Isoproterenol-stimulated cAMP Production Research Articles

Catestatin (Chromogranin A352–372) and Novel Effects on Mobilization of Fat from Adipose Tissue through Regulation of Adrenergic and Leptin Signaling

Chromogranin A knock-out (Chga-KO) mice display increased adiposity despite high levels of circulating catecholamines and leptin. Consistent with diet-induced obese mice, desensitization of leptin receptors caused by hyperleptinemia is believed to contribute to the obese phenotype of these KO mice. In contrast, obesity in ob/ob mice is caused by leptin deficiency. To characterize the metabolic phenotype, Chga-KO mice were treated with the CHGA-derived peptide catestatin (CST) that is deficient in these mice. CST treatment reduced fat depot size and increased lipolysis and fatty acid oxidation. In liver, CST enhanced oxidation of fatty acids as well as their assimilation into lipids, effects that are attributable to the up-regulation of genes promoting fatty acid oxidation (Cpt1α, Pparα, Acox, and Ucp2) and incorporation into lipids (Gpat and CD36). CST did not affect basal or isoproterenol-stimulated cAMP production in adipocytes but inhibited phospholipase C activation by the α-adrenergic receptor (AR) agonist phenylephrine, suggesting inhibition of α-AR signaling by CST. Indeed, CST mimicked the lipolytic effect of the α-AR blocker phentolamine on adipocytes. Moreover, CST reversed the hyperleptinemia of Chga-KO mice and improved leptin signaling as determined by phosphorylation of AMPK and Stat3. CST also improved peripheral leptin sensitivity in diet-induced obese mice. In ob/ob mice, CST enhanced leptin-induced signaling in adipose tissue. In conclusion, our results implicate CST in a novel pathway that promotes lipolysis and fatty acid oxidation by blocking α-AR signaling as well as by enhancing leptin receptor signaling.

Adenylyl Cyclase 6 Improves Calcium Uptake and Left Ventricular Function in Aged Hearts

This study tested the hypothesis that activation of adenylyl cyclase 6 (AC6) expression in cardiac myocytes improves calcium uptake and left ventricular (LV) function in aging mice. Aging hearts exhibit impaired β-adrenergic receptor signaling and LV dysfunction. Twenty-month-old mice with cardiac-directed and regulated AC6 expression were randomized into 2 groups, and AC6 expression was activated in 1 group (AC6-On) but not the other (AC6-Off). One month later, LV function and sarcoplasmic reticulum calcium uptake were assessed. AC6 expression was associated with increased LV contractility, as reflected by ejection fraction (p = 0.02), rate of pressure development (p = 0.002), and slope of the LV end-systolic pressure-volume relationship (p = 0.04). No changes in LV weight to tibial length ratio, LV fibrosis, and expression of fetal genes (atrial natriuretic factor, α-skeletal muscle actin, and β-myosin heavy chain) and collagens were observed between AC6-On and AC6-Off groups. However, LV samples from AC6-On mice showed increases in: isoproterenol-stimulated cAMP production (p = 0.04), cAMP-dependent protein kinase activity (p < 0.0004), phosphorylation of phospholamban (at Ser16 site; p = 0.04) and cardiac troponin I (at Ser23/24 sites; p = 0.01), velocity of sarcoplasmic reticulum calcium uptake (p < 0.0001), and sarcoplasmic reticulum calcium-ATPase2a (SERCA2a) affinity for calcium (p < 0.0001). Finally, we found that AC6 expression increased sarcoplasmic reticulum calcium storage in cardiac myocytes isolated from 23-month-old rats. In contrast, AC6 expression in 7-month-old mice did not change LV function and calcium uptake. These results indicate that activation of cardiac AC6 expression improves impaired function of aged hearts through improved calcium uptake.

