Suppression of -adrenergic responsiveness of L-type Ca2 current by ...

Suppression of b-adrenergic responsiveness of L-type Ca21 current by IL-1b in rat ventricular myocytes SHI J. LIU,1,2 WEIGUO ZHOU,3 AND RICHARD H. KENNEDY2 Departments of 1Biopharmaceutical Sciences, 2Pharmacology and Toxicology, and 3Anesthesiology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 Liu, Shi J., Weiguo Zhou, and Richard H. Kennedy. Suppression of b-adrenergic responsiveness of L-type Ca21 current by IL-1b in rat ventricular myocytes. Am. J. Physiol. 276 (Heart Circ. Physiol. 45): H141–H148, 1999.—The possible mechanism by which interleukin-1b (IL-1b) affects b-adrenergic responsiveness of L-type Ca21 current (ICa,L ) was examined in adult rat ventricular myocytes by use of whole cell patch-clamp techniques. In the presence of isoproterenol (Iso), exposure for 3 min to IL-1b suppressed the Iso-activated ICa,L. In the presence of IL-1b, the response of ICa,L to Iso was decreased, and the EC50 for Iso stimulation was increased. However, IL-1b had no effect on [3H]CGP12177 binding, displacement of [3H]CGP-12177 binding by Iso, or on basal and Iso-enhanced cAMP content. When ICa,L was activated by extracellular application of forskolin or 8-(4-chlorophenylthio)-cAMP, a membrane-permeable cAMP analog, or by intracellular dialysis with cAMP, IL-1b had little effect on ICa,L. In contrast, in the presence of cAMP, IL-1b still suppressed the Iso-enhanced ICa,L. These results show that the IL-1b-induced decrease in b-adrenergic responsiveness of ICa,L does not result from inhibition of b-adrenoceptor binding, adenylyl cyclase activity, or cAMP-mediated pathways, suggesting a cAMP-independent mechanism.

The direct autocrine and/or paracrine effects of IL-1b on ventricular cell function include decreases in basal L-type Ca21 channel current (ICa,L ) (26) and contractility (10, 20, 36). In addition, in neonatal rat cardiac myocytes, IL-1b decreases the b-adrenergic responsiveness of contractility by reducing isoproterenol (Iso)enhanced cAMP levels after a 72-h exposure (14). Studies in adult guinea pig ventricular myocytes suggest that preincubation with IL-1b for 1–5 h inhibits the b-adrenergic control of ICa,L via activation of nitric oxide synthase (NOS) (31). These data demonstrate a delayed effect of IL-1b on myocardial b-adrenergic responsiveness. However, available data have not addressed the possibility that IL-1b has an acute effect on adult ventricular myocyte responsiveness to b-adrenergic stimulation. In the present study we have examined the acute effect of IL-1b on b-adrenergic receptor binding and on intracellular cAMP content and ICa,L in adult ventricular myocytes. We show that IL-1b decreases the b-adrenergic responsiveness of ICa,L primarily via a cAMPindependent pathway.

