3-Hydroxy-3-methylglutaryl CoA reductase inhibitors up-regulate transforming growth factor-beta signaling in cultured heart cells via inhibition of geranylgeranylation of RhoA GTPase.

Transforming growth factor-beta (TGFbeta) signaling has been shown to play a role in cardiac development as well as in the pathogenesis of cardiovascular disease. Prior studies have suggested a relationship between cholesterol metabolism and TGFbeta signaling. Here we demonstrate that induction of the cholesterol metabolic pathway by growth of embryonic chicken atrial cells in medium supplemented with lipoprotein-depleted serum coordinately decreased the expression of the TGFbeta type II receptor (TGFbetaRII), TGFbeta(1), and TGFbeta signaling as measured by plasminogen activator inhibitor-1 (PAI-1) promoter activity. Inhibition of the cholesterol metabolic pathway by the hydrophobic 3-hydroxy-3-methylglutaryl CoA (HMGCoA) reductase inhibitors, simvastatin and atorvastatin, reversed the effect of lipoprotein-depleted serum and up-regulated TGFbetaRII expression, whereas the hydrophilic HMGCoA reductase inhibitor, pravastatin, had no effect. Simvastatin stimulated the expression of TGFbetaRII, TGFbeta(1), and PAI-1 at the level of transcription. Experiments using specific inhibitors of different branches of the cholesterol metabolic pathway demonstrated that simvastatin exerted its effect on TGFbeta signaling by inhibition of the geranylgeranylation pathway. C3 exotoxin, which specifically inactivates geranylgeranylated Rho GTPases, mimicked the effect of simvastatin on PAI-1 promoter activity. Cotransfection of cells with a PAI-1 promoter-reporter and a dominant-negative RhoA mutant increased PAI-1 promoter activity, whereas cotransfection with a dominant-active RhoA mutant decreased PAI-1 promoter activity. These data support the conclusion that TGFbeta signaling is regulated by RhoA GTPase and demonstrate a relationship between cholesterol metabolism and TGFbeta signaling. Our data suggest that in patients treated with HMGCoA reductase inhibitors, these agents may exert effects independent of cholesterol lowering on TGFbeta signaling in the heart.

Induction of the cholesterol metabolic pathway regulates the farnesylation of RAS in embryonic chick heart cells: a new role for ras in regulating the expression of muscarinic receptors and G proteins.

We propose a novel mechanism for the regulation of the processing of Ras and demonstrate a new function for Ras in regulating the expression of cardiac autonomic receptors and their associated G proteins. We have demonstrated previously that induction of endogenous cholesterol synthesis in cultured cardiac myocytes resulted in a coordinated increase in expression of muscarinic receptors, the G protein alpha-subunit, G-alphai2, and the inward rectifying K+ channel, GIRK1. These changes in gene expression were associated with a marked increase in the response of heart cells to parasympathetic stimulation. In this study, we demonstrate that the induction of the cholesterol metabolic pathway regulates Ras processing and that Ras regulates expression of G-alphai2. We show that in primary cultured myocytes most of the RAS is localized to the cytoplasm in an unfarnesylated form. Induction of the cholesterol metabolic pathway results in increased farnesylation and membrane association of RAS. Studies of Ras mutants expressed in cultured heart cells demonstrate that activation of Ras by induction of the cholesterol metabolic pathway results in increased expression of G-alphai2 mRNA. Hence farnesylation of Ras is a regulatable process that plays a novel role in the control of second messenger pathways.

Physiological and biochemical evidence for coordinate increases in muscarinic receptors and Gi during pacing-induced heart failure.