Gq-mediated Ca2+ signals inhibit adenylyl cyclases 5/6 in vascular smooth muscle cells

cAMP and Ca(2+) are antagonistic intracellular messengers for the regulation of vascular smooth muscle tone; rising levels of Ca(2+) lead to vasoconstriction, whereas an increase of cAMP induces vasodilatation. Here we investigated whether Ca(2+) interferes with cAMP signaling by regulation of phophodiesterases (PDEs) or adenylyl cyclases (ACs). We studied regulation of cAMP concentrations by Ca(2+) signals evoked by endogenous purinergic receptors in vascular smooth muscle cells (VSMCs). The fluorescence resonance energy transfer (FRET)-based cAMP sensor Epac1-camps allowed the measurement of cAMP levels in single-living VSMCs with subsecond temporal resolution. Moreover, in vitro calibration of Epac1-camps enabled us to estimate the absolute cytosolic cAMP concentrations. Stimulation of purinergic receptors decreased cAMP levels in the presence of the beta-adrenergic agonist isoproterenol. Simultaneous imaging of cAMP with Epac1-camps and of Ca(2+) with Fura 2 revealed a rise of intracellular Ca(2+) in response to purinergic stimulation followed by a decline of cAMP. Chelation of intracellular Ca(2+) and overexpression of Ca(2+)-independent AC4 antagonized this decline of cAMP, whereas pharmacological inhibition of Ca(2+)-activated PDE1 had no effect. AC assays with VSMC membranes revealed a significant attenuation of isoproterenol-stimulated cAMP production by the presence of 2 muM Ca(2+). Furthermore, small interfering RNA (siRNA) knockdown of AC5 and AC6 (the two ACs known to be inhibited by Ca(2+)), significantly reduced the decrease of cAMP upon purinergic stimulation of isoproterenol-prestimulated VSMCs. Taken together, these results implicate a Ca(2+)-mediated inhibition of AC5 and 6 as an important mechanism of purinergic receptor-induced decline of cAMP and show a direct cross talk of these signaling pathways in VSMCs.

β-Adrenergic Agonist Hyperplastic Effect Is Associated with Increased Fibronectin Gene Expression and Not Mitogen-Activated Protein Kinase Modulation in C2C12 Cells

Abstract. β-Adrenergic agonists (β-AA) enhance protein accretion in skeletal muscles. This stimulation is characterized by increased protein synthesis, increased expression of myofibrillar protein genes and a depression in protein degradation in animals, and increased proliferation and DNA synthesis in muscle cells in vitro. The mechanism or signal path in muscle whereby β-AA would elicit these physiological effects upon binding to the G protein–coupled β-adrenergic receptor (β-AR) is unclear. C2C12 myoblasts were used to determine β-AR ligand binding characteristics, cyclic AMP synthesis in response to isoproterenol (ISO) stimulation, and effects of ISO on DNA synthesis, mitogen activated protein kinase (MAPK), and fibronectin (FN) gene expression. Results showed that C2C12 cells possess β-AR which are specific, saturable, and of high affinity (Kd = 0.2 nM). Forskolin and ISO stimulated cAMP production by ≈ 20-fold (P < 0.001) and 17-fold (P < 0.001), respectively. ISO and the cAMP analog, 8-bromo-cAMP (8-BC) stimulated DNA synthesis in proliferating cells by 150% (P < 0.05) and 200% (P < 0.01), respectively, without modulating MAPK activity, whereas addition of fetal bovine serum to culture resulted in a 500% increase (P < 0.01) in DNA synthesis and MAPK activation. DNA synthesis in C2C12 cells treated with ISO, 8-BC, or FBS was abolished in the presence of 25 μM PD098059, an MAPK-kinase inhibitor, suggesting that an MAPK-dependent pathway is likely involved in C2C12 proliferation. During cAMP elevating agent stimulation, basal MAPK activity may be sufficient, in the presence of other putative signaling molecules, to support proliferation in these cells. ISO or 8-BC treatment increased FN mRNA by three- and seven-fold, respectively, in growing C2C12 cells implying a connection between increased DNA synthesis and FN gene expression.

Why FRET? Focus on “β-Adrenergic and muscarinic receptor-induced changes in cAMP activity in adult cardiac myocytes using a FRET-based biosensor”

fluorescence imaging of cAMP in single cells was first described in Roger Tsien's laboratory in 1991 ([1][1]). The ability to perform real-time imaging of signaling pathways in living cells has been a major advance in cell biology and physiology. The standard approach has been microinjection of

Thermal injury and sepsis modulates β-adrenergic receptors and cAMP reponses in monocyte-committed bone marrow cells

We have previously reported that adrenergic stimulation enhances monocytopoiesis following experimental burn injury and sepsis (BI/S). In the present work we measured β-adrenergic receptor number and affinity in bone marrow committed monocyte progenitor cells (CD59 +) following BI/S. We find that BI/S treatment significantly decreased monocyte progenitor cell β-adrenergic receptors but significantly increased receptor binding affinity and isoproterenol-stimulated cAMP production. CD14 expression in macrophages derived in vitro from CD59 + cells following BI/S was significantly increased by epinephrine and this change was blocked by β 2-adrenergic receptor antagonist. PCR analysis suggests the presence of β 2- but not β 1-adrenergic receptors. Enhanced adrenergic receptor signaling in CD59 + bone marrow cells following BI/S may be important in macrophage development.