cytokines; calcium channel; signal transduction; cardiac myocytes

METHODS

INTERLEUKIN-1b

(IL-1b), a 17-kDa proinflammatory cytokine, has been closely associated with immune- and injury-mediated changes in cardiovascular function (8, 9, 25, 33). Marked increases in plasma IL-1b concentration are observed during the cardiac dysfunction associated with myocardial infarction (13, 29), ischemiareperfusion (11, 15), myocarditis (6), acute septic cardiomyopathy (27), and allograft rejection (16). Studies using PCR techniques show that mRNAs for IL-1b and its receptor are expressed in endomyocardium of patients with inflammatory myocarditis (18) and dilated cardiomyopathy (32) and in hearts with acute viral myocarditis (24). The enhanced expression of IL-1b mRNAs was shown to occur primarily in ventricular myocytes (24). Moreover, these cardiac disorders are associated with an increased sympathetic nervous system activity (22) and altered adrenergic responsiveness of myocardial function (1, 6, 12, 14). The interaction between the increased IL-1b and the enhanced sympathetic tone under these pathophysiological conditions remains unclear.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Myocyte isolation. Single adult ventricular myocytes were isolated from the hearts of male Sprague-Dawley rats (250– 300 g) with use of protocols described previously (26). Briefly, hearts were rapidly excised and perfused at 37°C via the aorta with an oxygenated control buffer solution consisting of (in mM) 110 NaCl, 3.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 0.2 CaCl2, and 11 glucose (pH 7.4 in 95% O2-5% CO2 at 37°C) and for another 5 min with Ca21-free buffer solution. Hearts were then perfused for 20 min with a buffer solution containing 25 µM CaCl2 plus 0.5 mg/ml collagenase. The ventricles were removed, minced, rinsed with control buffer solution, and shaken in a water bath at 37°C for two to three periods of 10 min each. Isolated ventricular myocytes were then plated into 60-mm culture dishes (Falcon) containing antibiotic-free, bicarbonate-buffered culture medium 199 (60%; GIBCO, Grand Island, NY) with 36% Earle’s balanced salt solution composed of (mM) 116 NaCl, 4.7 KCl, 0.9 NaH2PO4, 0.8 MgSO4, 26 NaHCO3, and 5.6 glucose and 4% fetal bovine serum (GIBCO; pH 7.4 in 5% CO2-95% air at 37°C). Electrophysiological measurements. Ventricular myocytes were placed on the heated stage of an inverted microscope (Nikon Diaphot) and perfused with a normal Tyrode solution consisting of (in mM) 145 NaCl, 5.4 KCl, 0.8 MgCl2, 1.0 CaCl2, 5.6 glucose, 5.8 HEPES, and 4.2 Tris base (pH 7.4 at 37°C). Cells were patch clamped in the whole cell configuration by conventional techniques (17) with use of a patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, CA), as previously described (26). Briefly, patch electrodes were filled with a pipette solution; tip resistance was 2–5 MV. Recorded currents were filtered at 1–2 kHz through a fourpole low-pass Bessel filter and sampled at 5 kHz with a PC/AT

0363-6135/99 $5.00 Copyright r 1999 the American Physiological Society

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IL-1b REDUCES b-ADRENERGIC RESPONSIVENESS OF CA21 CHANNELS