BACKGROUND: It is not clear whether the increase in the myocardial guanylyl nucleotide inhibitory protein (Gi), frequently observed in heart failure, is associated with any functional effects. METHODS AND RESULTS: Eight sham-operated dogs and 10 dogs were studied with pacing-induced heart failure (240 bpm for 4 to 7 weeks), characterized by reduced (P<.05) left ventricular dP/dt (from 2926+/-99 to 1303+/-126 mm Hg/s). The muscarinic agonist acetylcholine (10 micrograms/kg IV) in the presence of ganglionic blockade reduced left ventricular dP/dt more (P<.05) in heart failure (-23+/-2%) than before heart failure (-8+/-2%), despite lesser reductions in arterial pressure. Gi alpha2 was increased by 55% in heart failure. Dose-response curves for carbachol (10-8 to 10-3 mol/L) inhibition of isoproterenol-stimulated adenylyl cyclase demonstrated significantly greater (P<.05) inhibition in heart failure compared with sham-operated dogs. These changes were associated with a coordinate increase in muscarinic receptor density, determined by antagonist binding with 3H-quinuclidinyl benzilate, in heart failure (153+/-6.2 fmol/mg protein) compared with sham-operated dogs (124+/-7.4 fmol/mg protein). Agonist binding with carbachol also revealed an increase in total muscarinic receptors in heart failure without a change in fraction of high- and low-affinity receptors. CONCLUSIONS: These data, in the aggregate, provide physiological and biochemical evidence to support the concept that the coordinate increases in muscarinic receptor number and Gi levels in heart failure are coupled to increased inhibition of adenylyl cyclase activity and an increased inhibition of myocardial contractility.

Specificity of coupling of muscarinic receptor isoforms to a novel chick inward-rectifying acetylcholine-sensitive K+ channel.

The G-protein-gated inward-rectifying K+ channel GIRK1 has been demonstrated in heart and brain. These tissues also both express the M2, M3, and M4, muscarinic acetylcholine receptors (mAChR) (Gadbut, A.P., and Galper, J.B. (1994),J. Biol. Chem. 269,25823-25829). Only the M2 mAChR has been demonstrated to couple to GIRK1 (Kubo, Y., Reuveny, E., Slesinger, P. A., Jan, Y. N., and Jan, L. Y. (1993) Nature 264, 802-806). In this study we determined the specificity of coupling of the M3 and M4 mAChR to a new GIRK1 cloned from a chick brain cDNA library. This clone codes for a 492-amino acid protein that is 93% identical to rat GIRK1 and is expressed in brain, atrium, and ventricle, but not skeletal muscle. In Xenopus laetis oocytes co-expression of GIRK1 with either the chick M2 or M4 mAChR gave carbamylcholine (10 microm)-stimulated K+ currents of 308 +/-26 nA and 298 +/-29 nA, respectively, which were both Ba2+- and pertussis toxin-sensitive. Activation of the M3 receptor produced 2382 +/-478 nA of current which was insensitive to Ba2+ and pertussis toxin, but was 85% inhabitable by the Cl channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (10-20 microm) consistent with coupling to an endogenous Ca2+-activated Cl- channel via a phosphatidylinositol-dependent mechanism. Co-expression of the cardiac inward rectifier CIR with chick M2 or M4 mAChR and GIRK1 increased currents more than 10-fold, but had no effect on specificity of coupling. These data demonstrate a new function for the M4 mAChR and a high degree of specificity for coupling of each receptor subtype to GIRK1.

Differential sensitivity of C2-C12 striated muscle cells to lovastatin and pravastatin.

One of the major side-effects of the use of HMG CoA reductase inhibitors for the treatment of hypercholesterolemia is the development of myositis and, in some patients undergoing concomitant immunosuppressive treatment, the development of rhabdomyolysis. Experiments outlined in these studies demonstrate that inhibitors of HMG-CoA reductase activity which differ primary in the substitution of a methyl group for a hydroxyl group have differential effects on both cholesterol levels and cell viability in a striated muscle cell model, the mouse C2-C12 myoblast. Thus, concentrations as high as 200 microM of pravastatin had little effect on total cholesterol level while 25 microM of lovastatin decreased cellular cholesterol by over 90%. Simvastatin and lovastatin decreased viability of C2-C12 myoblasts by nearly 50% at concentrations as low as 1 and 5 microM, respectively, and decreased viability by almost 90% at 10 and 15 microM respectively. However, 300 microM of pravastatin decreased cell viability by less than 50%. The order of potency for the effects on cell viability wassimvastatin>lovastatin>>>pravastatin. The possible relationship between effects on cell viability and the development of myositis is discussed.

