Calcium Entry Attenuates Adenylyl Cyclase Activity

laborator and animal investigations -' Calcium Entry Attenuates Adenylyl Cyclase Activity* A Possible Mechanism for Calcium-induced Catecholamine Resistance William B. Abernethy, MD;f John F. Butterworth IV, MD; Richard C. Prielipp, MD; Jian P. Leith, BS; and Gary P. Zaloga, MD, FCCP In experimental animals, coadministration of calcium (Ca) salts with ,B-adrenergic receptor agonists reduces the increased blood pressure and cyclic AMP (cAMP) produced by ,B-adrenergic receptor agonists alone. In patients, coadministration of these drugs reduces the increased cardiac output and blood glucose produced by selective administration of ,B-adrenergic agonists. The mechanism by which Ca might produce catecholamine resistance remains unclear. Healthy volunteers donated venous blood from which lymphocytes were isolated. The cAMP production was measured by radioimmunoassay under control conditions and after incubation with epinephrine or colforsin (forskolin) in the presence and absence of inhibitors. Epinephrine and colforsin produced concentration-dependent increases in cAMP production. Extracellular Ca concentration over the range from 0 to 8 mM did not inhibit basal cAMP production or that stimulated by either colforsin or epinephrine. The calcium channel agonist Bay K 8644 (50 ,uM) combined with normal extracellular Ca concentra-

n myocardial cells, phasic increases in free intracellular calcium (Ca) concentration couple excitation to contraction.' Many clinicians, particularly those who care for patients who have undergone cardiac surgery or patients with sepsis, administer Ca salts concurrently with f-adrenergic receptor agonists hoping to augment the positive inotropic effects of 3-adrenergic receptor stimulation.2'3 Yet, recent evidence suggests that Ca salts actually attenuate the cardiotonic effects of f-adrenergic agonists in normocalcemic animals4 and humans,5'6 and mild hypocalcemia does not impair catecholamine action.7 *From the Department of Anesthesia, the Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC. tNow with the Department of Medicine, University of Washington Medical Center, Seattle. Presented in part at "Ion Channels in the Cardiovascular System," Chantilly, Va, October 1992, conference sponsored by National Heart, Lung, and Blood Institute and the American Association for the Advancement of Science; the 1993 meeting of the International Anesthesia Research Society, San Diego, Calif; and the 1993 meeting of the American Society of Anesthesiologists, Washington, DC. Manuscript received June 1, 1994; revision accepted August 3. Reprint requests: Dr. Butterworth, Dept. of Anesthesia, Bowman Gray School of Medicine, Medical Center Blvd, WinstonSalem, NC 27157-1009

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tion significantly atten iuated colforsin-induced increases in cAMP production. ' When barium was substituted for Ca in the extracellulair fluid, the cAMP response to colforsin was restored, de spite Bay K 8644. Inhibition of Ca channel permeability with cadmium or cobalt ions partially restored colforssin-stimulated cAMP production, despite the presence oif extracellular Ca and Bay K 8644. These results suggest that entry of Ca ions through Ca channels attenuates adenylyl cyclase. The inhibition appears specific for C-a ions over other permeant divalent cations, and favo)rs a possible physiologic role for the recently cloned C :a-inhibited adenylyl cyclase. ,

(Chest 1995; 107:1420-25) Ca=calcium; cAMP=(cyclic adenosine monophosphate; DMSO=dimethylsufox cide; Gs=G-protein Key words: adenylyl cyclase; bay K 8644; calcium; colforsin (forskolin); ccyclic adenosine monophosphate-

(cAMP) How might Ca diminish the response to f-adrenergic agonists? We hypothesized that increased Ca entry may interfere with f-adrenergic receptor signaling mechanisms and reduce the potency of f-adrenergic receptor stimulation. The f-adrenergic receptor in heart muscle is coupled, via the stimulatory G-protein (Gs), to adenylyl cyclase, activation of which leads to increased intracellular concentrations of cyclic adenosine-3':5'-monophosphate (cAMP).8 Increased intracellular cAMP concentration triggers a cascade of events that increase Ca entry through activated voltage-gated Ca channels,9 and enhance release of Ca from intracellular stores in the sarcoplasmic reticulum.'0 The resulting increased availability of free Ca ions for binding to contractile proteins accounts for the positive inotropic effects of f-adrenergic agonists. Intracellular Ca accumulation, however, may act as a negative feedback regulator of the d-receptor signaling pathway and help prevent detrimental effects of excess Ca accumulation. Molecular cloning studies have identified a diversity of adenylyl cyclases," type 5 and 6 isoforms of which one characteristically was inhibited by [Ca]i.12'14 Laboratory and Animal Investigations

