Early activation of the cardiac CX3CL1/CX3CR1 axis delays ß-adrenergic-induced heart failure.

We recently highlighted a novel potential protective paracrine role of cardiac myeloid CD11b/c cells improving resistance of adult hypertrophied cardiomyocytes to oxidative stress and potentially delaying evolution towards heart failure (HF) in response to early ß-adrenergic stimulation. Here we characterized macrophages (Mφ) in hearts early infused with isoproterenol as compared to control and failing hearts and evaluated the role of upregulated CX3CL1 in cardiac remodeling. Flow cytometry, immunohistology and Mφ-depletion experiments evidenced a transient increase in Mφ number in isoproterenol-infused hearts, proportional to early concentric hypertrophy (ECH) remodeling and limiting HF. Combining transcriptomic and secretomic approaches we characterized Mφ-enriched CD45+ cells from ECH hearts as CX3CL1- and TNFα-secreting cells. In-vivo experiments, using intramyocardial injection in ECH hearts of either Cx3cl1 or Cx3cr1 siRNA, or Cx3cr1-/- knockout mice, identified the CX3CL1/CX3CR1 axis as a protective pathway delaying transition to HF. In-vitro results showed that CX3CL1 not only enhanced ECH Mφ proliferation and expansion but also supported adult cardiomyocyte hypertrophy via a synergistic action with TNFα. Our data underscore the in-vivo transient protective role of the CX3CL1/CX3CR1 axis in ECH remodeling and suggest the participation of CX3CL1-secreting Mφ and their crosstalk with CX3CR1-expressing cardiomyocytes to delay HF.

Tumor necrosis factor alpha and glutathione interplay in chronic heart failure.

The pro-inflammatory cytokine, tumor necrosis factor alpha (TNF alpha), in concert with neurohormones, contributes to chronic heart failure (CHF) progression. This implies that TNF a antagonism may constitute an important target for CHF therapy. However, clinical trials in CHF patients using compounds that trap TNF alpha, comprising infliximab, an antibody directed to TNF alpha, and etanercept, a soluble recombinant receptor of TNF alpha, gave disappointing results bringing back to light the dual, short-term beneficial and long-term harmful effect of TNF alpha. This review focuses on the dual, concentration- and time-related effects of TNF alpha, the yin and yang action of TNF alpha in cardiac ischemia/reperfusion and contraction. Importantly, the harmful effects of TNF a are related to glutathione deficiency, a common hallmark to several other chronic inflammatory diseases. Recently, in rat models of CHF, oral administration of the glutathione precursor, N-acetylcysteine (NAC), was shown to hinder pathways of TNF alpha harmful signalling and to rescue cardiac structure and function. These results suggest that glutathione deficiency in association with TNF alpha activation may play a role in the pathophysiology of CHF and that NAC may represent a potential therapy in CHF.

Beta(2)-adrenergic receptor agonists increase intracellular free Ca(2+) concentration cycling in ventricular cardiomyocytes through p38 and p42/44 MAPK-mediated cytosolic phospholipase A(2) activation.

We have recently reported that arachidonic acid mediates beta(2)-adrenergic receptor (AR) stimulation of [Ca(2+)](i) cycling and cell contraction in embryonic chick ventricular cardiomyocytes (Pavoine, C., Magne, S., Sauvadet, A., and Pecker, F. (1999) J. Biol. Chem. 274, 628-637). In the present work, we demonstrate that beta(2)-AR agonists trigger arachidonic acid release via translocation and activation of cytosolic phospholipase A(2) (cPLA(2)) and increase caffeine-releasable Ca(2+) pools from Fura-2-loaded cells. We also show that beta(2)-AR agonists trigger a rapid and dose-dependent phosphorylation of both p38 and p42/44 MAPKs. Translocation and activation of cPLA(2), as well as Ca(2+) accumulation in sarcoplasmic reticulum stores sensitive to caffeine and amplification of [Ca(2+)](i) cycling in response to beta(2)-AR agonists, were blocked by inhibitors of the p38 or p42/44 MAPK pathway (SB203580 and PD98059, respectively), suggesting a role of both MAPK subtypes in beta(2)-AR stimulation. In contrast, beta(1)-AR stimulation of [Ca(2+)](i) cycling was rather limited by the MAPKs, clearly proving the divergence between beta(2)-AR and beta(1)-AR signaling systems. This study presents the first evidence for the coupling of beta(2)-AR to cardiac cPLA(2) and points out the key role of the MAPK pathway in the intracellular signaling elicited by positive inotropic beta(2)-AR agonists in heart.