computer using pCLAMP 6.03 software (Axon Instruments) through an Axon Digidata 2000A acquisition system. Measurement of ICa,L has been described in our previous studies (26). The pipette solution for experiments measuring ICa,L consisted of (in mM) 100 CsOH, 70 aspartic acid, 11 CsCl, 15 tetraethylammonium chloride, 2 MgCl2, 5 MgATP, 10 EGTA, 0.1 CaCl2, 5 pyruvic acid, 5.6 glucose, 5 Tris2- phosphocreatine, 0.4 Li4GTP, and 10 HEPES-Tris base (pH 7.2 at 37°C). Myocytes were voltage clamped at 270 mV when the normal Tyrode solution was switched to an external solution consisting of (in mM) 140 N-methyl-D-glucamine chloride, 2 CaCl2, 0.8 MgCl2, 2 4-aminopyridine, and 10 HEPES-Tris base (pH 7.40 at 37°C). These conditions eliminated most membrane currents associated with Na1 and K1. After formation of the whole cell configuration, ICa,L was elicited by a single 250-ms pulse to 110 mV from the holding potential once every 15 s. The peak current-voltage relationship of ICa,L was constructed by applying 250-ms voltage pulses to potentials between 260 and 170 mV in 10-mV increments from the holding potential of 270 mV at 0.1 Hz. The magnitude of ICa,L was defined by the difference between the peak current and that at the end of the 250-ms pulse. All experiments were carried out at 37°C. Membrane preparation. b-Adrenoceptor binding was performed using partially purified membranes that were prepared at 4°C. Ventricular muscle from 8–10 rats was pooled, suspended in 50 mM Tris, 2 mM MgSO4, 0.1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride (pH 7.4), and initially homogenized for 30 s using a Polytron at a setting of 6. Further homogenization was achieved using seven strokes of a Dounce homogenizer. The homogenate was centrifuged at 800 g for 20 min, and the supernatant was subsequently centrifuged at 2,500 g for 20 min. The resultant supernatant was subjected to two sequential centrifugations at 30,000 g for 20 min with use of the homogenizing buffer to wash the pellet between centrifugations. The final pellet, a partially purified membrane preparation, was resuspended in a reaction solution (50 mM Tris and 2 mM MgSO4, pH 7.4) and stored at 280°C. Protein concentrations were determined by the method of Bradford (3), with BSA as the standard. Radioligand binding to membrane preparations. Binding assays with [3H]CGP-12177 (42.5 Ci/mmol; New England Nuclear Research Products, Boston, MA) were performed in polypropylene tubes containing reaction solutions in the presence or absence of 4 ng/ml IL-1b. In saturation experiments, final concentrations of the b-adrenergic antagonist [3H]CGP-12177 ranged from ,0.02 to 10 nM. In competition experiments, increasing levels of nonlabeled Iso were added to reaction solutions containing 1.0 nM [3H]CGP-12177 in the presence or absence of 100 µM 58-guanylyl imidodiphosphate guanosine (GppNHp). Nadolol (10 µM final concentration) was added to a parallel set of tubes to estimate nonspecific binding in all experiments. The reaction was initiated by addition of membrane protein to assay tubes, and the contents were incubated at 37°C for 30 min. Bound and free [3H]CGP-12177 were separated by filtration through GF/C filters, which were washed three times with ice-cold reaction solution. Filters were then immersed in scintillation fluid, and the retained radioactivity was determined by liquid scintillation spectrometry. Data were analyzed using a microcomputer version of LIGAND (28). Radioligand binding to ventricular myocytes. Effects of IL-1b on b-adrenoceptor binding were also monitored in intact ventricular myocytes (0.5 3 106 cells/ml normal Tyrode solution containing 0.5 nM [3H]CGP-12177) with and without 4 ng/ml IL-1b. Iso (100 nM) was included in some tubes, and nonspecific binding was determined using 1025 M nadolol.