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The present study investigates the molecular mechanism by which Ca may attenuate responses to f-adrenergic receptor agonists. Circulating human lymphocytes (which contain Gs-coupled 32-adrenergic receptors) were used as a model system'5 to examine the effects of Ca ion entry on adenylyl cyclase activity. The dihydropyridine agonist Bay K 8644 was used to modulate Ca permeability through Ca channels.'6 In some studies, colforsin (forskolin), rather than an adrenergic agonist, was used to eliminate the possibility of Ca effects on the coupling mechanisms between 3-adrenergic receptor binding and stimulation of adenylyl cyclase. Colforsin, unlike f-adrenergic agonists, directly activates adenylyl cyclase without the need for binding to a cell-surface receptor or for transduction via G, 17 METHODS AND MATERIALS Samples of 15 to 20 mL of venous blood were obtained from healthy volunteers (n=13). None of these volunteers had any signs, symptoms, or history of cardiovascular disease (eg, heart failure or hypertension) or were receiving medications (eg, phosphodiesterase inhibitors or 0-adrenergic agonists or antagonists) known to alter /-adrenergic receptor responses or cAMP metabolism. Blood from only one volunteer was used in any one lymphocyte preparation. Blood samples were immediately anticoagulated with heparin sodium and diluted 1:1 with phosphatebuffered saline solution. The phosphate-buffered saline solution contained (in mM): NaCl, 140; K2HPO4, 6; KCl, 2.7; and KH2PO4, 1.5. The pH was 7.4. Lymphocytes were isolated as described by B6yum'8 by applying the diluted blood to Ficol-Paque (Pharmacia LKB Biotechnology, Piscataway, NJ) and subjecting it to density-gradient centrifugation at 1,300 g for 35 min (4°C). The lymphocyte-rich fraction was then collected, washed twice in ice-cold phosphate-buffered saline solution, and resuspended in 140 mM NaCl. Cell counts were accomplished using a model cell counter (ZE, Coulter Electronics, Hialeah, Fla) set to recognize only mononuclear cells. Adenylyl cyclase activity was measured in an incubation mixture containing 140 mM NaCl, 1.5 mM CaCl2 (in most cases, except as noted), and 0.5 AM isobutylmethylxanthine (to inhibit cAMP degradation by phosphodiesterases). In some experiments, CdCl2 or CoCI2 was added, or barium (Ba2+) was substituted for Ca2+. In other experiments, [Ca2+] was adjusted between 0 and 8 mM. The reaction was initiated by addition of cells to the incubation mixture in a final reaction volume of 500 ML. Adenylyl cyclase was stimulated with either epinephrine (10-9 through 10-6 M) or colforsin (0.5 through 50 gM). Incubation was carried out for 10 min at 27°C, and terminated by addition of 1% (100 nM) cold perchloric acid with subsequent addition of 2% (187 ,M) KHCO3. The cAMP levels were determined by competitive protein binding radioimmunoassay (Kit APH2-0033; Diagnostics Products Corporation, Los Angeles) according to a method modified from Gilman.19 In brief, the above assay suspension was centrifuged at 2,000 g for 5 min. A 100-,uL aliquot of supernatant was incubated on ice for 90 min with 100 AL of [3H]-cAMP and 300 MuL of 50 mM Tris HCI buffer, pH 7.5, containing 4 mM edetic acid, and cAMP-dependent protein kinase. Standard solutions containing cAMP (0.13 to 64 pM) were incubated under similar conditions in parallel. At the end of incubation, free [3H]-cAMP was absorbed with 500 ,L of activated charcoal. The mixture was centrifuged at 2,000 g for 30 mn to pellet the charcoal. Bound

