Hypertrophic effect of selective beta(1)-adrenoceptor stimulation on ventricular cardiomyocytes from adult rat

Weinvestigated whether selective ₁-adrenoceptorstimulation causes hypertrophic growth on isolated ventricularcardiomyocytes from adult rat. As parameters for the induction ofhypertrophic growth, the increases of [¹⁴C]phenylalanineincorporation, protein and RNA mass, and cell size weredetermined. Isoproterenol (Iso, 10 µM) alone had no growtheffect. In the presence of the ₂-adrenoceptor antagonist ICI-118551 (ICI, 10 µM), Iso caused an increase in[¹⁴C]phenylalanine incorporation, protein and RNA mass,cell volume, and cross-sectional area. We showed for phenylalanineincorporation that the growth effect of Iso+ICI could be antagonized by₁-adrenoceptor blockade with atenolol (10 µM) ormetoprolol (10 µM), indicating that it was caused by selective₁-adrenoceptor stimulation. The growth response toIso+ICI was accompanied by an increase in ornithine decarboxylase (ODC)activity and expression. Inhibition of ODC by the ODC antagonistdifluoromethylornithine (1 mM) attenuated this hypertrophic response,indicating that ODC induction is causally involved. The growth responseto Iso+ICI was found to be cAMP independent but was sensitive togenistein (100 µM) or rapamycin (0.1 µM). The reaction was enhancedin the presence of pertussis toxin (10 µM). We conclude thatselective ₁-adrenoceptor stimulation causes hypertrophicgrowth of ventricular cardiomyocytes by a mechanism that is independentof cAMP but dependent on a tyrosine kinase and ODC.

     

Evidence for selective coupling of alpha 1-adrenergic receptors to phospholipase C-beta 1 in rat neonatal cardiomyocytes

Activation of phospholipase C (PLC) in neonatal rat cardiomyocytes (NCM) generates primarily inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) in response to rises in intracellular Ca(2+), or inositol 1,4-bisphosphate (Ins(1,4)P(2)) in response to norepinephrine (NE) (Matkovich, S. J. and Woodcock, E. A. (2000) J. Biol. Chem. 275, 10845-10850). To examine the PLC subtype mediating the alpha(1)-adrenergic receptor response, PLC-beta(1) and PLC-beta(3) were overexpressed in NCM using adenoviral infection (Ad-PLC-beta(1) NCM and Ad-PLC-beta(3) NCM, respectively) and PLC responses assessed from [(3)H]inositol phosphate (InsP) generation in the presence of 10 mm LiCl. The [(3)H]InsP response to NE (100 microm) was enhanced in Ad-PLC-beta(1) NCM relative to cells infected with blank virus (Ad-MX NCM), but was reduced in Ad-PLC-beta(3) NCM. In contrast, the [(3)H]InsP response to ATP (100 microm) was not elevated in Ad-PLC-beta(1) NCM, and was enhanced rather than diminished in Ad-PLC-beta(3) NCM, showing that effects of the two PLC-beta isoforms were specific for particular receptor types. PLC-delta(1) overexpression selectively reduced NE-induced [(3)H]InsP responses, without affecting the ATP stimulation. The reduced NE response was associated with a selective loss of PLC-beta(1) expression in Ad-PLC-delta(1) NCM. alpha(1)-Adrenergic receptor activation caused phosphorylation of PLC-beta(1) but not PLC-beta(3), whereas stimulation by ATP induced phosphorylation of PLC-beta(3) but not PLC-beta(1.) Taken together, these studies provide evidence that NE-stimulated InsP generation in NCM is primarily mediated by PLC-beta(1), despite the presence of both PLC-beta(1) and PLC-beta(3) isoforms.     