Adult cardiac myocytes survive and remain excitable during long-term culture on synthetic supports.

OBJECTIVE: Cardiomyocytes can be transplanted successfully into skeletal and cardiac muscle. Our goal was to determine the feasibility of grafting cardiomyocytes onto various synthetic supports to create an excitable and viable tissue for implantation. METHODS: Adult rat cardiomyocytes were cultured over an 8-week period onto different substitutes, including human glutaraldehyde-treated pericardium (n = 3), equine glutaraldehyde-treated pericardium (n = 3), polytetrafluoroethylene (n = 8), Dacron polyester (n = 16), and Vicryl polyglactin (n = 8). RESULTS: Only the cells seeded on the Dacron survived, with the synthetic fibers colonized at 8 weeks. On the other supports, the number of myocytes progressively decreased from the first week, with their density (number of cells per square millimeter) being, after 20 days, 17 +/- 2 on the polytetrafluoroethylene and 5 +/- 1 on the human or equine pericardium compared with 45 +/- 3 on the Dacron. After 8 weeks of culture on Dacron, the sarcomeric protein (sarcomeric alpha-actinin) was detected in all cells. In addition, the staining was regularly arranged and well aligned in a striated pattern. Spontaneous beating activity was obtained. Moreover, electrical stimulation of the cell preparation resulted in the generation of calcium transients, the frequency of which followed the frequency of the electrical stimulation. CONCLUSIONS: These results suggest that adult cardiac myocytes remain viable and excitable during long-term culture on a 3-dimensional Dacron support, which might constitute a new synthetic cardiac tissue.

Biological effects of C-type natriuretic peptide in human myofibroblastic hepatic stellate cells.

During chronic liver diseases, hepatic stellate cells (HSC) acquire a myofibroblastic phenotype, proliferate, and synthetize fibrosis components. Myofibroblastic HSC (mHSC) also participate to the regulation of intrahepatic blood flow, because of their contractile properties. Here, we examined whether human mHSC express natriuretic peptide receptors (NPR). Only NPR-B mRNA was identified, which was functional as demonstrated in binding studies and by increased cGMP levels in response to C-type natriuretic peptide (CNP). CNP inhibited mHSC proliferation, an effect blocked by the protein kinase G inhibitor 8-(4 chlorophenylthio)-cGMP and by the NPR antagonist HS-142-1 and reproduced by analogs of cGMP. Growth inhibition was associated with a reduction of extracellular signal-regulated kinase and c-Jun N-terminal kinase and with a blockade of AP-1 DNA binding. CNP and cGMP analogs also blunted mHSC contraction elicited by thrombin, by suppressing calcium influx. The relaxing properties of CNP were mediated by a blockade of store-operated calcium channels, as demonstrated using a calcium-free/calcium readdition protocol. These results constitute the first evidence for a hepatic effect of CNP and identify mHSC as a target cell. Activation of NPR-B by CNP in human mHSC leads to inhibition of both growth and contraction. These data suggest that during chronic liver diseases, CNP may counteract both liver fibrogenesis and associated portal hypertension.

Evidence for a beta2-adrenergic/arachidonic acid pathway in ventricular cardiomyocytes. Regulation by the beta1-adrenergic/camp pathway.

The signaling pathway mediating the contractile effect of beta2-adrenergic receptors (beta2-AR) in the heart is still matter of debate. By using embryonic chick ventricular cardiomyocytes that express both functional beta1-and beta2-ARs, we show here that the specific beta2-AR agonist, zinterol, increases the amplitude of Ca2+ transients and cell contraction of electrically stimulated cells. Zinterol, up to 10 microM, did not stimulate adenylyl cyclase activity, and its effect on Ca2+ transients was unmodified by the specific cAMP antagonist, (Rp)-cAMPS. In contrast, zinterol (10-100 nM) triggered arachidonic acid (AA) release from [3H]AA-loaded cells via the activation of the cytosolic phospholipase A2 (cPLA2). Stimulation of the Ca2+ transients by zinterol was abolished by the cPLA2 inhibitor, AACOCF3, and was mimicked by AA (0.3-3 microM). Both stimulations of [3H]AA release and of [Ca2+]i cycling by zinterol were abolished after treatment of the cardiomyocytes with pertussis toxin. Although cell responses to beta2-AR stimulation were mediated by AA, they were under cAMP control as follows: (i) the beta1-AR stimulation exerted a cAMP-mediated negative constraint on the beta2-AR/cPLA2 pathway; (ii) cAMP potentiated AA action downstream beta-AR stimulation. We conclude that, in cardiomyocytes, beta2-AR is coupled to cPLA2 activation via a pertussis toxin-sensitive G protein. These results demonstrate the involvement of the cPLA2/AA pathway in mediating positive inotropic effects, which could potentially compensate for a defective cAMP pathway.