[3H]-cAMP in the supernatant was measured by liquid scintillation spectrophotometry using a scintillation counter (LKB 1219 Rackbeta, Wallac, Gaithersburg, Md). Bay K 8644 was obtained from the manufacturer (Calbiochem, La Jolla, Calif) and added to the assays from a 1.5-mM stock in dimethylsulfoxide (DMSO). Control experiments with DMSO concentrations as high as 6% revealed no alterations in cAMP production. Colforsin, obtained from the manufacturer (Sigma Chemical, St. Louis) was also dissolved in DMSO as a stock solution. Other chemicals were obtained from another company (Fisher Scientific, Pittsburgh) and were reagent grade or better. The dose-response experiments, measuring the effects of increasing concentrations of Ca2+ and increasing concentrations of colforsin or epinephrine on cAMP production, were analyzed using a univariate, doubly repeated measures analysis of variance. Huyn-Feldt adjustments were made for nonsphericity of data. The data from these experiments are presented as means ± SEM. For the experiments testing the interacting effects of colforsin, Bay K 8644, Co2 , Cd2 , and Ba2+ on cAMP production, Fisher's sign test was used to make treatment comparisons. Residual analyses did not allow parametric analysis of this data. Raw data from individual experiments are presented. All statistical analyses were accomplished using a computer program (SAS program, Release 6.03, SAS Institute, Cary, NC). Our study was reviewed and approved by the Clinical Research Practices Committee of the Bowman Gray School of Medicine of Wake Forest University (approval date, November 23, 1993). All blood donors gave their written, informed consent.

RESULTS

Effects of Extracellular [Ca2+] on cAMP Production Colforsin increased cAMP production in a concentration-dependent manner to a maximum of 10.4 ± 1.3 pmol/106 cells/10 min at 1 mmol extracellular [Ca2+] (Fig 1). Epinephrine also produced concentration-dependent increases in cAMP production (Fig 2). Epinephrine (10-6 M) increased cAMP production to 5.7 ± 1.1 pmol/106 cells/10 min with physiologic extracellular [Ca2+]. Basal and epinephrine-stimulated production of cAMP was not signif-

icantly influenced (p=0.39) by extracellular [Ca2+] over the range from 0 to 8 mM (Figs 1 and 2). However, colforsin-stimulated cAMP production was increased by higher, nonphysiologic (>2 mM) concentrations of extracellular [Ca2+] (p=0.0001) (Fig 1). Effects of Bay K 8644 on cAMP Production Colforsin, 50 ,uM, significantly increased (n=11, p=0.0001) cAMP production from a median basal level of 2.1 pmol/106 cells/10 min to a median 6.7 pmol/106 cells/10 min. Addition of Bay K 8644 50 ,M diminished the response to colforsin by 55% to a median 3.6 pmol/106 cells/lO min (n= 11, p=0.0005). The interaction of Bay K 8644 with colforsin is also depicted in Figures 3 and 4.

Effects of Ca Channel Blockade on cAMP Production In other experiments, the effects of added Cd2+ 1 CHEST / 107 / 5 / MAY, 1995

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FIGURE 1. Production of cAMP under basal conditions and after stimulation with colforsin (forskolin) in lymphocytes incubated with varying concentrations of extracellular [Ca2+]. Colforsin increased cAMP production concentration dependently (p=O.OOOl). Extracellular [Ca2+] did not inhibit either basal or colforsinstimulated cAMP production. Higher, nonphysiologic, concentrations of [Ca2+] tended to increase cAMP production stimulated by colforsin (p=O.OOO1). Data are presented as the means + SEM of four determinations; p values were determined using a univariate, doubly repeated measures analysis of variance.

FIGURE 2. Production of cAMP after stimulation with epinephrine in lymphocytes incubated with varying concentrations of extracellular [Ca2+]. Epinephrine increased cAMP production concentration dependently (p=0.0005). Extracellular [Ca2+] had no significant (p=0.39) effect on epinephrine-stimulated cAMP production. Data are presented as means ± SEM of five determinations; p values were determined using a univariate, doubly repeated measures analysis of variance.