Decreased adenylate cyclase activity and expression of Gs alpha in human myocardium after orthotopic cardiac transplantation.

We studied several aspects of guanine nucleotide-stimulated adenylate cyclase function in patients after orthotopic cardiac transplantation. In 28 patients, adenylate cyclase activity was measured in endomyocardial biopsy samples obtained just before and at monthly intervals after cardiac transplantation. In biopsies obtained > or = 6 months after transplantation, basal adenylate cyclase activity was decreased by 67% (n = 12; P < .05), GTP gamma S-stimulated adenylate cyclase activity was decreased by 78% (n = 12; P < .05), Mn+2+forskolin-stimulated adenylate cyclase activity was decreased by 80% (n = 8; P < .05), and Mn+2-stimulated adenylate cyclase activity (a measure of activity of the catalytic subunit of adenylate cyclase) was decreased by 83% (n = 8, P < .05). Western blot analysis demonstrated that 6 months after cardiac transplantation, the level of Gs alpha protein was decreased by 61 +/- 12% (n = 8; P < .001). There was no change in the level of Gi alpha as assessed by pertussis toxin-catalyzed ADP-ribosylation (n = 4; P = NS). With the use of the quantitative polymerase chain reaction, a 50 +/- 10% (n = 6; P < .001) reduction in the steady-state level of Gs alpha mRNA was observed. There was no change in the level of mRNA for Gi-3 alpha. Thus, after orthotopic cardiac transplantation in humans, guanine nucleotide-stimulated adenylate cyclase activity is decreased in parallel with decreased levels of Gs alpha protein and mRNA.

Low density lipoproteins induce parasympathetic responsiveness in embryonic chick ventricular myocytes in parallel with a coordinate increase in expression of genes coding for the M2 muscarinic receptor, G alpha i2, and the acetylcholine-sensitive K+ channel.

Growth of chick atrial cells in medium supplemented with lipoprotein-depleted serum has been shown to result in an increase in total cell cholesterol, and an increase in the negative chronotropic response to muscarinic stimulation in parallel with an increase in levels of muscarinic receptors and the G-protein alpha-subunits alpha i and alpha o (Haigh, L. S., Leatherman, G. F., O'Hara, D. S., Smith, T. W., and Galper, J. B. (1988) J. Biol. Chem. 263, 15608-15618). In this study we determined whether growth of chick ventricular cells in medium supplemented with lipoprotein depleted serum could alter levels of muscarinic receptors and G-protein alpha-subunits and induce a negative chronotropic response to muscarinic stimulation. We further determined whether levels of mRNA coding for muscarinic receptors, G-proteins, and the acetylcholine-sensitive K+ channel were coordinately regulated. Growth of embryonic chick ventricular cells from hearts 14 days in ovo in medium supplemented with lipoprotein depleted serum resulted in a 21 +/- 5% (n = 3, +/- S.E.) increase in muscarinic receptor number as demonstrated by [3H]quinuclidinyl benzilate binding and a 4.7 +/- 1.0 (+/- S.E., n = 4)-fold increase in G alpha i2 as demonstrated by Western blot analysis. These changes in receptor and G-protein were associated with a coordinate increase in levels of mRNA coding for the M2 muscarinic receptor, G alpha i2 and the acetylcholine sensitive K+ channel as determined by RNase protection. These increases were reversed by addition of 30 microM mevinolin, an inhibitor of HMG-CoA reductase activity. Carbamylcholine (0.1 mM) had no effect on beat rate in ventricular cells grown in medium supplemented with fetal calf serum. Cells grown in medium supplemented with lipoprotein depleted serum demonstrated a 40 +/- 8% (+/- S.E., n = 10, p < 0.0001) decrease in beat rate in response to 0.1 mM carbamylcholine which was reversed by the addition of 30 microM mevinolin. These data suggest that, during growth in medium supplemented with lipoprotein depleted serum, a component of the cholesterol biosynthetic pathway plays a role in the coordinate induction of mRNAs coding for receptors, G-proteins, and an effector (ion channel) that results in the induction of a parasympathetic response in the ventricular cell characteristic of the atrial phenotype.