After 30 min of incubation at 37°C, bound and free [3H]CGP12177 were separated by filtration through GF/C filters, which were washed three times with ice-cold Tyrode solution. Radioactivity was determined as described above. Data were analyzed using a microcomputer version of LIGAND (28). Chemicals and solutions. Most reagents were purchased from Sigma Chemical (St. Louis, MO). Nucleotides were directly added to pipette or bath solutions. The stock solution of human recombinant IL-1b [106 U/ml (Promega, Madison, WI) and 5 µg/ml (R & D Systems, Minneapolis, MN)] was made in the normal Tyrode solution containing 0.1% BSA. The effect on ICa,L of the final concentration of 1,000 U/ml IL-1b (or 4 ng/ml) from Promega is equivalent to that of 5 ng/ml IL-1b from R & D Systems. Statistics. Values are means 6 SE. Statistical significance was evaluated by the two-tailed Student’s t-test or, when more than two conditions are compared, by one-way ANOVA with Duncan’s multiple range test. Differences with P , 0.05 were considered significant. RESULTS

Suppression of b-adrenergic responsiveness of ICa,L by IL-1b in adult rat ventricular myocytes. Initial experiments were designed to examine the effect of IL-1b on ICa,L during stimulation by Iso. In Fig. 1A, exposure of a myocyte to 50 nM Iso caused an 80% increase in ICa,L that was further enhanced (,60%) by subsequent exposure to 1 µM Iso. In the presence of 1 µM ISO a 3-min exposure to 0.4 ng/ml IL-1b resulted in an ,30% decrease in peak ICa,L. The ICa,L recovered after removal of IL-1b and return to 50 nM Iso. Similar experiments show that, in the presence of 1 µM Iso, 0.4 and 4 ng/ml, IL-1b decreased Iso-activated ICa,L by 33.2 6 4.7% (n 5 6) and 43.1 6 1.4% (n 5 5; see also Fig. 6A), respectively. The 3-min exposure duration was chosen because, during this period of time, desensitization of b-adrenergic responsiveness was minimal (,7%; Fig. 1B). Figure 1B shows the temporal change in ICa,L in the presence and absence of IL-1b. The result indicates that the IL-1b-induced decrease in Iso-activated ICa,L was greater than that observed in the continued presence of Iso. Figure 2 demonstrates the effect of IL-1b on b-adrenergic responsiveness of ICa,L in adult rat ventricular myocytes. Figure 2A shows results from a myocyte that was pretreated with 0.4 ng/ml IL-1b for 3 min before exposure to increasing concentrations of Iso (1 nM–1 µM). In the presence of 0.4 ng/ml IL-1b, 1 µM Iso induced a 58% increase in ICa,L, a level less than that observed in the absence of IL-1b. The maximal stimulation of ICa,L by 1 µM Iso was 192 6 8% (n 5 35) and 150 6 3% (n 5 24) of control in the absence and presence of 0.4 ng/ml IL-1b, respectively. Figure 2B shows the concentration-dependent effects of Iso on ICa,L by plotting relative peak ICa,L (as measured at 110 mV) to the value at 1 µM Iso vs. Iso concentrations in the absence and presence of 0.4 ng/ml IL-1b. IL-1b caused a rightward shift in the dose-response curve, with the EC50 for Iso being increased from 50.5 to 156 nM. Thus IL-1b reduced the maximal stimulation (efficacy) and the potency of Iso.


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population of high-affinity binding sites with a dissociation constant (Kd ) of 0.29 6 0.03 nM and a maximal binding site density (Bmax ) of 54.2 6 12.6 fmol/mg protein (n 5 3), values similar to those reported by others (5). Figure 3 also shows that IL-1b (4 ng/ml) had no effect on Kd (0.36 6 0.04 nM, n 5 3) or Bmax (56.6 6 12.0 fmol/mg protein, n 5 3). Figure 4 shows results from experiments examining competitive displacement of [3H]CGP-12177 binding by increasing concentrations of Iso. Figure 4A shows that,

Fig. 1. Effect of interleukin 1b (IL-1b) on isoproterenol (Iso)activated L-type Ca21 channel current (ICa,L ) in adult rat ventricular myocytes. A: exposure of a myocyte to 50 nM (j) and 1 µM Iso (l) resulted in 80 and 140% increases in ICa,L, respectively. Subsequent addition of 0.4 ng/ml IL-1b caused a 30% reduction of ICa,L in presence of 1 µM Iso (T). ICa,L recovered when IL-1b was removed and solution was returned to 50 nM Iso. h, Control Inset: superimposed current traces where indicated; calibration bars, 10 pA/pF (vertical) and 5 ms (horizontal); dashed line, 0-current level. Cell membrane capacitance was 190 pF. B: time-dependent changes in Iso-activated ICa,L in absence and presence of IL-1b. After peak ICa,L reached a steady state in 1 µM Iso (designated time 0), current was monitored over time. Time-dependent decay was obtained by plotting currents that were normalized to magnitude of peak ICa,L at time 0 as a function of time and fit by a straight line with linear regression. Slope of decline in Iso-activated peak ICa,L in absence of IL-1b was 0.01 min21 (light dashed line, 21 experiments). Exposure to 0.4 ng/ml IL-1b caused a significant decrease in Iso-activated ICa,L, with a slope of 0.08 min21 (dark dashed line, 13 experiments).

Effect of IL-1b on cardiac b-adrenergic receptor binding. In attempts to determine the mechanism of the IL-1b-induced decrease in b-adrenergic responsiveness, we first examined whether IL-1b affects b-adrenoceptor binding. Figure 3 shows representative Scatchard plots from saturation binding assays with use of [3H]CGP-12177, a b-adrenergic antagonist, in partially purified membranes prepared from rat ventricular myocardium. Results from repeated assays were analyzed by the nonlinear curve-fitting program LIGAND (28) and consistently showed the best fit to be a single