Caveolae-Independent Activation of Protein Kinase A in Rat Neonatal Myocytes

Cardiomyocytes express both β1- and β2-adrenergic receptors, and these receptors play a differential role in chronotropic and inotropic effects of the heart. Caveolae are known as an important regulator of G-protein-coupled receptor signaling. In the present report, we examined whether caveolae have a role in β-adrenergic receptor-stimulated cAMP production and protein kinase A activation in neonatal myocytes. Isoproterenol-stimulated cAMP production was mediated by β1- and β2-subtypes, which depends on the receptor number of each subtype. However, protein kinase A activation was exclusively mediated by the β1-subtype. Disruption of caveolae by methyl-β-cyclodextrin treatment did not affect the relative contribution of subtypes to isoproterenol-stimulated cAMP production. β1-Subtype-mediated protein kinase A activation was also not affected by the disruption of caveolae. These results suggest that β1-adrenergic receptor-mediated protein kinase A activation is compartmentalized and independent of caveolae.

Regulation of the rainbow trout ( Oncorhynchus mykiss) hepatic β 2-adrenoceptor by adrenergic agonists

The characteristics of hepatic β 2-adrenoceptors (AR) were examined in rainbow trout ( Oncorhynchus mykiss) chased once per day to exhaustion for up to 7 days or fed the repartitioning agents clenbuterol (CLEN) or ractopamine (RACT) that function in mammals as β-agonists. A one-day chase and feeding the CLEN for 37 days resulted in a significant 27% and 33% decrease, respectively, in the number of CGP-binding sites ( B max) with no significant change in affinity ( K d ) of hepatic β 2-ARs. Despite the significant decrease in β 2-AR numbers with CLEN feeding, no significant differences were found for either β 2-AR mRNA levels or adenylyl cyclase (ACase) activities. In addition, CLEN displayed only partial agonist activities as it was found to be more effective at blocking isoproterenol-stimulated cAMP production in isolated hepatocytes than stimulating cAMP production. The small affects of RACT may be related to its low active stereoisomer content and low affinity for the trout β 2-AR. Agonist regulation of the trout hepatic β 2-ARs may involve down-regulation of the receptors without affecting responsiveness.

Propofol attenuates beta-adrenoreceptor-mediated signal transduction via a protein kinase C-dependent pathway in cardiomyocytes.

Activation of beta adrenoreceptors by catecholamines is an important mechanism for increasing the inotropic state of the heart. The objectives of the current study were to investigate the effects of propofol on beta-adrenoreceptor-mediated increases in cardiomyocyte intracellular Ca2+ concentration ([Ca2+]i), cell shortening, L-type Ca2+ current (ICa) and cyclic adenosine monophosphate (cAMP) accumulation. The authors also investigated the site of action of propofol in the beta-adrenoreceptor signaling pathway, as well as the role of protein kinase C (PKC), and tested the hypothesis that propofol would inhibit the beta-adrenoreceptor signaling pathway via a PKC-dependent mechanism. Freshly isolated ventricular myocytes were obtained from adult rat and guinea pig hearts. Myocyte shortening (video edge detection) and [Ca2+]i (fura-2, 340/380 ratio) were monitored simultaneously in individual cells. Conventional whole cell patch clamp analysis was used to measure the ICa in individual myocytes. cAMP production was assessed in suspensions of myocytes using an enzyme immunoassay kit. Propofol (0.1-10 mum) had no effect on steady state [Ca2+]i, cell shortening, ICa, or cAMP production. In contrast, propofol caused dose-dependent decreases in isoproterenol-stimulated increases in [Ca2+]i, shortening, ICa, and cAMP. Forskolin-induced increases in [Ca2+]i, shortening, and cAMP production were not altered by propofol. PKC activation with phorbol myristate acetate attenuated isoproterenol-stimulated cAMP production. Inhibition of PKC with bisindolylmaleimide (broad range inhibitor) or Gö 6976 (inhibitor of Ca2+-dependent PKC isoforms) abolished propofol-induced inhibition of isoproterenol-stimulated increases in [Ca2+]i, shortening, and cAMP production. Clinically relevant concentrations of propofol attenuate beta-adrenergic signal transduction in cardiac myocytes via inhibition of cAMP production. The inhibitory site of action of propofol is upstream of adenylyl cyclase and involves activation of PKC alpha.