Pharmacological and molecular characterisation of beta-adrenoceptors in adult rat diaphragm muscle.

Using pharmacological and molecular approaches to investigate beta-adrenoceptor (beta-AR) subtype expression in adult rat diaphragm, we found that adenylyl cyclase (AC) was potently stimulated by the beta2-AR-selective agonist fenoterol, weakly stimulated by the beta1-AR-selective agonist prenalterol and unaffected by the beta3-AR agonist CGP12177. AC activity in response to a submaximal isoproterenol concentration was potently inhibited by the beta2-AR-selective antagonist ICI118551, whereas the beta1-AR-selective antagonist CGP20712A was effective only in very high concentrations. (-)-[125I]-cyanopindolol ([125I]-CYP) saturation binding experiments indicated a single affinity component (dissociation constant (Kd) = 22 +/- 2 pM) for beta-AR sites (maximal beta -AR density (Bmax) = 14 +/- 2 fmol/ mg). Eadie-Hofstee analysis of [125I]-CYP displacement curves by beta1-, beta2- or beta3-AR-selective ligands allowed to characterise a homogeneous population of beta2-AR sites. Finally, reverse transcriptase-polymerase chain reaction analysis of beta-AR subtype mRNAs identified beta2-AR transcripts but no beta1- and beta3-AR mRNAs. Our results demonstrate that beta2-AR is the only beta-AR subtype expressed in the diaphragm.

Xanthine affects [Ca2+]i and contractile responses of ventricular cardiocytes to electrical stimulation.

Xanthine, a major purine by-product of ATP, accumulates during myocardial ischemia. In the present study, we show that xanthine (0.5-1 mM) impaired the occurrence of cytosolic Ca2+ concentration ([Ca2+]i) transients, visualized in fura 2-loaded cells, and twitches of contraction in ventricular cardiocytes in response to electrical stimulation. This effect of xanthine was independent of superoxide anion production. That it was a result of decreased membrane excitability was supported by the following: 1) it was reversed by increasing either the amplitude of the stimulus voltage required to stimulate cardiocytes or the extracellular concentration of NaCl; and 2) xanthine reversed the depolarization following electrical stimulation in cardiocytes loaded with the voltage-sensitive dye bis-oxonol. P2 purinergic-agonists, including ATP (10 microM), but not P1 purinergic agonists reproduced the effects seen with xanthine. In addition, a lack of additivity between xanthine and ATP at maximal concentrations was observed. We conclude that xanthine, through activation of a P2 purinoceptor, may contribute to myocardial arrhythmia occurring during ischemia-reperfusion injury.

Arachidonic acid drives mini-glucagon action in cardiac cells.

Recent studies have shown that glucagon is processed by cardiac cells into its COOH-terminal (19-29) fragment, mini-glucagon, and that this metabolite is an essential component of the contractile positive inotropic effect of glucagon (Sauvadet, A., Rohn, T., Pecker, F. and Pavoine, C. (1996) Circ. Res. 78, 102-109). We now show that mini-glucagon triggers arachidonic acid (AA) release from [3H]AA-loaded embryonic chick ventricular myocytes via the activation of a phospholipase A2 sensitive to submicromolar Ca2+ concentrations. The phospholipase A2 inhibitor, AACOCF3, prevented mini-glucagon-induced [45Ca2+] accumulation into the sarcoplasmic reticulum, but inhibitors of lipoxygenase, cyclooxygenase, or epoxygenase pathways were ineffective. AA applied exogenously, at 0. 3 microM, reproduced the effects of mini-glucagon on Ca2+ homeostasis and contraction. Thus AA: (i) caused [45Ca2+] accumulation into a sarcoplasmic reticulum compartment sensitive to caffeine; 2) potentiated caffeine-induced Ca2+ mobilization from cells loaded with Fura-2; 3) acted synergistically with glucagon or cAMP to increase both the amplitude of Ca2+ transients and contraction of electrically stimulated cells. AA action was dose-dependent and specific since it was mimicked by its non-hydrolyzable analog 5,8,11,14-eicosatetraynoic acid but not reproduced by other lipids such as, arachidic acid, linolenic acid, cis-5,8,11,14,17-eicosapentaenoic acid, cis-4,7,10,13,16, 19-docosahexaenoic acid, or arachidonyl-CoA, even in the micromolar range. We conclude that AA drives mini-glucagon action in the heart and that the positive inotropic effect of glucagon on heart contraction relies on both second messengers, cAMP and AA.