Homologous regulation of adenylate cyclase-coupled receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes

The effect of agonists on VIP receptor regulation has been investigated in mononuclear human blood leucocytes. VIP receptor number and affinity, as well as VIP-stimulated cyclic AMP accumulation were measured after pretreatment with VIP, PHM-27 or secretin. Pretreatment for 30 min with 0.1 μM VIP caused 28% (S.E.M. = 15) reduction in specific binding, and 52% (S.E.M. = 12) reduction in cyclic AMP accumulation, while 3 h of pretreatment caused 59% (S.E.M. = 10) and 68% (S.E.M. = 12) reduction. Only VIP concentrations at the nanomolar level and higher were shown to have any effect. B_max of the high-affinity receptor was reduced by 66% (S.E.M. = 8) after 30 min, and 95% (S.E.M. = 3) after 3 h of exposure to 0.1 μM VIP. No significant change was observed in receptor affinity, in B_max of the low-affinity receptor, in ED₅₀, or in ED₁₀₀ of VIP-stimulated cyclic AMP accumulation. Pretreatment with PHM-27 (0.1 μM, 3 h) caused 24% reduction in [¹²⁵I]VIP binding and 25% reduction in cyclic AMP accumulation, while no effect was detected after pretreatment with secretin (0.1 μM, 3 h).     

Post-translational modifications of tubulin and microtubule stability in adult rat ventricular myocytes and immortalized HL-1 cardiomyocytes

Little is known about the subcellular distribution and the dynamics of tubulins in adult cardiac myocytes although both are modified during cardiac hypertrophy and heart failure. Using confocal microscopy, we examined post-translational modifications of tubulin in fully differentiated ventricular myocytes isolated from adult rat hearts, as well as in immortalized and dividing HL-1 cardiomyocytes. Detyrosinated Glu-alpha-tubulin was the most abundant post-translationally modified tubulin found in ventricular myocytes, while acetylated- and delta2-alpha-tubulins were found in lower amounts or absent. In contrast, dividing HL-1 cardiomyocytes exhibited high levels of tyrosinated or acetylated alpha-tubulins. A mild nocodazole treatment (0.1 microM, 1 h) disrupted microtubules in HL-1 myocytes, but not in adult ventricular myocytes. A stronger treatment (10 microM, 2 h) was required to disassemble tubulins in adult myocytes. Glu-alpha-tubulin containing microtubules were more resistant to nocodazole treatment in HL-1 cardiomyocytes than in ventricular myocytes. Endogenous activation of the cAMP pathway with the forskolin analog L858051 (20 microM) or the beta-adrenergic agonist isoprenaline (10 microM) disrupted the most labile microtubules in HL-1 cardiomyocytes. In contrast, isoprenaline (10 microM), cholera toxin (200 ng/ml, a G(S)-protein activator), L858051 (20 microM) or forskolin (10 microM) had no effect on the microtubule network in ventricular myocytes. In addition, intracellular Ca2+ accumulation induced either by thapsigargin (2 microM) or caffeine (10 mM) did not modify microtubule stability in ventricular myocytes. Our data demonstrate the unique stability of the microtubule network in adult cardiac myocytes. We speculate that microtubule stability is required to support cellular integrity during cardiac contraction.     

Arachidonic acid incorporation in cardiomyocytes,endothelial cells and fibroblast-like cells isolated from adult rat heart

The incorporation of radiolabeled arachidonic acid (3[H]-AA) in normoxic cardiomyocytes (MC), cardiac endothelial cells (EC) and fibroblast-like cells (FL) isolated from adult rat heart was studied. Deposition of 3[H]-AA in the cellular lipid pool was assessed with biochemical and autoradiographic techniques. Extraction and subsequent analysis of lipids from the three different cell types revealed that MC contained significantly more triacylglycerols than EC and FL. The proportion of (unlabeled) AA was also higher in MC triacylglycerols than in EC and FL. The quantity of phospholipids did not differ among the three cell types studied. However, the content of (unlabeled) AA in the MC phospholipid pool was twice as high as in EC and FL. The amount of 3[H]-AA incorporated in the cellular lipid pool of MC, EC and FL depended on the concentration of AA in the incubation medium and the incubation time. In EC and FL incorporation of 3[H]-AA was highest in the cellular phospholipid pool (0.01 microM AA, 30 min incubation). With increased concentration of AA and longer incubation times, the cellular triacylglycerol pool became more important as site of 3[H]-AA incorporation. In MC, comparable amounts of 3[H]-AA were incorporated in the cellular triacylglycerol and phospholipid pools (0.01 and 1 microM AA). At higher AA concentrations (10 microM) the triacylglycerol pool was the preferred site of 3[H]-AA deposition. Autoradiographic analysis at the light microscopic level revealed that the extra-nuclear space was readily stained when the three cell types were incubated with 3[H]-AA. These findings indicate that all cellular lipid pools and membranes are most likely site of deposition of radiolabeled arachidonic acid.     