Adenylyl cyclase isoforms and signal integration in models of vascular smooth muscle cells.

Adenylyl cyclases present a potential focal point for signal integration in vascular smooth muscle cells (VSMC) influencing contractile state and cellular responses to vessel wall injury. In the present study, we examined the influence of the vasoactive peptide arginine vasopressin (AVP) on cAMP regulation in primary cultures of rat aortic VSMC and in the A7r5 arterial smooth muscle cell line. In cultured VSMC and A7r5 cells, AVP had no effect on basal cAMP but differentially affected beta-adrenergic receptor-induced activation of adenylyl cyclase. AVP synergistically increased (twofold) isoproterenol-stimulated cAMP production in VSMC but inhibited the effect of isoproterenol (50%) in the A7r5 cell line. The effects of AVP in both preparations were blocked when cells were pretreated with a selective V(1) vasopressin receptor antagonist. Moreover, the actions of AVP in both models were dependent on release of intracellular Ca(2+) and were mimicked by elevation of Ca(2+) with the ionophore A23187, suggesting that the responses to AVP involve Ca(2+)-mediated regulation of adenylyl cyclase stimulation. Adenylyl cyclase types I, III, and VIII are stimulated by Ca(2+)/calmodulin, whereas types V and VI are directly inhibited by Ca(2+). RNA blot analysis for effector isotypes indicated that both VSMC and A7r5 cells expressed types III, V, and VI. VSMC also expressed mRNA for type IV and VIII effectors, which could account for the cell-specific responses to peptide hormone and Ca(2+).

Selective inhibition of beta(2)-adrenergic receptor-mediated cAMP generation by activation of the P2Y(2) receptor in mouse pineal gland tumor cells.

Rhythmic noradrenergic signaling from the hypothalamic clock in the suprachiasmatic nucleus to the pineal gland causes an increase in intracellular cAMP which regulates the circadian fluctuation of melatonin synthesis. The activation of phospholipase C (PLC)-coupled P2Y(2) receptors upon treatment with ATP and UTP exclusively inhibited the isoproterenol-stimulated cAMP production in mouse pineal gland tumor cells. However, the activation of other PLC-coupled receptors including P2Y(1) and bombesin receptors had little or no effect on the isoproterenol-stimulated cAMP production. Also, ATP did not inhibit cAMP production caused by forskolin, prostaglandin E(2), or the adenosine analog NECA. These results suggest a selective coupling between signalings of P2Y(2) and beta(2)-adrenergic receptors. The binding of [(3)H]CGP12177 to beta(2)-adrenergic receptors was not effected by the presence of ATP or UTP. Ionomycin decreased the isoproterenol-stimulated cAMP production, whereas phorbol 12-myristate 13-acetate slightly potentiated the isoproterenol response. Chelation of intracellular Ca(2+), however, had little effect on the ATP-induced inhibition of cAMP production, while it completely reversed the ionomycin-induced inhibition. Treatment of cells with pertussis toxin almost completely blocked the inhibitory effect of nucleotides. Pertussis toxin also inhibited the nucleotide-induced increase in intracellular Ca(2+) and inositol 1,4,5-trisphosphate production by 30-40%, suggesting that the ATP-mediated inhibition of the cAMP generation and the partial activation of PLC are mediated by pertussis toxin-sensitive G(i)-protein. We conclude that one of the functions of P2Y(2) receptors on the pineal gland is the selective inhibition of beta-adrenergic receptor-mediated signaling pathways via the inhibitory G-proteins.