Fig. 2. Effects of IL-1b on b-adrenergic responsiveness of ICa,L. A: b-adrenergic responsiveness of ICa,L was examined by exposing a myocyte to increasing concentrations of Iso after a 3-min preincubation with 0.4 ng/ml IL-1b. Inset: superimposed current traces; calibration bars, 10 pA/pF (vertical) and 5 ms (horizontal); dashed line, 0-current level. Cell membrane capacitance was 175 pF. B: concentration-response curves showing effects of Iso on ICa,L were obtained in control conditions and in presence of 0.4 ng/ml IL-1b. Data were curve fit by Hill equation: I 5 Imax · h [Iso]h/([Iso]h 1 EC50 ), where I is peak current normalized to that at 1 µM Iso, Imax is peak current at maximum effective concentration, [Iso], is Iso concentration, h is Hill coefficient, and EC50 is concentration of Iso producing a half-maximal effect. Values are means 6 SE; number of experiments is in parentheses. Values for EC50 and h were 50.5 6 2.7 nM and 1.1 in control and 156 6 29 nM and 1.3 in presence of 0.4 ng/ml IL-1b (estimated value 6 SE of 95% confidence of curve fitting).

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Fig. 3. Representative Scatchard plots of [3H]CGP-12177 binding to partially purified membrane preparations from rat cardiac ventricular muscle in absence and presence of 4 ng/ml IL-1b. Data were analyzed by LIGAND (with a linear regression of r2 . 0.92). Binding capacities and apparent dissociation constants were 57.9 fmol/mg protein and 0.3 nM in control (light dashed line) and 60.9 fmol/mg protein and 0.33 nM in IL-1b (dark dashed line), respectively.

in the absence of guanine nucleotides, Iso competition for [3H]CGP-12177 binding was best characterized by a two-binding-site model with relative affinities (Ki ) of 5.8 6 1.8 3 1028 M (representing 80.3 6 3.0% of total specific [3H]CGP-12177 binding) and 1.4 6 0.5 3 1025 M (19.6 6 2.8% of total specific binding). Analysis by LIGAND always suggested that a two-site model was a better fit than a single-site model; however, in two of the six experiments, the F value did not indicate significant differences between the two models. The presence of 4 ng/ml IL-1b did not significantly alter the effect of Iso on [3H]CGP-12177 binding; observed Ki values were 6.9 6 2.7 3 1028 M (77.3 6 4.3% of total specific binding) and 1.0 6 0.4 3 1025 M (22.7 6 4.3% of total specific binding). Figure 4B shows that, in the presence of 100 µM GppNHp, Iso antagonized [3H]CGP12177 binding from a single population of binding sites with Ki values of 0.71 6 0.31 3 1026 M in control conditions (n 5 3) and 0.82 6 0.23 1026 M in the presence of 4 ng/ml IL-1b (n 5 3). LIGAND analysis demonstrated a single-site model to be the best fit in all experiments. These findings were supported by data obtained from binding studies with intact ventricular myocytes. Table 1 shows that 4 ng/ml IL-1b did not alter specific [3H]CGP-12177 binding (0.5 nM final concentration) to adult rat ventricular myocytes in the presence or absence of 0.1 µM Iso. In summary, results of the binding studies indicated that IL-1b does not affect agonist binding to b-adrenoceptors. Effects of IL-1b on cAMP levels. Studies in neonatal rat cardiac myocytes have shown that cytokines decrease b-adrenergic responsiveness by suppressing the Iso-induced increase in cell cAMP concentration (14). We determined whether this is the case in adult rat ventricular myocytes by examining cell cAMP levels in

Fig. 4. Effects of IL-1b on Iso displacement of [3H]CGP-12177 binding to rat cardiac ventricular membranes. A: effects of Iso on [3H]CGP-12177 binding in absence (light dashed line) and presence (dark dashed line) of 4 ng/ml IL-1b. B: experiments performed in A with 100 µM 58-guanylyl imidodiphosphate guanosine. Data were normalized to specific [3H]CGP-12177 binding observed in absence of Iso and presented as means 6 SE for 3 experiments in each group. Data were curve fit using LIGAND (see METHODS ).

response to Iso in the absence and presence of IL-1b. The basal intracellular cAMP concentration in adult rat ventricular myocytes was 4.73 6 0.33 pmol/105 cells (n 5 13) or 8.03 6 0.56 pmol/mg protein, a value comparable to that reported by other investigators (37). Table 1. Effects of IL-1b on specific [3H]CGP-12177 binding to adult rat ventricular myocytes in the presence and absence of Iso [3H]CGP-12177 Binding, fmol/106 cells IL-1b, ng/ml

Control

10.1 µM Iso

0 4

205.4 6 12.2 214.5 6 12.0

142.3 6 5.7 138.2 6 10.3

Values are means 6 SE from 3 experiments per condition. IL-1b, interleukin-1b; Iso, isoproterenol. Final concentration of [3H]CGP12177 was 0.5 nM.