Modulation of cardiac A1-adenosine receptors in rats following treatment with agents affecting heart rate

Effects of chronic treatment affecting heart rate on A₁ adenosine receptor levels and their functions were studied. Treatment of rats with isoproterenol for 10 days accelerated heart rate and increased the level of adenosine receptors, in both the atria and ventricles. Negative dromotropic response of isolated heart to adenosine was enhanced in isoproterenol-treated rats. Similar results were obtained following treatment with atropine sulfate, or swimming training but not after treatment with thyroxine. On the other hand, treatment with amiodarone, which normally causes a decrease in heart rate, also increased the level of adenosine receptors in both atria and ventricles. The sensitivity of the isolated heart to the negative dromotropic and chronotropic effects of adenosine was not enhanced in the amiodarone treated rats. Similar results were obtained following treatment with propranolol, while treatment with PTU (6-n-propyl-2-thiouracil) increased adenosine sensitivity in the isolated heart. It was concluded that the levels of A₁ adenosine receptors in the heart correspond to heart rate, and to cardiac efficiency. While an increase in heart rate was followed by up-regulation of A₁ adenosine receptors, a decrease in heart rate caused a moderate elevation of these receptors.     

Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes

Endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors have been demonstrated in intact heart and cardiac membranes. ET(A) receptors have been demonstrated on adult ventricular myocytes. The aim of the present study was to determine the presence of ET(B) and the relative contribution of this receptor subtype to total endothelin-1 (ET-1) binding on adult ventricular myocytes. Saturation binding experiments indicated that ET-1 bound to a single population of receptors (Kd = 0.52 +/- 0.13 nM, n = 4) with an apparent maximum binding (Bmax) of 2.10 +/- 0.25 sites (x 10(5))/cell (n = 4). Competition experiments using 40 pM [125I]ET-1 and nonradioactive ET-1 revealed a Ki of 660 +/- 71 pM (n = 10) and a Hill coefficient (nH) of 0.99 +/- 0.10 (n = 10). A selective ET(A) antagonist, BQ610, displaced 80% of the bound [125I]ET-1. No displacement was observed by concentrations of an ET(B)-selective antagonist, BQ788, up to 1.0 microM. However, in the presence of 1.0 microM BQ610, BQ788 inhibited the remaining [125I]ET-1 binding. Similarly, in the presence of 1.0 microM BQ788, BQ610 inhibited the remaining specific [125I]ET-1 binding. Binding of an ET(B1)-selective agonist, [125I]IRL-1620, confirmed the presence of ET(B). ET(B) bound to ET-1 irreversibly, whereas binding to ET(A) demonstrated both reversible and irreversible components, and BQ610 and BQ788 bound reversibly. Reducing the incubation temperature to 0 degrees C did not alter the irreversible component of ET-1 binding. Hence, both ET(A) and ET(B) receptors are present on intact adult rat ventricular myocytes, and the ratio of ET(A):ET(B) binding sites is 4:1. Both receptor subtypes bind to ET-1 by a two-step association involving the formation of a tight receptor-ligand complex; however, the kinetics of ET-1 binding to ET(A) versus ET(B) differ.     