[Calcium signal and contraction]. / Signal calcium et contraction.

The calcium ion plays a unique role as a messenger and a cofactor in cardiac contraction. This role relies on the strict control by the cell of Ca homeostasis, the components of which are described in this review. During the few last years, tools for the measurement of free intracellular Ca in living cells have been developed which include: probes (aequorin, Fura 2, Indo 1, Fluo 3...), tools for the loading of the cells (microinjection and AM-probes) and systems to analyze the signal (photometers, microfluorimeters, confocal microscopy). Those tools allowed the analysis of calcium signal in cardiomyocytes. In the cardiac cell, activation of a Ca influx through L type Ca channels is usually considered as the pathway initializing Ca mobilization and leading to contraction. It has now been demonstrated that this pathway is activated by beta 1-adrenergic agonists via cyclic AMP. However, amplification of contraction may involve other targets. Thus, the positive inotropic effect of beta 2-adrenergic agonists is also associated with a rise in cytosolic Ca but is not linked to cyclic AMP increase. The alpha 1-adrenergic pathway involves a sensitization of myofilaments for Ca, and increases contraction without an increase in cytosolic Ca. Finally, the positive inotropic effect of glucagon combines the cyclic AMP pathway with a cyclic AMP independent pathway triggered by the metabolite mini-glucagon.

Synergistic actions of glucagon and miniglucagon on Ca2+ mobilization in cardiac cells.

It has been recently shown that the physiological processing of glucagon into its C-terminal (19-29) fragment, miniglucagon, by cardiac cells was essential for the contractile positive inotropic effect of the hormone. However, the mechanisms underlying the effects of miniglucagon remained undetermined. In the present study, we assessed the effects of miniglucagon on Ca2+ homeostasis in embryonic chick ventricular myocytes. In quiescent cells, short-term applications of 0.1 nmol/L miniglucagon markedly increased the accumulation of 45Ca into intracellular compartments resistant to digitonin lysis and sensitive to caffeine. Ca2+ accumulation into the sarcoplasmic reticular (SR) store was further attested by fura 2 imaging studies on quiescent or prestimulated cells: miniglucagon potentiated Ca2+ release from the SR compartment triggered by caffeine and evoked a rise in cytosolic Ca2+ when applied on cells pretreated with 1 mumol/L thapsigargin, a specific inhibitor of the SR Ca2+ pump. Glucagon alone produced a small cytosolic Ca2+ signal that was considerably amplified by miniglucagon. The action of glucagon was mimicked by 8-bromo-cAMP and was blocked by isradipine, suggesting that it relied on the activation of L-type Ca2+ channels, via phosphorylation. We conclude that the combined actions of miniglucagon and glucagon on Ca2+ accumulation into SR stores and Ca2+ release from the same stores are likely to support the positive inotropic effect elicited in vivo by glucagon on heart contraction.

Erythro-9-(2-hydroxy-3-nonyl)adenine inhibits cyclic GMP-stimulated phosphodiesterase in isolated cardiac myocytes.