In Fig. 5, a 5-min incubation in 0.1 µM Iso approximately doubled the intracellular cAMP concentration. Incubation for 5 or 10 min with 5 ng/ml IL-1b had no effect on basal or Iso-enhanced cAMP levels. These results suggest that the IL-1b-induced decrease in b-adrenergic responsiveness did not result from alterations in Iso-stimulated intracellular cAMP accumulation. Effects of IL-1b on the cAMP-dependent activation of ICa,L. Because IL-1b had no effect on Iso-enhanced cAMP content, we then determined whether IL-1b alters ICa,L activated by forskolin (Fsk). Figure 6A shows results from a control experiment in which 4 ng/ml IL-1b attenuated Iso-enhanced ICa,L by 42%. In Fig. 6B, 1 µM Fsk caused an ,77% increase in ICa,L, a level similar to that induced by 1 µM Iso. However, in contrast to the effect observed with Iso, a 3-min exposure to 4 ng/ml IL-1b did not significantly affect peak ICa,L in the presence of Fsk. A subsequent exposure to IL-1b also had no effect on ICa,L, further supporting the lack of its effect on Fsk-enhanced ICa,L (Fig. 6B). Average peak ICa,L in the Fsk and Fsk 1 IL-1b conditions were 167 6 5% (n 5 5) and 168 6 5% (n 5 5) of control, respectively. These results showed that IL-1b has no effect on Fsk-activated adenylyl cyclase activity or downstream effects of cAMP. To further determine whether IL-1b interferes with cAMP-mediated activation of ICa,L, we examined the effect of IL-1b on ICa,L in myocytes internally dialyzed with cAMP or extracellularly perfused with 8-(4chlorophenylthio)-cAMP (CPT-cAMP), a membranepermeable analog of cAMP. In Fig. 7A, 10 µM cAMP in the pipette solution almost doubled the peak ICa,L, a level similar to that induced by 1 µM Iso. Exposure of the myocyte to 4 ng/ml IL-1b had no significant effect on the cAMP-enhanced ICa,L. Averaged current magni-

Fig. 5. Effect of IL-1b on intracellular cAMP content in adult rat ventricular myocytes in absence and presence of Iso. Left: cAMP contents were measured in myocytes in control conditions and in presence of 5 ng/ml IL-1b, 0.1 µM Iso, or IL-1b 1 Iso for 5 min. Right: cells were treated with Iso alone for 5 min or IL-1b alone for 10 min or preincubated with 5 ng/ml IL-1b for 5 min and then treated with 0.1 µM Iso for another 5 min. Values are means 6 SE; number of experiments is in parentheses. * P , 0.05 compared with control.

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Fig. 6. Effect of IL-1b on forskolin (Fsk)-activated ICa,L. A: control experiment. Addition of 4 ng/ml IL-1b 3 min after incubation of a myocyte with 1 µM Iso (j) caused a 40% decrease in ICa,L (T). B: 1 µM Fsk caused a 77% increase in ICa,L (j). Two subsequent exposures to 4 ng/ml IL-1b had no effect on Fsk-activated ICa,L (T). ICa,L recovered fully after removal of Fsk (k). Insets: superimposed current traces; calibration bars in A and B, 10 pA/pF (vertical) and 5 ms (horizontal); dashed lines, 0-current level. Cell membrane capacitance was 176 pF (A) and 194 pF (B).