Cloning of cDNAs coding for the G alpha i1 and G alpha i2 G-proteins from chick brain.

We have cloned and characterized several cDNAs coding for G-protein inhibitory alpha subunits (G alpha i) from a chick brain cDNA library. Based on homology to G alpha subunits from other eukaryotes, these clones were designated chick G alpha i1 and G alpha i2. On the deduced amino-acid level, G alpha i1 and G alpha i2 were found to be 98 and 95% identical to rat G alpha i1 and G alpha i2, respectively. Using RNase protection analysis, the G alpha i1 and G alpha i2 mRNAs were found to be expressed in chick atria, ventricle, lung, liver, brain and kidney.

A novel M3 muscarinic acetylcholine receptor is expressed in chick atrium and ventricle.

Prior studies have suggested that heart expresses only the M2 isoform of the muscarinic receptor (Peralta, E.G., Ashkenazi, A., Winslow, J.W., Smith, D.H., Ramachandran, J., and Capon, D.J. (1987) EMBO J. 6, 3923-3929). Tietje and Nathanson (Tietje, K.M., and Nathanson, N. M. (1991) J. Biol. Chem. 266, 17382-17387) have recently demonstrated that the chick heart may be unique since it expresses both the M2 and M4 isoforms of the muscarinic receptor. In this study, in order to determine whether other isoforms of the muscarinic receptor were present in the chick heart, a chick M3 muscarinic receptor receptor was cloned, characterized, and its expression in chick tissues determined. Using a human M3 muscarinic receptor cDNA as a probe, a 2.4-kilobase pair cDNA was isolated from a chick brain cDNA library which contained an open reading frame coding for a 639 amino acid protein. This protein demonstrated an 87 and 86% homology to the human and rat M3 muscarinic receptor, respectively. Chinese hamster ovary (CHO-GRA) cells were stably transfected with the chick M3 muscarinic receptor and one clone (CHO-CM3) expressed the M3 receptor, as measured by the binding of quinuclidinly benzilate at 116 +/- 14 (+/- S.E., n = 3) fmol/mg protein with a Kd of 76 +/- 17 pM. This receptor demonstrated a rank order of potency for muscarinic antagonist binding characteristic for the M3 receptor: with high affinity binding for hexahydrosiladifenidol, Kd: 16 +/- 2 nM (+/- S.E., n = 3); intermediate affinity for pirenzepine, Kd: 383 +/- 47 nM, and low affinity for methoctramine, Kd: 533 +/- 185 nM (+/- S.E., n = 3). Carbamylcholine stimulation of CHO-CM3 cells resulted in a 1.6-fold increase in cyclic AMP accumulation and a 3.5-fold increase in a pertussis toxin-insensitive inositol phosphate release. These data demonstrate that the chick M3 muscarinic receptor has the properties characteristic of M3 receptors from other species. RNase protection studies demonstrated the presence of M3 muscarinic receptor mRNA in the brain, atria, and ventricle of chicks 17 days in ovo. Hence, the chick heart appears to have the unique capacity to express mRNAs coding not only for the M2 and M4 muscarinic receptors but also for the M3 muscarinic receptor.

Cloning and developmental expression of the chick type II and type III TGF beta receptors.

To address the role of peptide growth factors in chick organogenesis, we have focused on TGF beta 2 and have cloned the chick Type II and Type III TGF beta receptors. The chick Type II receptor is a serine/threonine kinase with a ligand binding profile identical to the human receptor and a divergent N-terminus when compared to the mammalian receptors. The chick Type III receptor is a beta-glycan that demonstrates a binding profile identical to the rat receptor and contains a single transmembrane spanning domain and short cytoplasmic tail that are highly conserved when compared to the mammalian receptors. Both the Type II and Type III TGF beta receptors are coexpressed during chick embryogenesis in the developing heart, lung, and eye, and are developmentally upregulated in parallel in the heart and lung. Levels of both receptor proteins and mRNAs also increase in cardiocytes cultured from different developmental stages, in agreement with the increase in Type II and Type III receptor mRNA levels observed in the developing heart. Although exhibiting different temporal or spatial profiles from the receptors, TGF beta 2 is also expressed in the developing heart, lung, and eye. These findings are consistent with recent data indicating that co-expression of both the Type II and Type III TGF beta receptors is required for high affinity binding of TGF beta 2 by the Type II receptor and suggest that TGF beta 2 and the Type II and Type III TGF beta receptors participate in heart, lung, and eye development.