Recently, an inhibitor of adenosine deaminase, erythro-9-(2-hydroxyl-3-nonyl)adenine (EHNA), was shown to selectively block the activity of purified cGMP-stimulated phosphodiesterase (PDE) (cGS-PDE, or PDE2) in human and porcine heart [J. Mol. Cell. Cardiol. 24 (Suppl. V):102 (1992)]. Because cGS-PDE was found to mediate the cGMP-induced inhibition of L-type Ca2+ current (Ica) in frog ventricular cells, we tested the effects of EHNA in this preparation. Ica was measured using the whole-cell patch-clamp technique and a perfusing pipette. EHNA (0.3-30 microM) had no significant effect on either basal Ica or isoprenaline (1 nM)- or cAMP (10 microM)-elevated Ica. However, EHNA dose-dependently (IC50 approximately 3 microM) reversed the inhibitory effect of cGMP on cAMP-stimulated Ica. EHNA (30 microM) also blocked the inhibitory effect of NO donors, such as sodium nitroprusside (1 mM) and 3-morpholinosydnonimine (30 microM), on isoprenaline-stimulated Ica. In addition, EHNA dose-dependently (IC50 approximately 4-5 microM) inhibited the cGMP-induced stimulation of PDE activity in frog ventricle particulate fraction, as well as purified soluble cGS-PDE. However, EHNA (up to 30 microM) did not modify the activities of three other purified soluble PDE isoforms. Moreover, EHNA did not change the Ka (40 nM) for cGMP activation of cGS-PDE, which suggests that EHNA does not inhibit cGS-PDE by displacing cGMP from the allosteric regulator site. Because adenosine did not mimic the effects of EHNA on Ica or PDE activity, it is unlikely that the effects of EHNA are due to adenosine deaminase inhibition. We conclude that EHNA acts primarily to inhibit cGS-PDE in intact cardiac myocytes. This compound should be useful in evaluating the physiological role of cGS-PDE in various tissues.

Inhibition of the sarcolemmal Ca2+ pump in embryonic chick heart cells by mini-glucagon.

The effect of mini-glucagon, the metabolite (19-29) of glucagon was examined on the sarcolemmal (SL) Ca2+ pump activity measured in situ, in single quiescent embryonic chick heart ventricular cells loaded with Fura-2. The method consisted in triggering limited cytosolic Ca2+ concentration ([Ca2+]i) pulses by the addition of the Ca2+ ionophore 4-bromo-A23187. [Ca2+]i decays, imposed by the addition of EGTA, were monitored in conditions in which only the SL Ca2+ pump could ensure [Ca2+]i removal, i.e. in the presence of the sarcoplasmic reticular (SR) Ca2+ pump specific inhibitor, thapsigargin, substituting NaCI by LiCI in the external medium in order to quench the Na+/Ca2+ exchanger, and under null Ca2+ gradient. Mini-glucagon elicited a dose-dependent inhibition of the SL Ca2+ pump, maximal 80% inhibition being observed with 1 nM mini-glucagon. In addition to its effect on the SL Ca2+ pump, mini-glucagon evoked a delayed onset of a [Ca2+]i oscillatory response in cells incubated in normal conditions. Both effects of mini-glucagon were mimicked by vanadate tested at 2 microM, a concentration at which it acts as a specific inhibitor of the SL Ca2+ pump. These results define the contribution of the cardiac sarcolemmal Ca2+ pump to Ca2+ homeostasis in situ and its role as a target for mini-glucagon action.

Nitric oxide regulates cardiac Ca2+ current. Involvement of cGMP-inhibited and cGMP-stimulated phosphodiesterases through guanylyl cyclase activation.

The effects of the nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) on the L-type Ca2+ current (ICa) were examined in frog ventricular myocytes under basal and phosphorylated conditions. SIN-1 was found to exert insignificant effects on basal ICa but to induce a biphasic action on stimulated ICa. Indeed, in the nanomolar range of concentrations (0.1-10 nM), SIN-1 induced a pronounced (approximately 40%) stimulation of ICa elevated by a non-maximal concentration of forskolin (0.3 microM). However, the stimulatory effects of SIN-1 on ICa were not additive with those of maximal concentrations (10 microM) of forskolin or intracellular cAMP. In contrast, at higher concentrations (100 nM to 1 mM), SIN-1 strongly reduced ICa (by up to 85%) which had been previously stimulated by cAMP, forskolin, or isoprenaline. All the effects of SIN-1 appeared to be mediated by the liberation of NO since they were suppressed by methylene blue and LY83583 and were not mimicked by SIN-1C, a metabolite of SIN-1. The stimulatory or inhibitory effects of SIN-1 were absent, respectively, in the presence of milrinone (10 microM) or when the hydrolysis-resistant cAMP analog 8-bromo-cAMP was used instead of cAMP to stimulate ICa. In addition to its effects on ICa, SIN-1 induced a dose-dependent stimulation of guanylyl cyclase activity in the cytosolic and membrane fractions of frog ventricle. The membrane form of guanylyl cyclase displayed a higher sensitivity to SIN-1 than the cytosolic form, which correlated with SIN-1 sensitivity of ICa. Our data suggest that the activatory and inhibitory effects of NO donors on ICa result from an inhibition of the cGMP-inhibited cAMP-phosphodiesterase and an activation of the cGMP-stimulated cAMP-phosphodiesterase, respectively, both linked to the activation of guanylyl cyclase, possibly a membrane form of the enzyme.

