inhibited Csk catalytic activity towards poly(Glu, Tyr) in the presence of Mn2+, but not in the presence of Mg2+. Phosphorylation of a physiological substrate (Lck) and autophosphorylation of Csk was not inhibited by phosphate, indicating that the phosphate-dependent inhibition of Csk activity was substrate specific.

High-affinity binding of epidermal growth factor (EGF) to EGF receptor is disrupted by overexpression of mutant dynamin (K44A).

Activation of the epidermal growth factor receptor (EGFR) kinase was analyzed in cells conditionally defective for clathrin-dependent endocytosis by overexpression of mutant dynamin (K44A). EGF-induced autophosphorylation of the EGFR on ice was strongly reduced in cells overexpressing mutant dynamin, and consistently, binding analyses showed that high-affinity EGFRs were lost. In the absence of mutant dynamin the cells displayed both high- and low-affinity EGFR. At 4 degreesC EGF-EGFR localized mainly outside coated pits regardless of expression of mutant dynamin. However, also low-affinity EGFR efficiently moved to coated pits upon incubating cells at 37 degreesC. Thus, expression of mutant dynamin disrupts high-affinity binding of EGF, but not ligand-induced recruitment of EGFR to clathrin-coated pits.

Selective activation of cAMP-dependent protein kinase type I inhibits rat natural killer cell cytotoxicity.

The present study examines the expression and involvement of cAMP-dependent protein kinase (PKA) isozymes in cAMP-induced inhibition of natural killer (NK) cell-mediated cytotoxicity. Rat interleukin-2-activated NK cells express the PKA alpha-isoforms RIalpha, RIIalpha, and Calpha and contain both PKA type I and type II. Prostaglandin E2, forskolin, and cAMP analogs all inhibit NK cell lysis of major histocompatibility complex class I mismatched allogeneic lymphocytes as well as of standard tumor target cells. Specific involvement of PKA in the cAMP-induced inhibition of NK cell cytotoxicity is demonstrated by the ability of a cAMP antagonist, (Rp)-8-Br-adenosine 3',5'-cyclic monophosphorothioate, to reverse the inhibitory effect of complementary cAMP agonist (Sp)-8-Br-adenosine 3',5'-cyclic monophosphorothioate. Furthermore, the use of cAMP analog pairs selective for either PKA isozyme (PKA type I or PKA type II), shows a preferential involvement of the PKA type I isozyme, indicating that PKA type I is necessary and sufficient to completely abolish killer activatory signaling leading to NK cell cytotoxicity. Finally, combined treatment with phorbol ester and ionomycin maintains NK cell cytotoxicity and eliminates the cAMP-mediated inhibition, demonstrating that protein kinase C and Ca2+-dependent events stimulate the cytolytic activity of NK cells at a site distal to the site of cAMP/PKA action.

Structure, function, and regulation of human cAMP-dependent protein kinases.

A large number of hormones, neurotransmitters, and other signaling substances that bind to G-protein-coupled cell-surface receptors have their signals converge at one sole second messenger, cAMP. The question of how specificity can be maintained in a signal-transduction system in which many extracellular signals leading to a vast array of intracellular responses are all mediated through one second-messenger system has been the subject of thorough investigation and a great deal of speculation. An increasing number of cAK isozymes, consisting of homo- or heterodimers of R subunits (RIalpha, RIbeta, RIIalpha, RIIbeta) with associated catalytic subunits (C alpha, Cbeta, Cgamma), may, at least in part, explain this specificity. The various cAK isozymes display distinct biochemical properties, and the heterogeneous subunits of cAK reveal cell-specific expression and differential regulation at the level of gene transcription, mRNA stability, and protein stability in response to a wide range of hormones and other signaling substances. The existence of a number of anchoring proteins specific to either RIIalpha or RIIbeta, and which localize cAKII isozymes toward distinct substrates at defined subcellular loci, strongly supports the idea that specific functions can be assigned to the various cAK isozymes. The demonstration that selective activation of cAKI is necessary and sufficient for cAMP-mediated inhibition of T-cell proliferation, and the observation that T-cell activation is associated with redistribution and colocalization of cAKI to the TCR, is also compatible with the notion of isozyme-specific effects.