Isolation and expression of a novel chick G-protein cDNA coding for a G alpha i3 protein with a G alpha 0 N-terminus.

We have cloned cDNAs coding for G-protein alpha subunits from a chick brain cDNA library. Based on sequence similarity to G-protein alpha subunits from other eukaryotes, one clone was designated G alpha i3. A second clone, G alpha i3-o, was identical to the G alpha i3 clone over 932 bases on the 3' end. The 5' end of G alpha i3-o, however, contained an alternative sequence in which the first 45 amino acids coded for are 100% identical to the conserved N-terminus of G alpha o from species such as rat, mouse, human, bovine and hamster. Both clones were found to be expressed in all tissues studied. The unusual alpha o-alpha i3-like G-protein chimera, G alpha i3-o, was found to be expressed at significantly lower levels than G alpha i3. In vitro transcription and translation of the G alpha i3-o cDNA clone gave a protein of approx. 41 kDa which stably bound guanosine 5'-[gamma-thio]triphosphate. G alpha i3-o appears to be the first G-protein alpha subunit cloned which contains ends that are homologous to two different alpha subunit isoforms, G alpha o and G alpha i3.

Effect of low-density lipoproteins, mevinolin, and G proteins on Ca2+ response in cultured chick atrial cells.

Growth of cells from atria of embryonic chick hearts 14 days in ovo in medium supplemented with lipoprotein-depleted serum (LPDS) results in an increase in total cell cholesterol, enhanced parasympathetic responsiveness (7), and decreased sympathetic responsiveness (1). These effects were reversed by the hydroxymethyl glutaryl CoA reductase inhibitor, mevinolin. In these studies, comparison of cell growth in medium supplemented with fetal calf serum (FCS) and LPDS demonstrated that, after growth with LPDS, the ability of Ca2+ and the Ca2+ channel agonist, BAY K 8644, to enhance the amplitude of contraction decreased by 25 and 50%, respectively. These effects of growth in LPDS were reversed by incubation with mevinolin. LPDS had no effect on either Ca2+ channel number as measured by (+)-[5-methyl-3H]PN200-110 binding or Ca2+ current density as measured by the whole cell patch method. Treatment of cells grown in LPDS with pertussis toxin, which inactivates alpha o and alpha i, returned the contractile response to 10(-7) M BAY K 8644 to control levels. Pertussis toxin had no effect on the contractile response or adenosine 3',5'-cyclic monophosphate levels in control cells grown in FCS alone. These data suggest that alterations in the relative levels of alpha o and alpha s in cells grown in LPDS may play a role in regulating the contractile response to Ca2+ channel agonists and to exogenous Ca2+.

Co-culture of embryonic chick heart cells and ciliary ganglia induces parasympathetic responsiveness in embryonic chick heart cells.

We have developed a system for the co-culture of embryonic chick heart cells obtained from embryos at 3.5 days in ovo with ciliary ganglia from chick embryos at 7 days in vivo. After 3 days of co-culture, removal of the ciliary ganglia resulted in complete degeneration of axons within 6-8 h, leaving the post-innervated heart cell culture devoid of neurons. Embryonic chick heart cells at 3.5 days in ovo are unresponsive to muscarinic stimulation. However, following 3 days of co-culture with ciliary ganglia, the heart cells developed a negative chronotropic response to muscarinic stimulation (paired t test, P < 0.02) which persisted for at least 24 h after removal of the ciliary ganglion. The development of muscarinic responsiveness was associated with an increase in the levels of specific alpha-subunits of the guanine nucleotide binding proteins (G-proteins), with a 3-fold increase in the level of alpha 39 (39 kDa subunit) and a 2.5-fold increase in the level of alpha 41. The level of the G-protein subunit alpha s remained unchanged. Culture of embryonic chick heart cells at 3.5 days in ovo with medium conditioned by the growth of embryonic chick heart cells and ciliary ganglia had an effect on the chronotropic response to muscarinic stimulation and on alpha 39 and alpha 41 levels identical to that of co-culture. These data suggest that a soluble factor released during the co-culture of embryonic chick heart cells and ciliary ganglia is capable of inducing muscarinic responsiveness. These studies suggest that innervation of the heart may induce parasympathetic responsiveness by increasing the availability of G-proteins which couple the muscarinic receptor to a physiological response.