Nanoscale organization of junctophilin-2 and ryanodine receptors within peripheral couplings of rat ventricular cardiomyocytes

The peripheral distributions of the cardiac ryanodine receptor (RyR) and a junctional protein, junctophilin-2 (JPH2), were examined using single fluorophore localization-based super-resolution microscopy in rat ventricular myocytes. JPH2 was strongly associated with RyR clusters. Estimates of the colocalizing fraction of JPH labeling with RyR was ~90% within 30 nm of RyR clusters. This is comparable to fractions estimated from confocal data (~87%). Similarly, most RyRs were associated with JPH2 labeling in super-resolution images (~81% within 30 nm of JPH2 clusters). The shape of associated RyR clusters and JPH2 clusters were very similar, but not identical, suggesting that JPH2 is dispersed throughout RyR clusters and that the packing of JPH2 into junctions and the assembly of RyR clusters are tightly linked.     

Lipopolysaccharide induces apoptosis in adult rat ventricular myocytes via cardiac AT(1) receptors

Lipopolysaccharide (LPS) from gram-negative bacteria circulates in acute, subacute, and chronic conditions. It was hypothesized that LPS directly induces cardiac apoptosis. In adult rat ventricular myocytes (isolated with depyrogenated digestive enzymes to minimize tolerance), LPS (10 ng/ml) decreased the ratio of Bcl-2 to Bax at 12 h; increased caspase-3 activity at 16 h; and increased annexin V, propidium iodide, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining at 24 h. Apoptosis was blocked by the caspase inhibitor benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone (Z-VAD-fmk), captopril, and angiotensin II type 1 receptor (AT(1)) inhibitor (losartan), but not by inhibitors of AT(2) receptors (PD-123319), tumor necrosis factor-alpha (TNFRII:Fc), or nitric oxide (N(G)-monomethyl-L-arginine). Angiotensin II (100 nmol/l) induced apoptosis similar to LPS without additive effects. LPS in vivo (1 mg/kg iv) increased apoptosis in left ventricular myocytes for 1-3 days, which dissipated after 1-2 wk. Losartan (23 mg. kg(-1). day(-1) in drinking water for 3 days) blocked LPS-induced in vivo apoptosis. In conclusion, low levels of LPS induce cardiac apoptosis in vitro and in vivo by activating AT(1) receptors in myocytes.     

Activity of amylin at CGRP1-preferring receptors coupled to positive contractile response in rat ventricular cardiomyocytes

Calcitonin gene-related peptide (CGRP) exerts a positive contractile response directly in rat ventricular cardiomyocytes. This response is mediated by receptors of the CGRP₁-subtype. Amylin is 46% homologous with CGRP and binds to receptors selective for CGRP in a range of tissues. The ability of amylin to influence ventricular contractility has been assessed using cardiomyocytes isolated from the ventricles of adult rats. Cardiomyocytes were subjected to biphasic electrical stimulation at 0.5 Hz. CGRP produced a concentration-dependent positive contractile response which became maximal 4 min after initial stimulation. CGRP increased the contractile amplitude maximally at 1 nM and to a value which was 23.3% greater than in the absence of peptide (EC₅₀ VALUE = 21 pM). Amylin increased the contractile amplitude maximally at 20 nM and to a value which was 17.3% greater than in the absence of peptide (EC₅₀ VALUE = 216 pM). In the presence of amylin (20 nM), the concentration-dependence of the contractile response to CGRP was shifted to the left, so that the response became maximal when CGRP was present at 50 pM. In the presence of CGRP_8–37 (100 nM), a selective antagonist at CGRP₁-preferring receptors, the concentration-dependence of the contractile response to CGRP was shifted to the right (dose RATIO = 54). Similarly, in the presence of CGRP_8–37 (100 nM), the contractile response to amylin was inhibited significantly (P ≤ 0.01). Amylin_8–37 (100 nM) did not inhibit the concentration-dependence of the contractile responses to CGRP and amylin significantly (dose RATIOS = 4.2 and 2.4, respectively). In conclusion, these data indicate that amylin exerts a contractile response directly in rat ventricular cardiomyocytes via CGRP₁-preferring receptors. This effect could assume greater significance in non-insulin-dependent diabetes mellitus and in hypertensive states, in which the concentration of amylin is elevated in plasma.     