tude in IL-1b was 0.96 6 0.02 (n 5 4) and 0.97 6 0.03 (n 5 4) of the control peak ICa,L in the presence of 1 and 10 µM cAMP, respectively. Similarly, in a representative experiment in Fig. 7B, extracellular perfusion of a myocyte with 0.3 mM CPT-cAMP caused a 240% increase in peak ICa,L. Subsequent exposure to 5 ng/ml IL-1b did not significantly alter the cAMP-activated ICa,L. Averaged current magnitude in IL-1b was 0.94 6 0.02 (n 5 4) of the control peak ICa,L in the presence of CPT-cAMP. These results suggested that the IL-1binduced suppression of Iso-enhanced ICa,L is not mediated by inhibition of the cAMP-dependent activation of Ca21 channels. We then examined whether IL-1b suppresses Iso-enhanced ICa,L in the presence of cAMP. Figure 8 shows an increase in ICa,L of 150% in the presence of 0.1 mM CPT-cAMP that was further enhanced by addition of 1 µM Iso. Under these conditions, exposure to 5 ng/ml IL-1b caused an ,43% inhibition of Iso-stimulated ICa,L. Results from five experiments show that IL-1b reduced ICa,L by 21.9 6 5.2% in the presence of CPT-cAMP and Iso. Similarly, in myocytes internally

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dialyzed with 10 µM cAMP, 5 ng/ml IL-1b decreased Iso-stimulated ICa,L by 24.6 6 6.1% (n 5 3). These data further support the suggestion that the IL-1b-induced suppression of b-adrenergic responsiveness of ICa,L is mainly mediated by a cAMP-independent mechanism rather than antagonism of the cAMP-induced activation of Ca21 channels. DISCUSSION

We previously showed that IL-1b reduces basal peak ICa,L in rat ventricular myocytes (26). The present study demonstrates that IL-1b decreases the b-adrenergic responsiveness of ICa,L by suppressing the maximal effect of Iso and increasing its EC50. IL-1b does not alter basal or Iso-induced cAMP levels, b-adrenoceptor binding, or Fsk-stimulated ICa,L and has little, if any, effect on cAMP-activated ICa,L. These results suggest that the IL-1b-induced acute inhibition of Iso’s effects on ICa,L is mediated primarily at a site other than the b-adrenoceptor-adenylyl cyclase-protein kinase A pathway.

Fig. 7. Effect of IL-1b on cAMP-activated ICa,L. A: ICa,L magnitude in a myocyte internally dialyzed with 10 µM cAMP in pipette solution was approximately twice that observed under control conditions (cf. k in B). IL-1b (4 ng/ml) had no effect on cAMP-activated ICa,L (T). B: ICa,L in a myocyte extracellularly perfused with 0.3 mM 8-(4chlorophenylthio)-cAMP (CPT-cAMP) was increased by 240% (j). Subsequent addition of 5 ng/ml IL-1b did not significantly affect cAMP-activated ICa,L (T). Insets: superimposed current traces; calibration bars in A and B, 10 pA/pF (vertical) and 5 ms (horizontal); dashed lines, 0-current level. Cell membrane capacitance was 132 pF (A) and 130 pF (B).

Fig. 8. Effect of IL-1b on Iso-activated ICa,L in presence of cAMP. Activation of ICa,L of 2.5-fold in presence of 0.1 mM CPT-cAMP was further enhanced by addition of 1 µM Iso. Subsequent exposure to 5 ng/ml IL-1b suppressed Iso-activated ICa,L by 43%. ICa,L recovered after solution was returned to 0.1 mM CPT-cAMP. Inset: superimposed current traces; calibration bars, 10 pA/pF (vertical) and 10 ms (horizontal); dashed line, 0-current level. Cell membrane capacitance was 155 pF.