Direct contact between sympathetic neurons and rat cardiac myocytes in vitro increases expression of functional calcium channels.

To test the hypothesis that direct contact between sympathetic neurons and myocytes regulates expression and function of cardiac Ca channels, we prepared cultures of neonatal rat ventricular myocytes with and without sympathetic ganglia. Contractile properties of myocytes were assessed by an optical-video system. Contractility-pCa curves showed a 60% greater increase in contractility for innervated myocytes compared with control cells at 6.3 mM [Ca]0 (n = 8, P less than 0.05). Cells grown in medium conditioned by growth of ganglia and myocytes were indistinguishable physiologically from control cells. [Bay K 8644]-contractility curves revealed a 60 +/- 10% enhancement of the contractility response at 10(-6) M for innervated cells compared with control cells. The increased response to Bay K 8644 was not blocked by alpha- or beta-adrenergic antagonists. Moreover, increased efficacy of Bay K 8644 was maintained for at least 24 h after denervation produced by removal of ganglia from the culture. Dihydropyridine binding sites were assessed with the L channel-specific radioligand 3[H]PN200-110. PN200-110 binding sites were increased by innervation (51 +/- 5 to 108 +/- 20 fmol/mg protein, P less than 0.01), with no change in KD. Peak current-voltage curves were determined by whole-cell voltage clamp techniques for myocytes contacted by a neuron, control myocytes, and myocytes grown in conditioned medium. Current density of L-type Ca channels was significantly higher in innervated myocytes (10.5 +/- 0.4 pA/pF, n = 5) than in control myocytes (5.9 +/- 0.3 pA/pF, n = 8, P less than 0.01) or myocytes grown in conditioned medium (6.2 +/- 0.2 pA/pF, n = 10, P less than 0.01). Thus, physical contact between a sympathetic neuron and previously uninnervated neonatal rat ventricular myocytes increases expression of functional L-type calcium channels as judged by contractile responses to Ca0 and Bay K 8644, as well as by electrophysiological and radioligand binding properties.

Effect of manganese (II) bis(glycinate)dichloride on Ca2+ channel function in cultured chick atrial cells.

Manganese (II) bis(glycinate)dichloride (Mn(glycinate)2) is a coordination complex of manganese with application as a contrast enhancement agent for magnetic resonance imaging in the heart. To determine the cardioactivity of the manganese ion in this chelation cage, the effects of Mn(glycinate)2 on Ca channel function in the cultured chick atrial cell was studied. Mn(glycinate)2 decreased amplitude of contraction in chick atrial cells from embryos 14 days in ovo with complete inhibition of beating at 1 mM and half-maximal effect at 0.1 mM. Under control conditions, Bay K 8644, a Ca channel activator increased amplitude of contraction by 86% with a half maximal effect at 3.2 x 10(-7) M. In the presence of 0.025 mM Mn(glycinate)2, a concentration which had no effect on the amplitude of contraction, the maximum response to Bay K 8644 was decreased to 31%. Mn(glycinate)2 had no effect on the EC50 for the response to Bay K 8644, 1.7 +/- 0.1 x 10(-9) M (S.E.M., n = 4) in control cells compared to 2.2 +/- 0.4 x 10(-9) M (S.E.M., n = 4) in cells incubated with Mn(glycinate)2. 45Ca2+ uptake over 5 min in cultured chick atrial cells decreased from 2.0 nmol/mg protein in control cells to 1.5 nmol/mg protein in the presence of 10(-5) M PN200-110, a Ca2+ channel blocker, a decrease of 28%. 45Ca2+ uptake decreased to 0.94 nmol/mg protein (53%) in the presence of 1 nmol Mn(glycinate)2. Effects of Mn(glycinate)2 and PN200 were not additive. These data demonstrate that Mn(glycinate)2 exerts its negative inotropic effect, at least partially, by interfering with the function of the L-type Ca channels at high concentrations.