Evidence for functional sodium and calcium ion channels in the membrane of cultured cardiomyocytes of the adult rat

Characteristics are reported for electrical activity of adult rat cardiomyocytes in long-term primary culture. Cells in vitro for 12 to 28 days have mean membrane potential of -53 mV, are electrically excitable, and some are spontaneously contractile. The action potential of these cells has a slow rate of depolarization and is abolished by methoxyverapamil (D-600) but not by tetrodotoxin (TTX). When cells are hyperpolarized by passage of an inward current, spontaneous action potentials cease and action potentials evoked by depolarizing pulses are then TTX sensitive. Fetal bovine serum is a constituent of the culture medium. Its temporary removal causes spontaneous contractility to cease but the cells remain electrically excitable.     

A role for focal adhesion kinase in phenylephrine-induced hypertrophy of rat ventricular cardiomyocytes

A variety of agonists including phenylephrine (PE) induce hypertrophy in neonatal ventricular cardiomyocytes. Here we report that signals provided by extracellular matrix proteins (ECM) augment the PE-induced hypertrophic response of cardiomyocytes and provide evidence that ECM-dependent signaling is mediated in part by the protein tyrosine kinase, focal adhesion kinase (FAK). Addition of PE to cultured neonatal cardiomyocytes stimulated sarcomeric organization, increased cell size, and induced atrial natriuretic factor in cardiomyocytes plated on the ECM protein laminin or fibronectin. In contrast, cardiomyocytes plated on the non-adhesive substrate gelatin exhibited a reduced capacity to undergo these PE-stimulated hypertrophic changes. In cardiomyocytes cultured on ECM, PE stimulated a rapid increase in tyrosine phosphorylation of focal adhesion proteins including FAK, paxillin, and p130 Crk-associated substrate and subsequent formation of peripheral focal complexes. Inhibition of the PE-induced hypertrophic response by genistein and herbimycin-A indicated a requirement for protein tyrosine kinases in PE signaling. To determine whether activation of FAK is required for PE-induced hypertrophy, a dominant-interfering mutant form of FAK, termed FRNK (FAK-related non-kinase), was ectopically expressed in cardiomyocytes using a replication-defective adenovirus expression system. FRNK expression attenuated PE-stimulated hypertrophy as assessed by cell size, sarcomeric organization, and induction of atrial natriuretic factor. These data indicate that the signal transduction pathways leading to cardiomyocyte hypertrophy are strongly influenced by and/or dependent upon an integrin-mediated signaling process requiring FAK.     

Cocultures of fetal and adult cardiomyocytes yield rhythmically beating rod shaped heart cells from adult rats

Summary Different models of isolated cardiomyocytes are generally used for biochemical, biophysical, and pharmacological studies. Fetal cardiomyocytes can be easily cultured for several weeks regaining their ability for rhythmical and synchronous contractions. For investigations, differentiated myocytes derived from adult hearts are closer to the in situ situation. Unfortunately, these cells at best exhibit irregular and asynchronous contractions at very low frequencies. Already 1 d after seeding calcium-tolerant rod-shaped adult cardiomyocytes on a suitable substrate, the differentiated cells begin to dedifferentiate forming a confluent monolayer. After 7–10 d their beating activities are like those of fetal cells. Therefore, we tried to combine the advantages of both cell types to achieve fully differentiated cardiomyocytes, rod-shaped and rhythmically beating, isolated from adult hearts. Using contractile fetal cells as a substrate for the adult cardiomyocytes, freshly seeded differentiated adult myocytes are paced by the contraction frequency of the fetal monolayer. As a consequence, the rod-shaped adult cardiomyocytes reach frequencies of more than 140 cycles/min without external electrical stimulation. This model enables us to study cardiomyocytes in a state very similar to the in situ situation with respect to morphology, integrity, and contractile behavior. An abstract of this article was previously published in Eur. J. Cell Biol. 57 (Suppl.36): 86; 1992.     