Comparison with findings observed in other cardiac myocytes. Studies with neonatal rat cardiac myocytes have shown that a 72-h incubation with activated splenocyte-conditioned medium or IL-1b inhibits the b-adrenergic responsiveness of contractility by suppressing the Iso-enhanced cAMP level (14). A consecutive study showed that b-adrenoceptor binding was unaltered; however, Fsk-stimulated cAMP concentrations were enhanced, whereas Iso-stimulated cAMP content was decreased by 7% after a 24-h treatment (6). In contrast, the present study in adult rat ventricular myocytes shows no effect of IL-1b on basal or Isoenhanced cAMP levels. The discrepancy in these results could be due to numerous factors, such as different developmental stages, duration of incubation with IL-1b, or different cell populations in primary culture of neonatal cardiac myocytes. Developmental differences in the b-adrenergic and Fsk responsiveness of ICa,L have been shown in rat (23) and rabbit ventricular myocytes (30). Studies in adult guinea pig ventricular myocytes have shown that IL-1b does not alter the b-adrenergic responsiveness of ICa,L unless the exposure duration of the cytokine is .1 h (31). This IL-1b-induced inhibition of b-adrenergic responsiveness of ICa,L was attenuated by replacement of L-arginine with D-arginine and by incubation with an inhibitor of NOS, suggesting the involvement of the NOS pathway (31). These investigators did not provide information about whether IL-1b affects basal or Iso-enhanced cAMP content; however, they did show that IL-1b has no effect on Fsk-activated ICa,L, as indicated by our data (Fig. 6B). In addition, the present results obtained from adult rat ventricular myocytes show that the IL-1b-induced inhibition of b-adrenergic responsiveness of ICa,L occurs after only a couple minutes of cytokine exposure (Figs. 1A and 6A), when cGMP production is not significantly altered (unpublished data). These data suggest that the NOS pathway is not involved in this action. The cause of the


discrepancy between these two studies is unclear but could be attributed to differences in species and/or experimental conditions. Species variations in the b-adrenergic and Fsk responsiveness of ICa,L in ventricular myocytes have been reported (23, 30). IL-1b and cAMP. Studies in vascular smooth cells showed that IL-1b stimulates cAMP but not cGMP production within 1 h (2). The increased cAMP has been suggested to mediate the stimulation of expression of inducible NOS and production of nitrite that causes vasodilatation (2, 35). Similarly, cAMP has been shown to upregulate IL-1b-induced inducible NOS mRNA expression and nitrite production in neonatal rat cardiac myocytes (21). In contrast, a study in decidual cells showed that low concentrations of IL-1b increase the production of cAMP during a 24-h exposure, whereas low concentrations of the cytokine (1 ng/ml) inhibit cAMP production (7). This study suggested that cAMP does not mediate the IL-1b-induced stimulation of prostaglandin production. In addition, in astrocytoma cells, IL-1b induces IL-6 release without altering cAMP formation (4). Our present study showed that IL-1b does not affect the basal or Iso-induced cAMP production in adult rat ventricular myocytes. Therefore, data suggest that the role of cAMP in the signal transduction mechanisms for IL-1b varies among species and cell types. Possible mechanisms. Our results show that IL-1b has no effect on Iso-stimulated cAMP content, b-adrenoceptor binding, or ICa,L in the presence of Fsk. The minor inhibitory effect (,5%) of IL-1b in the presence of intracellular or extracellular cAMP suggests that a cAMP-independent mechanism is involved in the IL-1binduced inhibition of Iso-activated ICa,L. This is supported by results showing that, in the presence of cAMP, ICa,L is further increased by additional exposure to 1 µM Iso, and the Iso-induced increase in ICa,L is decreased by addition of IL-1b. It has been suggested that Iso can stimulate ICa,L via a cAMP-independent pathway that involves direct regulation via a G protein in adult rat ventricular myocytes (23). However, because the relative contribution of this direct G protein cAMP-independent effect of Iso on ICa,L has been questioned (19), the role of this proposed pathway for the observed IL-1b-induced suppression of b-adrenergic responsiveness of ICa,L requires further investigation. We previously showed that IL-1b stimulates the production of ceramide and that ceramide mediates the IL-1binduced suppression of basal ICa,L in adult rat ventricular myocytes (34). It is very likely that ceramide may be involved in this cAMP-independent pathway that suppresses the Iso-activated ICa,L. In summary, IL-1b suppresses b-adrenergic responsiveness of ICa,L via a cAMP-independent pathway. This pathway may include IL-1b-stimulated ceramide production, which counterbalances, rather than disrupts, the Iso-stimulated cAMP-dependent pathway. This action may play an important role in the reduced myocardial function observed during various cardiac disorders associated with cell injury and immune and inflammatory responses. It is also possible that the IL-1b-

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