mM or Co2+ on basal cAMP production was assessed. Both these ions selectively inhibit Ca permeability through Ca channels. There was no significant effect of these ions on basal cAMP production (1.71 ± 0.49 vs 1.46 ± 0.49 and 1.44 ± 0.42 pmol/106 cells/10 min comparing means ± SE in the absence and presence of Cd2+ and Co2+, respectively in seven experiments (data not shown). However, when Cd2+ (n=10) was added in the presence of Bay K 8644, cAMP production by colforsin was restored from a median 3.5 pmol/106 cells/10 min to a median 5.6 pmol/106 cells/10 min (Fig 3) (p=0.001). In similar fashion, after Bay K 8644 significantly (p=0.016) reduced the response to colforsin, Co2+ ions (n=6) restored (p=0.016) colforsin-stimulated cAMP production from a median 2.7 pmol/106 cells/10 min to a median 5.0 pmol/106 cells/10 min (not shown).

responsible for Ca modulation of 3-adrenergic receptor agonists. Calcium ions are essential for sustained cardiac membrane depolarization, excitationcontraction coupling, and actin-myosin interaction during muscle contraction. Because of the wellknown positive inotropic effects of Ca and ,B-adrenergic agonists. clinicians often coadminister these two

Effects of Ba Substitution for Ca on cAMP Production Substituting Ba (2 mM) for Ca had no effect on basal cAMP production (0.81±0.15 vs 0.82±0.18 pmol/106 cells/10 min, comparing means±SE for Ca and Ba, respectively, in five experiments. When Ba was substituted for Ca, colforsin-stimulated cAMP production was restored from a median 3.6 pmol/106 cells/10 min to 5.7 pmol/106 cells/10 min despite the presence of Bay K 8644 (n=9, p=0.002 comparing experiments with Ca to those with Ba) (Fig 4). DISCUSSION

This study investigated the molecular mechanism 1 422

10

eE X8 0.o

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o

01~~~ Basal

Col

Col+Bay Cd

FIGURE 3. Effect of Ca channel blockade with Cd t on cAMP production. Colforsin (forskolin) significantly (p=0.OO1, n=10) increased cAMP production. Bay K 8644 (Col+Bay) significantly reduced the response to colforsin (Col) (p=O.OOl, n= 10). Addition of Cd2+ (Cd) restored colforsin-stimulated cAMP production, despite the presence of Bay K 8644 (p=O.OOl, n=10), comparing colforsin+Bay K 8644+Cd (Cd on the figure) to colforsin+Bay K 8644 (Col+Bay on the figure). Basal=baseline; Col=colforsin (forskolin), 50 AM; Bay=Bay K 8644, 50 gM; Cd=Cd2+, 2 mM. The raw data from individual experiments are presented; p values were determined using Fisher's sign test. Laboratory and Animal Investigations

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lymphocyte f-adrenergic-receptor density in patients with heart failure and tolerance to the 13-adrenergic agonist pirbuterol. N Engl J Med 1981; 305:185-90 Hui KK, Connolly ME. Increased numbers of beta receptors in orthostatic hypotension due to autonomic dysfunction. N Engl J Med 1981; 304:1473-76 Thomas JA, Marks BH. Plasma norepinephrine in congestive heart failure. Am J Cardiol 1978;41:233-43 Lewis RS, Cahalan MD. Ion channels and signal transduction in lymphocytes. Annu Rev Physiol 1990; 52:415-30 Bechem M, Hebisch S, Schramm M. Ca agonists: new, sensitive probes for Ca channels. Trends Pharmacol Sci 1988; 9:257-61 Bean BP. Two kinds of calcium channels in canine atrial cells: differences in kinetics, selectivity, and pharmacology. J Gen Physiol 1985; 86:1-30

31 Osterriede W, Brum G, Hescheler J, et al. Injection of subunits of cAMP-dependent protein kinase into cardiac myocytes modulates Ca2+ current. Nature 1982; 298:576-78 32 Morad M, Cleeman L. Role of Ca2+ channel in development of tension in heart muscle. J Mol Cell Cardiol 1987; 19:527-53 33 Katz AM. Interplay between inotropic and lusitropic effects of cyclic adenosine monophosphate on the myocardial cell. Circulation 1990; 82:1-7-I-1l 34 Katz AM. Cardiomyopathy of overload: a major determinant of prognosis in congestive heart failure. N Engl J Med 1990; 322:100-10 35 Benovic JL, Bouvier M, Caron MG, et al. Regulation of adenylyl cyclase-coupled 3-adrenergic receptors. Ann Rev Cell Biol 1988; 4:405-28