Rearrangement of tubulin, actin, and myosin in cultured ventricular cardiomyocytes of the adult rat

Antitubulin, phalloidin, and antimyosin were used to study the distribution of microtubules, microfilaments, and myofibrils in cultured adult cardiomyocytes. These cells undergo a stereotypic sequence of morphological change in which myotypic features are lost and then reconstructed during a period of polymorphic growth. Microtubules, though rearranged during these events in culture, are always present in an organized network. Myosin and actin structures, on the other hand, initially degenerate. This initial degeneration is reversed when a cell attaches to the culture substratum. Upon attachment, new microtubules are laid down as a cortical network adjacent to the sarcolemma and, subsequently, as a network in the basal part of the cell. Actin and then myosin filament bundles appear next, in a pattern corresponding to the pattern of the microtubules. Finally, striated myofibrils are formed, first in the central part of the cell, and subsequently in the outgrowing processes of the cell. A mechanism is suggested by which the eventual polymorphic shape of a cell is related to the shape of its initial area of contact with the culture substratum. Finally, a model of myofibrillogenesis is proposed in which microtubules participate in the insertion of myosin among previously formed actin filament bundles to produce myofibrils.     

S100A1 decreases calcium spark frequency and alters their spatial characteristics in permeabilized adult ventricular cardiomyocytes

S100A1, a Ca2+-sensor protein of the EF-hand type, exerts positive inotropic effects in the heart via enhanced cardiac ryanodine receptor (RyR2) activity. Here we report that S100A1 protein (0.1microM) interacts with the RyR2 in resting permeabilized cardiomyocytes at free Ca2+-levels comparable to diastolic Ca2+-concentrations ( approximately 150nM). Alterations of RyR2 function due to S100A1 binding was assessed via analysis of Ca2+-spark characteristics. Ca2+-spark frequency, amplitude and duration were all reduced upon perfusion with 0.1microM S100A1 protein by 38%, 14% and 18%, respectively. Most likely, these effects were conveyed through the S100A1 C-terminus (S100A1-ct; amino acids 75-94) as the corresponding S100A1-ct peptide (0.1microM) inhibited S100A1 protein binding to the RyR2 and similarly attenuated frequency, amplitude and duration of Ca2+-sparks by 52%, 8% and 26%, respectively. Accordingly, the sarcoplasmic reticulum (SR) Ca2+-content was slightly increased but the stoichiometry of other accessory RyR2 modulators (sorcin/FKBP12.6) remained unaltered by S100A1. Hence, we propose S100A1 as a novel inhibitory modulator of RyR2 function at diastolic Ca2+-concentrations in rabbit ventricular cardiomyocytes.     

Characteristics of insulin receptors in the heart muscle Binding of insulin to isolated muscle cells from adult rat heart

Adult rat heart muscle cells obtained by perfusion of the heart with collagenase have been used to characterize the insulin receptors by equilibrium binding and kinetic measurements. Binding of ¹²⁵I-labelled insulin to heart cells exhibited a high degree of specificity; it was dependent on pH and temperature, binding at steady increased with decreasing temperatures. About 70% of the radioactivity bound at equilibrium at 25°C could be dissociated by addition of an excess of unlabelled insulin. 54 and 40% of ¹²⁵I-labelled insulin was degraded by isolated heart cells after 2 h at 37°C and 4 h at 25°C, respectively. This degrading activity was effectively inhibited by high concentration of albumin.Equilibrium binding studies were conducted at 25°C using insulin concentrations ranging from 2.5 · 10^?11 mol/l to 10^?6 mol/l. Scatchard analysis of the binding data resulted in a curvilinear plot (concave upward), which was further analyzed using the average affinity profile. The empty site affinity constant was calculated to be 9.5 · 10⁷ l/mol with a total receptor concentration of 3.4 · 10⁶ sites per cell.The presence of site-site interactions of the negative cooperative type among the insulin receptors has been confirmed by kinetic experiments. The rate of dilution induced dissociation was enhanced in the presence of native insulin (5 · 10^?9 mol/l), both, under conditions of low and high fractional saturation of receptors.     