Functional β2-Adrenergic Receptors Are Present in Nonstrial Tissues of the Lateral Wall in the Gerbil Cochlea

Recently, we have demonstrated that functional β₁-adrenergic receptors are the dominant β-adrenergic receptor subtype in the stria vascularis and that β₁-adrenergic receptors stimulate K⁺ secretion in strial marginal cells. The goal of the present study was to determine whether nonstrial tissues in the cochlear lateral wall contain β-adrenergic receptors and if so which subtype is present. Pharmacological tools were used to identify receptors in functional studies where cAMP production was measured. Further, receptors were identified as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. Experiments were performed on gerbil nonstrial lateral wall tissues. Tissues stimulated with 10^–5M isoproterenol produced 0.42 ± 0.22 pmol cAMP per ear within 12 min (n = 14). The selective β-adrenergic receptor agonist isoproterenol stimulated cAMP production with an EC₅₀ of (2 ± 3) × 10^–7M (n = 7). Isoproterenol-stimulated cAMP production was inhibited by the β₂-adrenergic receptor antagonist ICI 118551 with an IC₅₀ of (7 ± 7) × 10^–6M, which corresponds to an affinity constant of 1 × 10^–7M (pK_DB = 6.89 ± 0.23, n = 3). Isoproterenol-stimulated cAMP production was not inhibited by the highly selective β₁-adrenergic receptor antagonist CGP 20712A. The IC₅₀ and the affinity constant for CGP 20712A were estimated to be >3 × 10^–4 and >6 × 10^–6M, respectively. RT-PCR of total RNA isolated from nonstrial lateral wall tissues using primers specific for β₁-, β₂- and β₃-adrenergic receptors revealed products of the predicted sizes for the β₁- and β₂- but not for the β₃-subtype. Sequence analysis confirmed that amplified cDNA fragments encoded gene-specific nucleotide sequences. These results demonstrate that nonstrial lateral wall tissues contain transcripts for β₁- and β₂- but not for β₃-adrenergic receptors and that the β₂-adrenergic receptor is the dominant functional receptor subtype. The cellular localization and function of the β₂-adrenergic receptors remains to be determined.

Genetic Correlations Between Initial Sensitivity to Ethanol and Brain cAMP Signaling in Inbred and Selectively Bred Mice

Several lines of evidence have suggested a role for cAMP (adenosine 3',5'-cyclic monophosphate) signaling in the acute and chronic effects of ethanol. This study investigated whether there is a genetic correlation between cAMP synthesis in the brain and the acute effects of ethanol [alcohol sensitivity or acute functional tolerance (AFT)]. By using nine inbred strains of mice, we measured initial sensitivity and AFT to ethanol with a test of balance on a dowel. Initial sensitivity was defined by the blood ethanol concentration (BEC0) at the loss of balance on a dowel after an ethanol injection [1.75 g/kg intraperitoneally (ip)]. When mice were able to regain balance on the dowel, BEC1 was determined, and a second ethanol injection was given (2 g/kg ip). Upon final regaining of balance, BEC2 was determined. AFT was defined by the difference between BEC1 and BEC2 (AFT = DeltaBEC = BEC2 - BEC1). Cyclic AMP synthesis was measured in whole-cell preparations in the cerebellum and other brain areas of mice of the nine inbred strains. Significant differences in BEC0 and AFT were seen among the mice of the nine inbred strains. Cerebellar basal and forskolin- and isoproterenol-stimulated cAMP production differed significantly between the strains, and BEC0 was found to correlate significantly with forskolin- and isoproterenol-stimulated cAMP accumulation in the cerebellum (r = 0.70 and 0.94, respectively). When we measured cAMP production in mesencephalic and telencephalic tissue in three strains of mice that differed significantly in isoproterenol-stimulated cAMP accumulation in the cerebellum, significant differences between strains were found only in telencephalic tissue. The relative relationship between the rank order of the three strains for cAMP accumulation in the telencephalon and initial sensitivity to ethanol was identical to that seen with the cerebellum. However, AFT did not correlate with cAMP accumulation in the cerebellum or any other brain area tested. These results suggest that cAMP-generating systems of the cerebellum and possibly the brain areas contained in telencephalic tissues (e.g., basal ganglia) may have an important relationship to an animal's initial sensitivity to the incoordinating effects of ethanol.

K+ secretion in strial marginal cells is stimulated via beta 1-adrenergic receptors but not via beta 2-adrenergic or vasopressin receptors.