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unlike heart cells, Ca channels are mitogen-activated rather than voltage-gated and Ca flux is not increased with agonist binding to f-receptors.28 However, Bay K 8644 promotes Ca entry by prolonging open times of both L-type Ca channels of myocardial cells and the receptor-operated Ca channel in lymphocytes.16'29 Thus, in our study, lymphocytes had advantages over heart cells because they did not require voltage clamp to control Ca entry. With Bay K 8644, Ca entry into the lymphocytes could be regulated independently of the f-receptor pathway. In addition, since the publication of this data in abstract form, another group has achieved similar results showing Ca channel activation attenuates adenylyl cyclase activity in chick ventricular myocytes.14 Agonist binding to /-adrenergic receptors dramatically increases Ca flux across myocardial cell plasma membranes30 through production of cAMP and activation of cAMP-dependent protein kinase A.31 Calcium channel activation plays a crucial role in tension development and contraction in myocardial cells32 and agents that increase cellular cAMP, as do 0-adrenergic agonists, have powerful positive inotropic and lusitropic effects.33 Yet, sustained f-adrenergic agonist stimulation can be detrimental, lead to cardiomyopathy, and contribute to arrhythmogenesis.34 Thus, f-adrenergic receptor responses are subject to dynamic regulation at several levels, including the receptor, stimulatory and inhibitory G proteins, and intracellular concentrations of cofactors essential for adenylyl cyclase action.35 The relative contribution of each mechanism to reduced adrenergic responses (ie, catecholamine resistance) in patients remains controversial. Inhibition of adenylyl cyclase by Ca flux across the plasma membrane may help prevent Ca overload of the myocyte and represents one possible molecular mechanism for the clinical observation that calcium administration blunts the cardiotonic effects of ,B-agonists in experimental animals and patients. A fuller understanding of the regulation of ,B-adrenergic receptor responsiveness should lead to strategies that might improve the effectiveness of drug treatment. However, this study supports human studies documenting Ca-induced catecholamine resistance; therefore, we do not recommend routine coadministration of Ca salts with 0-adrenergic receptor agonists. Also, we would predict that overzealous administration of Ca salts during resuscitation might inhibit the ability of catecholamines and similar agents to increase oxygen delivery to tissues. ACKNOWLEDGMENT: We thank Robert James, MS, MStat, for performing the statistical analysis of our data.

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Laboratory and Animal Investigations

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lymphocyte f-adrenergic-receptor density in patients with heart failure and tolerance to the 13-adrenergic agonist pirbuterol. N Engl J Med 1981; 305:185-90 Hui KK, Connolly ME. Increased numbers of beta receptors in orthostatic hypotension due to autonomic dysfunction. N Engl J Med 1981; 304:1473-76 Thomas JA, Marks BH. Plasma norepinephrine in congestive heart failure. Am J Cardiol 1978;41:233-43 Lewis RS, Cahalan MD. Ion channels and signal transduction in lymphocytes. Annu Rev Physiol 1990; 52:415-30 Bechem M, Hebisch S, Schramm M. Ca agonists: new, sensitive probes for Ca channels. Trends Pharmacol Sci 1988; 9:257-61 Bean BP. Two kinds of calcium channels in canine atrial cells: differences in kinetics, selectivity, and pharmacology. J Gen Physiol 1985; 86:1-30

31 Osterriede W, Brum G, Hescheler J, et al. Injection of subunits of cAMP-dependent protein kinase into cardiac myocytes modulates Ca2+ current. Nature 1982; 298:576-78 32 Morad M, Cleeman L. Role of Ca2+ channel in development of tension in heart muscle. J Mol Cell Cardiol 1987; 19:527-53 33 Katz AM. Interplay between inotropic and lusitropic effects of cyclic adenosine monophosphate on the myocardial cell. Circulation 1990; 82:1-7-I-1l 34 Katz AM. Cardiomyopathy of overload: a major determinant of prognosis in congestive heart failure. N Engl J Med 1990; 322:100-10 35 Benovic JL, Bouvier M, Caron MG, et al. Regulation of adenylyl cyclase-coupled 3-adrenergic receptors. Ann Rev Cell Biol 1988; 4:405-28

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