Inhibition by glucagon of the cGMP-inhibited low-Km cAMP phosphodiesterase in heart is mediated by a pertussis toxin-sensitive G-protein.

We have recently reported that glucagon activated the L-type Ca2+ channel current in frog ventricular myocytes and showed that this was linked to the inhibition of a membrane-bound low-Km cAMP phosphodiesterase (PDE) (Méry, P. F., Brechler, V., Pavoine, C., Pecker, F., and Fischmeister, R. (1990) Nature 345, 158-161). We show here that the inhibition of membrane-bound PDE activity by glucagon depends on guanine nucleotides, a reproducible inhibition of 40% being obtained with 0.1 microM glucagon in the presence of 10 microM GTP, with GTP greater than GTP gamma S, while GDP and ATP gamma S were without effect. Glucagon had no effect on the cytosolic low-Km cAMP PDE, assayed with or without 10 microM GTP. Glucagon inhibition of membrane-bound PDE activity was not affected by pretreatment of the ventricle particulate fraction with cholera toxin. However, it was abolished after pertussis toxin pretreatment. Mastoparan, a wasp venom peptide known to activate G(i)/G(o) proteins directly, mimicked the effect of glucagon. PDE inhibition by glucagon was additive with the inhibition induced by Ro 20-1724, but was prevented by milrinone. This was correlated with an increase by glucagon of cAMP levels in frog ventricular cells which was not additive with the increase in cAMP due to milrinone. We conclude that glucagon specifically inhibits the cGMP-inhibited, milrinone-sensitive PDE (CGI-PDE). Insensitivity of adenylylcyclase to glucagon and inhibition by the peptide of a low-Km cAMP PDE were not restricted to frog heart, but also occurred in mouse and guinea pig heart. These results confirm that two mechanisms mediate the action of glucagon in heart: one is the activation of adenylylcyclase through Gs, and the other relies on the inhibition of the membrane-bound low-Km CGI-PDE, via a pertussis toxin-sensitive G-protein.

Role of G protein beta gamma subunits in the regulation of the plasma membrane Ca2+ pump.

In Zajdela hepatoma cells (ZHC) the plasma membrane Ca2+ pump displayed no sensitivity to glucagon (19-29) (mini-glucagon), whereas in hepatocyte this metabolite of glucagon evoked a biphasic regulation of the Ca2+ pump system via a cholera toxin-sensitive G protein. Analysis of G protein subunits in ZHC membranes indicated the presence of cholera toxin-sensitive Gs alpha and G beta gamma proteins, whose functionality was manifested by GTP and NaF stimulation of adenylylcyclase activity, and pertussis toxin-catalyzed ADP-ribosylation of Gi alpha, respectively. However, immunoblotting experiments suggested a lower content in beta gamma subunits in ZHC as compared with hepatocyte plasma membranes. Complementation of ZHC or hepatocyte plasma membranes with purified beta gamma subunits from transducin (T beta gamma) caused inhibition of the basal activity of the Ca2+ pump at 10 and 300 ng/ml, respectively, and revealed (in ZHC) or increased (in hepatocytes) sensitivity of the system to mini-glucagon. After cholera toxin treatment of ZHC, T beta gamma no longer reconstituted the response of the Ca2+ pump to mini-glucagon, suggesting that the mechanism of beta gamma action is dependent on an association with the alpha subunit of a cholera toxin-sensitive G protein. It is concluded that G beta gamma subunits control both the basal activity of the plasma membrane Ca2+ pump and its inhibition by mini-glucagon.

Miniglucagon [glucagon-(19-29)] is a component of the positive inotropic effect of glucagon.