Muscarinic cholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells. Evidence for a role of two guanine-nucleotide-binding proteins.

These studies demonstrate a novel mechanism for the coupling of the muscarinic receptor to phospholipase C activity in embryonic chick atrial cells. In monolayer cultures of atrial cells from hearts of embryonic chicks at 14 days in ovo, carbamylcholine stimulated the sequential appearance of InsP3, InsP2 and InsP1 with an EC50 (concn. causing 50% of maximal stimulation) of 30 microM. In the presence of 15 mM-Li, a 5 min exposure to carbamylcholine (0.1 mM) increased InsP3 levels to a maximum of 47 +/- 12% over basal, InsP2 to 108 +/- 13% over basal and InsP1 to 42 +/- 5% over basal. This effect was blocked by 5 microM-atropine. Incubation of these cells with pertussis toxin (15 h; 0.5 ng/ml) inhibited carbamylcholine-stimulated InsP3, InsP2 and InsP1 formation by 42 +/- 7%, 30 +/- 3% and 48 +/- 7% respectively. The IC50 (concn. causing 50% inhibition) for pertussis toxin inhibition of all three inositol phosphates was 0.01 ng/ml, with a half-time of 6 h at 0.5 ng/ml. This partial sensitivity to pertussis toxin was not due to incomplete ADP-ribosylation of the guanine-nucleotide-binding protein (G-protein), since autoradiography of polyacrylamide gels of cell homogenates incubated with [32P]NAD+ in the presence of pertussis toxin demonstrated that incubation of cells with 0.5 ng of pertussis toxin/ml for 15 h resulted in complete ADP-ribosylation of pertussis toxin substrates by endogenous NAD+. In cells permeabilized with saponin (10 micrograms/ml), 0.1 mM-GTP[S] (guanosine 5'-[gamma-thio]triphosphate) stimulated InsP1 by 102 +/- 15% (mean +/- S.E.M., n = 4), InsP2 by 421 +/- 67% and InsP3 by 124 +/- 33% above basal. Incubation of cells for 15 h with 0.5 ng of pertussis toxin/ml decreased GTP[S]-stimulated InsP1 production in saponin-treated cells by 30 +/- 10% (n = 3), InsP2 production by 45 +/- 7% (n = 4) and InsP3 production by 49 +/- 6% (n = 4). These data demonstrate that in embryonic chick atrial cells at least two independent G-proteins, a pertussis toxin-sensitive G-protein and a pertussis toxin-insensitive G-protein, play a role in coupling muscarinic agonist binding to phospholipase C activation and to inositol phosphate production.

Development of muscarinic-cholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells. Evidence for a switch in guanine-nucleotide-binding protein coupling.

We have demonstrated that muscarinic stimulation of inositol phosphate production in cultured atrial cells from chicks at 14 days in ovo is partially sensitive to inhibition by pertussis toxin. In these cells, muscarinic agonist binding is coupled to phospholipase C activity via at least two guanine-nucleotide-binding proteins (G-proteins), one sensitive to pertussis toxin and the other (Gp) insensitive to pertussis toxin [Barnett, Shamah, Lassegue, Griendling & Galper (1990) Biochem. J. 271, 437-442]. In the current study we demonstrate that during embryonic development of the chick heart, muscarinic stimulation of inositol phosphate production decreases by 50% between days 5 and 14 in ovo in cells cultured from both atrium and ventricle. In atrial cells, however, pertussis toxin-sensitive muscarinic stimulation of inositol phosphate production increased from undetectable levels at day 5 in ovo to 40% of total stimulation at day 12 in ovo. Muscarinic stimulation of inositol phosphate production in the ventricle did not become sensitive to pertussis toxin at any age studied. In permeabilized atrial cells from embryonic chicks at 5 days in ovo, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated InsP1 levels by 40 +/- 10% (mean +/- S.E.M., n = 3), InsP2 levels by 117 +/- 18% and InsP3 levels by 51 +/- 8%, suggesting that at day 5 in ovo all of the muscarinic-stimulated inositol phosphate production was coupled to phospholipase C via Gp. H.p.l.c. analysis demonstrated that, in spite of these changes in coupling of phospholipase C to different G-proteins, no changes could be demonstrated in the isomers of InsP3 produced in response to carbamylcholine at both days 5 and 14 in ovo. These data demonstrate that embryonic development of the chick atrium is associated with a switch in coupling of muscarinic receptors to phospholipase C from Gp to a pertussis toxin substrate. This developmental switch in coupling of G-proteins may be related to possible developmental switches in levels of muscarinic receptor isoforms or switches in the subtype of phospholipase C.