Three major forms of adenylate kinase from adult and fetal rat tissues.

The major enzymatic forms of adenylate kinase have been purified to homogeneity from fetal liver and adult brain of the rat. The two enzymes differ with respect to isoelectric points, Km (ATP), Km (AMP), and Ka (citrate). Antibody to adult liver adenylate kinase does not inhibit either enzyme, while entibody to adult skeletal muscle enzyme inhibits the brain enzyme but not the fetal liver enzyme. It is therefore probable that there are three major forms of adenylate kinases in fetal and adult rat tissues.     

Evidence for alpha-1 adrenergic receptor regulation of atriopeptin release from the isolated rat heart

Atrial myocardium is the source of a recently described peptide hormone termed atriopeptin. Atriopeptin is thought to have a role in the regulation of systemic arterial pressure, fluid balance and plasma electrolyte homeostasis. Isolated rat hearts release atriopeptin into the coronary effluent, and we have found that this release is stimulated by the administration of norepinephrine, a compound with alpha and beta adrenergic properties. Infusion of the pure beta-receptor agonist, isoproterenol, failed to stimulate the release; however, the alpha-1 receptor agonist phenylephrine induced the release in a dose-dependent manner. The stimulation of atriopeptin release by norepinephrine and phenylephrine was inhibited by alpha-blockade with phentolamine. Administration of BHT-920, a selective alpha-2 agonist, had no effect on atriopeptin release. We conclude that atriopeptin secretion by the atrial myocyte is stimulated by activation of the alpha-1 adrenergic receptor. This finding suggests an involvement of the sympathetic nervous system in the physiologic regulation of the secretion of this hormone.     

PKC alpha-dependent regulation of the IGF1 receptor in adult and embryonic rat cardiomyocytes

In both, the adult rat ventricular cardiomyocytes and the embryonic rat heart cell line, H9c2, acute exposure to IGF1 resulted in activation of the IGF1 receptor’s internal tyrosine kinase, and this was completely blocked by the PKC alpha inhibitor, Gö6976. In addition, RNA interference using siRNA mediated gene silencing of PKC alpha–inhibited IGF1 receptor activity and blocked PKC alpha expression in H9c2 cells. Biochemical experiments demonstrate that PKC alpha is associated with the IGF1R (beta subunit) only after acute IGF1 exposure, and this may suggest that there is a direct interaction and possibly a PKC alpha phosphorylation site within the internal IGF1 receptor domain. The downstream effects of blocking PKC alpha activity by exposure to Gö6976 include inhibition of IGF1-stimuated PI3 kinase activity and reduced IGF1-stimulated c-fos expression in the adult cardiomyocytes. Previously, the laboratory has reported that IGF1 activates PKC alpha in adult rat cardiomyocytes, and that PKC alpha activity is required for IGF1-dependent Erk/Erk2 activity and protein synthesis. Here, it is shown that IGF1-dependent protein synthesis is completely blocked by PD98059, indicating that the Raf-Mek-Erk cascade is required for IGF1’s anabolic activity. Pretreatment with LY294002, a specific inhibitor of PI3 kinase, blocked IGF1-stimulated Erk1/Erk2 activity; therefore, PI3 kinase may also be required for IGF1-dependent protein synthesis. In H9c2 cells, coincubation with PMA lead to an increase in the rate of the IGF1 receptor activation, and this may further implicate a role for PKC in regulating the IGF1R. In conclusion, PKC alpha plays an essential role in the IGF1-signaling cascade, including the regulation of key signaling proteins involved in cell signaling and gene expression, and this may primarily be due to PKC alpha directly regulating the IGF1R.Both contributed equally to the experiments.