Pharmacologic tools were used to identify receptors in functional studies by measuring either transepithelial current (I(sc)) in strial marginal cells (SMC) or cAMP production in stria vascularis (SV). Further, receptors were identified in SV as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. Experiments were performed using tissues isolated from gerbils unless specified otherwise. I(sc) under control conditions was 1090 +/- 21 microA/cm(2) (n = 213) in gerbil SMC and 2001 +/- 95 microA/cm(2) (n = 6) in murine SMC. Direct stimulation of adenylate cyclase with 10(-5) m forskolin but not with 10(-5) m 1,9-dideoxy-forskolin resulted in an increase in the I(sc) by a factor of 1.14 +/- 0.01 (n = 6). The vasopressin-receptor agonist 10(-8) m Arg(8)-vasopressin had no significant effect on I(sc) in gerbil and murine SMC. The beta-adrenergic agonists isoproterenol, norepinephrine and epinephrine stimulated I(sc) with an EC(50) of (6 +/- 2) x 10(-7) m (n = 28), (3 +/- 1) x 10(-6) m (n = 40) and (7 +/- 2) x 10(-6) m (n = 38), respectively. Isoproterenol stimulated cAMP production in SV with an EC(50) of (5 +/- 2) x 10(-7) m (n = 8). The beta-antagonist 10(-4) m propanolol completely inhibited 2 x 10(-5) m isoproterenol-induced stimulation of I(sc). The beta-antagonists atenolol, ICI118551 and CGP20712A inhibited isoproterenol-induced stimulation of I(sc) with a K(DB) of 1 x 10(-7) m (pK(DB) = 6.96 +/- 0.15, n = 14), 1 x 10(-7) m (pK(DB) = 7. 01 +/- 0.14, n = 15), 2 x 10(-9) m (pK(DB) = 8.73 +/- 0.13, n = 19), respectively. CGP20712A inhibited isoproterenol-induced cAMP production with a K(DB) of 1 x 10(-10) m (pK(DB) = 9.94 +/- 0.55, n = 9). RT-PCR of total RNA isolated from SV using primers specific for the beta(1)-, beta(2)- and beta(3)-adrenergic receptors revealed products of the predicted sizes for the beta(1)- and beta(2)- but not the beta(3)-adrenergic receptor. Sequence analysis confirmed that amplified cDNA fragments encoded gene-specific nucleotide sequences. These results demonstrate that K(+) secretion in SMC is under the control of beta(1)-adrenergic receptors but not beta(2)-adrenergic or vasopressin-receptors and that the beta(1)-subtype is the primary beta-adrenergic receptor in SV although SV contains transcripts for both beta(1)- and beta(2)-adrenergic receptors.

Beta-adrenergic agonist hyperplastic effect is associated with increased fibronectin gene expression and not mitogen-activated protein kinase modulation in C2C12 cells.

Beta-adrenergic agonists (beta-AA) enhance protein accretion in skeletal muscles. This stimulation is characterized by increased protein synthesis, increased expression of myofibrillar protein genes and a depression in protein degradation in animals, and increased proliferation and DNA synthesis in muscle cells in vitro. The mechanism or signal path in muscle whereby beta-AA would elicit these physiological effects upon binding to the G protein-coupled beta-adrenergic receptor (beta-AR) is unclear. C2C12 myoblasts were used to determine beta-AR ligand binding characteristics, cyclic AMP synthesis in response to isoproterenol (ISO) stimulation, and effects of ISO on DNA synthesis, mitogen activated protein kinase (MAPK), and fibronectin (FN) gene expression. Results showed that C2C12 cells possess beta-AR which are specific, saturable, and of high affinity (Kd = 0.2 nM). Forskolin and ISO stimulated cAMP production by = 20-fold (P<0.001) and 17-fold (P<0.001), respectively. ISO and the cAMP analog, 8-bromo-cAMP (8-BC) stimulated DNA synthesis in proliferating cells by 150% (P<0.05) and 200% (P<0.01), respectively, without modulating MAPK activity, whereas addition of fetal bovine serum to culture resulted in a 500% increase (P<0.01) in DNA synthesis and MAPK activation. DNA synthesis in C2C12 cells treated with ISO, 8-BC, or FBS was abolished in the presence of 25 microM PD098059, an MAPK-kinase inhibitor, suggesting that an MAPK-dependent pathway is likely involved in C2C12 proliferation. During cAMP elevating agent stimulation, basal MAPK activity may be sufficient, in the presence of other putative signaling molecules, to support proliferation in these cells. ISO or 8-BC treatment increased FN mRNA by three- and seven-fold, respectively, in growing C2C12 cells implying a connection between increased DNA synthesis and FN gene expression.