The development of physiologic responsiveness to muscarinic stimulation in embryonic chick heart. Relationship to increased levels of pertussis toxin substrates.

Studies of the development of parasympathetic responsiveness in embryonic chick hearts have demonstrated that between days 2.5 and 10 in ovo the ability of muscarinic agonists to inhibit adenylate cyclase activity increases 10-fold in parallel with a 2.7-fold increase in the level of alpha i and alpha o. Thus, muscarinic inhibition of adenylate cyclase increases in parallel with an increase in alpha o and alpha i. These data suggest that changes in levels of guanine nucleotide regulatory proteins control, at least in part, the appearance of a parasympathetic response in the heart during embryonic development of the chick.

Effects of low density lipoproteins and mevinolin on sympathetic responsiveness in cultured chick atrial cells. Regulation of beta-adrenergic receptors and alpha s.

We have previously demonstrated that cultures of myocytes from embryonic chick atria grown in media supplemented with fetal calf serum from which lipoproteins have been removed demonstrate a nearly 10-fold increase in sensitivity of beating to the muscarinic cholinergic agonist carbamylcholine compared with cells grown with control medium. This increased response to carbamylcholine was associated with a 1.4-fold increase in total cell cholesterol, a 2-fold increase in the number of muscarinic receptors which bind agonist with high affinity, and a 2-fold increase in the levels of the alpha subunits of Go and Gi (Haigh, L. S., Leatherman, G. F., O'Hara, D. S., Smith, T. W., and Galper, J. B. (1988) J. Biol. Chem. 263, 15608-15618). In the studies reported here, we determined the responsiveness of cells grown in lipoprotein-depleted serum (LPDS) to beta-adrenergic stimulation. Isoproterenol stimulated a contractile response of 58% measured as an increase in amplitude of contraction with a half-maximal effect at 3 x 10(-7) M for cells grown in fetal calf serum, but had no significant effect on amplitude of contraction on cells grown in LPDS. In cells grown in media supplemented with fetal calf serum, isoproterenol (1 x 10(-3) M) stimulated adenylate cyclase activity 100% over basal with an EC50 of 7 x 10(-6) M compared with an increase of 32% in cells grown in media supplemented with LPDS. beta-Adrenergic receptor number as measured by the binding of 125I-pindolol decreased from 24 +/- 3 (+/- S.E., n = 6) fmol/mg protein in cells grown under control conditions to 12 +/- 2 (n = 6) fmol/mg protein in media supplemented with LPDS. The level of alpha s as measured both by ADP-ribosylation with cholera toxin in the presence of 32P-NAD and by immunoblotting with specific antibody to alpha s decreased by 3-fold in cells grown in media supplemented with LPDS compared with control. All of these effects of growth of cells in LPDS were reversed by incubating cells with LPDS plus 30 microM mevinolin, an inhibitor of endogenous cholesterol synthesis. These studies indicate that growth of cells in media supplemented with LPDS results in a coordinate decrease in the levels of beta-adrenergic receptors and alpha s. Taken together with our previous studies these data support the hypothesis that the receptors and guanine nucleotide-binding proteins which mediate sympathetic and parasympathetic responsiveness in the heart are reciprocally regulated.