Glucagon is well known for its cardiotonic effect, but its mechanism of action remains undetermined. In the present study, we showed that glucagon, under minimal degradation conditions, had no effect on the amplitude of contractility of beating chick embryo ventricular cells. This raised the question of the contribution of the active metabolite of glucagon, glucagon-(19-29), referred to as miniglucagon, to the positive inotropic effect of glucagon. Incubation of glucagon with heart cells led to its rapid conversion into miniglucagon, as measured by radioimmunoassay. Accumulation of the metabolite was maximal after 8 min and remained stable until 15 min. reaching 6% of the initial glucagon concentration. Bacitracin inhibited this processing of glucagon into miniglucagon. Miniglucagon, from 0.1 pM to 1 nM, exerted a potent negative inotropic action. The most striking observation was a 45% increase in the amplitude of cell contractility elicited by the combination of 30 nM glucagon with 1 nM miniglucagon. A similar effect was obtained when glucagon was replaced by a low concentration (75 microM) of 8-bromoadenosine 3',5'-cyclic monophosphate. We conclude that glucagon processing into miniglucagon may be essential for the positive inotropic effect of glucagon on heart contraction.

Glucagon-(19-29), a Ca2+ pump inhibitory peptide, is processed from glucagon in the rat liver plasma membrane by a thiol endopeptidase.

Glucagon-(19-29) is 1000-fold more potent that glucagon as an inhibitor of the liver plasma membrane calcium pump, which suggests that this peptide fragment is naturally occurring. Since glucagon-(19-29) is undetectable in plasma, the processing of glucagon into its (19-29) fragment may occur upon interaction of glucagon with its target tissues. The use of a specific radioimmunoassay for glucagon-(19-29) in association with the separation and identification of peptides by high performance liquid chromatography revealed that, upon incubation at 37 degrees C with hepatic plasma membranes, glucagon is processed into its (19-29) C-terminal fragment. The identity of the fragment was confirmed by amino acid sequencing. The processing activity was inhibited by reagents of the thiol group and by 1,10-phenanthroline, suggesting that a thiol endopeptidase containing a catalytically active metal is involved in this processing. Following its production, glucagon-(19-29) was degraded with a half-life of less than 10 s. This degradation was inhibited by bacitracin and by the aminopeptidase inhibitors bestatin and amastatin. When glucagon was incubated with liver plasma membranes in the absence of inhibitors, the accumulation of glucagon-(19-29) reached a maximum at 2 min (1% of initial glucagon), followed by a slow decline. In the presence of bacitracin and bestatin, the amounts of glucagon-(19-29) obtained from glucagon increased continuously, 1 and 2% of glucagon being transformed after 10 and 30 min, respectively. The production of glucagon-(19-29) did not appear to be associated with the binding of glucagon to its receptors, since (i) guanosine 5'-(3-O-thio)triphosphate, a compound which decreases the glucagon-receptor interaction, could not decrease the conversion of glucagon into glucagon-(19-29); (ii) a glucagon analogue which displays a strongly decreased affinity for the hepatic glucagon receptors was processed similarly to glucagon. The conversion also occurs upon incubation with intact hepatoma cells in monolayer culture. These observations suggest that, under physiological conditions, glucagon is processed in liver by cleavage of the Arg17-Arg18 basic doublet, leading to the production of a fragment which is known to display an original biological specificity, namely the modulation of the hepatocyte plasma membrane calcium pump.

Activation of Na+/Ca2+ exchange by adenosine in ewe heart sarcolemma is mediated by a pertussis toxin-sensitive G protein.

We studied the effect of adenosine on Na+/Ca2+ exchange activity in ewe heart ventricular sarcolemmal vesicles. Adenosine was found to stimulate Na+/Ca2+ exchange activity in a dose-dependent manner from 0.1 nM to 10 microM, with maximal stimulation (40%) at 0.1 microM adenosine. The Vmax of Na+/Ca2+ exchange was increased, but the Km for Ca2+ was not altered. The effect of adenosine was specific since 1 microM adenine, inosine, and guanosine led to less than 15% stimulation, and adenosine diphosphate had no effect. Caffeine antagonized the activation of Na+/Ca2+ exchange by adenosine, and the order of potency of adenosine analogs was N6-(L-2-phenylisopropyl)adenosine = N6-cyclohexyladenosine = 5'-(N- ethylcarboxamido)adenosine much greater than N6-(D-2-phenylisopropyl)adenosine, indicating the involvement of A1 subclass receptors. The effect of adenosine was mimicked by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and blocked by pertussis toxin treatment. Taken together, these results suggest that A1 subclass receptors coupled to a pertussis toxin-sensitive G protein mediate the activation of Na+/Ca2+ exchange activity by adenosine. We conclude that the negative inotropic effect of adenosine in ventricular muscle, antagonistic toward cyclic AMP, may involve activation of Na+/Ca2+ exchange.