Nitric Oxide Modulates β2-Adrenergic Receptor Palmitoylation and Signaling

To determine whether nitric oxide (NO) modulates the beta-adrenergic signaling pathway, we treated cells expressing beta(2)-adrenergic receptors (beta(2)AR) with the NO donors, 3-morpholinosydnonimine (SIN-1) and 1,2,3,4-oxatriazolium, 5-amino-3-(3-chloro-2-methylphenyl)chloride and determined the intracellular production of cAMP after exposure to beta-adrenergic receptor agonists, cholera toxin and forskolin. NO significantly decreased the potency of the beta-adrenergic agonist, isoproterenol, to stimulate cAMP production without affecting the stimulatory action of forskolin and cholera toxin, which directly activate adenylyl cyclase and G(s), respectively. Treatment with the NO donor increased the guanyl nucleotide-sensitive high affinity constant for the agonist, isoproterenol, thus suggesting that it reduced functional coupling between the receptor and G(s). Stimulation of endogenous NO production by lipopolysaccharide in RAW 264.7 macrophages also caused a significant increase in the EC(50) for isoproterenol-stimulated cAMP production. SIN-1 treatment also led to a reduction in both basal and isoproterenol-stimulated incorporation of [(3)H]palmitate into the beta(2)AR. Signaling through the nonpalmitoylated, Gly(341)beta(2)AR mutant was unchanged by SIN-1 treatment. Given the link between beta(2)AR palmitoylation and its responsiveness to agonist, these results suggest that the primary action of NO was depalmitoylation of the beta(2)AR resulting in decreased signaling through the beta(2)AR.

Cultured astrocytes express messenger RNA for multiple serotonin receptor subtypes, without functional coupling of 5-HT 1 receptor subtypes to adenylyl cyclase

The literature describing the expression of 5-HT receptor subtypes by astrocytes is controversial and incomplete. It is clear that primary cultures of astrocytes express receptors of the 5-HT 2 family coupled to phospholipase C and of the 5-HT 7 receptor family positively coupled to adenylyl cyclase. Cultured astrocytes have also been reported to express receptors of the 5-HT 1 family, although the exact subtypes present are unknown. In the present study we have investigated which of the known rat G-protein coupled 5-HT receptor mRNAs are expressed by cultured astrocytes. Reverse transcriptase–polymerase chain reaction (RT-PCR) revealed expression of 5-HT 1A, 5-HT 1B, 5-HT 1D, 5-HT 1F, 5-HT 2A, 5-HT 2B, 5-HT 2C, 5-HT 5B, 5-HT 6 and 5-HT 7 receptor mRNAs in astrocytes derived from 2-day old rats and cultured for 10–12 days. Messenger RNAs for 5-HT 4 and 5-HT 5A receptors were not detected. The functional expression of 5-HT 1 receptor subtypes was investigated by measuring the ability of 5-HT 1 receptor agonists: 8-OH-DPAT (5-HT 1A receptors), RU24969 (5-HT 1A, 5-HT 1B, 5-HT 1D, and 5-HT 1F receptors) or sumatriptan (5-HT 1B, 5-HT 1D, and 5-HT 1F receptors) to modulate forskolin or isoproterenol stimulated cAMP production. These compounds, at concentrations up to 10 μM, did not significantly attenuate cAMP production. These results indicate that although astrocytes express mRNA for each of the five 5-HT 1 receptor subtypes which have been isolated from the rat, these receptors are not coupled to the inhibition of adenylyl cyclase.

Persistent alterations of the autonomic nervous system after noncardiac surgery.

BACKGROUND. Changes in the sympathetic nervous system may be a cause of postoperative cardiovascular complications. The authors hypothesized that changes in both beta-adrenergic receptor (betaAR) function (as assessed in lymphocytes) and in sympathetic activity (assessed by plasma catecholamines and by heart rate variability [HRV] measurements obtained from Holter recordings) occur after operation. The HRV parameters were measured in 28 patients having thoracotomy (n = 14) or laparotomy (n = 14) before and for as long as 6 days after operation. Transthoracic echocardiography was performed before and on postoperative day 2. Lymphocytes were also isolated from blood obtained before anesthesia and again on postoperative days 1, 2, 3, and 5 (or 6). They were used to examine betaAR number (Bmax) and cyclic adenosine monophosphate (cAMP) production after stimulation with isoproterenol and prostaglandin E1. In addition, plasma epinephrine, norepinephrine, and cortisol concentrations were determined at similar intervals. After abdominal and thoracic surgery, most time and all frequency indices of HRV decreased significantly, as did Bmax and basal and isoproterenol-stimulated cAMP production. The decrements in HRV correlated with those of Bmax and isoproterenol-stimulated cAMP throughout the first postoperative week and inversely correlated with the increase in heart rate. Plasma catecholamine concentrations did not change significantly from baseline values, but plasma cortisol levels did increase after operation in both groups. Left ventricular ejection fraction was normal in both groups and unaffected by surgery. Persistent downregulation and desensitization of the lymphocyte betaAR/adenylyl cyclase system correlated with decrements in time and frequency domain indices of HRV throughout the first week after major abdominal or thoracic surgery. These physiologic alterations suggest the continued presence of adaptive autonomic regulatory mechanisms and may explain why the at-risk period after major surgery appears to be about 1 week or more.

Enhanced vasorelaxation by overexpression of beta 2-adrenergic receptors in large arteries.

This study was designed to determine if adenoviral-mediated delivery of a transgene encoding the beta 2-adrenergic receptor (beta 2-AR) to the carotid arterial wall could result in alterations in in vivo vascular function. De-endothelialized rat carotid arteries were infused in vivo with 0.1 mg/ml elastase and adenovirus [6 x 10(9) plaque forming units (PFU)] containing either the marker gene beta-galactosidase (Adeno-beta-gal), DNA encoding the human beta 2-AR (Adeno-beta 2-AR), or no transgene. This low concentration of elastase increased the water permeability (5.2 +/- 0.6 v 1.9 +/- 0.4 x 10(-8) cm/s/mmHg, n = 4, P < 0.0001) without affecting either the vasomotor responsiveness or the morphology of the arterial wall. A transfection efficiency of 73% was achieved with Adeno-beta-gal (n = 3). beta-gal expression was associated with infrequent appearance of T and B lymphocytes, or neutrophil infiltration. Five days after infection with Adeno-beta 2-AR, the total beta-AR density increased six-fold (67.8 +/- 3.4 v 397.0 +/- 155.5 fmol/mg protein, n = 5, P < 0.01); isoproterenol-induced vasorelaxation at transmural pressures from 10-110/mmHg increased (P < 0.01) compared to arteries exposed to control virus (empty adenovirus), n = 4; and isoproterenol-stimulated cAMP production was increased by 65% (n = 5). Thus, adenoviral-mediated delivery of beta 2-ARs into large artery walls results in enhanced beta-AR-mediated vasorelaxation via augmentation in cAMP levels in vascular smooth muscle cells.

The influence of dietary cholesterol on cardiac and hepatic beta-adrenergic receptors in egyptian sand rats

We examined the effects of dietary cholesterol on cardiac and hepatic beta-adrenergic receptor functioning. Age-matched adult desert rodents (Psammomys obesus) were randomized to either a 5% cholesterol diet (CD, n = 20), or normal rabbit chow (RC, n = 18). After a 2-month exposure to the diets, animals were sacrificed and tissue from both heart and liver were retained for radioligand bindings studies. In heart tissue, cholesterol fed animals, relative to controls, showed an increased production of adenosine 3,5>-cyclic monophosphate (cAMP) in response to isoproterenol. Cholesterol supplementation was not associated with an increase in heart beta-adrenergic receptor number. Animals fed the 5% cholesterol diet showed significant increases in the number of beta-adrenergic receptor sites in hepatic tissue (M = 13.2 vs. 10.4 pmol/mg protein, CD and RC, respectively). The increased number of receptor sites in the liver was accompanied by a significant increase in isoproterenol-stimulated cAMP production. Results are supportive of the hypothesis that dietary cholesterol contributes to an upregulation of beta-adrenergic receptor function in cardiac, as well as hepatic tissue. These findings may be relevant to the observations of excessive stress-induced cardiovascular reactivity in persons with high cholesterol levels.