Persistent activation by and receptor reserve for an irreversible A1-adenosine receptor agonist in DDT1 MF-2 cells and in guinea pig heart

Abstract

The p- and m-isothiocyanate adenosine derivatives N6-[4-[[[4-[[[[2-[[[(p-(m)-isothiocyanatophenyl)amino]thiocarbonyl ]am ino]ethyl]amino]carbonyl]methyl]anilino]carbonyl]methyl]phenyl] adenosine (p- and m-DITC-ADAC) were examined for irreversible agonist effects at the A1-adenosine receptor (A1-AdoR) in DDT1 MF-2 (DDT) cells and a functional A1-AdoR response in the guinea pig isolated heart. The p- and m-DITC-ADAC inhibited (-)-isoproterenol stimulated cAMP accumulation in DDT cells in the low nanomolar range, and the maximal responses elicited by both compounds were similar to that for N6-cyclopentyladenosine. Once established, the p-DITC-ADAC-mediated inhibition of cAMP accumulation in DDT cells was not affected by the addition of the AdoR antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX). Pretreatment of DDT cells with p-DITC-ADAC (1 microM), followed by washing, reduced [3H]CPX binding to the A1-AdoR by 44% without altering the Kd value for the radioligand to the remaining receptors. The relationship between irreversible A1-AdoR occupancy by p-DITC-ADAC and inhibition of cAMP accumulation revealed a relatively large receptor reserve (64%) for the maximal response. In guinea pig isolated hearts, m-DITC-ADAC (5 microM) prolonged the stimulus to His bundle (SH) interval by 2.1-fold; this response could be prevented by the antagonist 8-cyclopentyltheophylline (5 microM). However, after the SH interval prolongation was established, extensive washout or the addition of 8-cyclopentyltheophylline had little reversal effect on the m-DITC-ADAC response. Binding of [3H]CPX to the guinea pig ventricular membranes after m-DITC-ADAC treatment and washing was reduced by 35%. The A1-AdoR occupancy response relationship for m-DITC-ADAC to prolong the SH interval indicated a small (10-20%) receptor reserve. Both p -and m-DITC-ADAC seem to be irreversible full agonists at the A1-AdoR and may prove to be useful probes to further investigate A1-AdoR structure-function relationships.

Fig. 1

Structure of p- and m-DITC-ADAC.

Fig. 2

Effects of p- and m-DITC-ADAC on inhibition of (−)-isoproterenol-stimulated cAMP accumulation in DDT cells. Intact DDT cells were incubated with 1 μM (−)-isoproterenol, 50 μM rolipram, and the indicated concentrations of CPA or p- or m- DITC-ADAC for 10 min at 37°. At the end of the incubation, the cAMP accumulated was determined as described in Experimental Procedures. Values are the mean ± standard error of four separate experiments, each performed in duplicate. The basal cAMP was 5.0 ± 1.7 pmol/mg of protein/min, and the cAMP accumulated in the presence of 5 μM (−)-isoproterenol alone was 60.0 ± 9.0 pmol/mg of protein/min.

Fig. 3

Time course effect of p-DITC-ADAC on the inhibition of (−)-isoproterenol-stimulated cAMP accumulation in DDT cells. DDT cells were incubated with 50 μM rolipram and 1 μM (−)-isoproterenol and without (Δ) or with 1 μM p-DITC-ADAC (○) or 1 μM CPA (□) at 37°. After 4 min of incubation with p-DITC-ADAC (●) or CPA (■), 1 μM CPX was added, and the accumulated cAMP was determined at the indicated time points. X, Basal cAMP levels. Points, mean ± standard error of three separate determinations assayed in duplicate; arrow, addition of CPX.

Fig. 4

Representative Rosenthal plots of [³H]CPX binding to DDT cell membranes after pretreatment of intact cells with p-DITC-ADAC. DDT cells were incubated with 1 μM p-DITC-ADAC or 1 μM CPA at 37° for 1 hr followed by six washes with HBSS. Cell membranes were then prepared, and the A₁-AdoRs were determined by [³H]CPX (0.06–4 nM) binding in DDT cell membranes. Points, mean of triplicate determinations.

Fig. 5

Time course of p-DITC-ADAC-induced loss of specific [³H]CPX binding to DDT cells. DDT cells were incubated without (F) or with (f) 1 μM p-DITC-ADAC for the indicated times followed by six washes with HBSS. The cells were then homogenized in 50 mM Tris-HCl buffer, pH 7.4, containing 5 mM MgCl₂, and the suspension was centrifuged at 48,000 × g for 15 min. The membranes were resuspended in buffer, and the A₁-AdoR content was determined with 4 nM [³H]CPX as described in Experimental Prcedures. Points, mean ± standard error of three separate experiments assayed in triplicate. The control specific [³H]CPX binding was 212 ± 30 fmol/mg of protein.

Fig. 6

The relationship between the inhibition of (−)-isoproterenol-stimulated cAMP accumulation and receptor occupancy by p-DITC-ADAC in DDT cells. Cells were pretreated with 0.1, 0.5, 1.0, or 2.0 μM p-DITC-ADAC for 1 hr at 37° followed by six washes with HBSS. The inhibition of cAMP accumulation by the A₁-AdoRs occupied by pretreatment with p-DITC-ADAC was determined by incubating the cells with 50 μM rolipram, 1 μM CPX, and 5 μM (−)-isoproterenol at 37° for 10 min. The accumulated cAMP was determined as described in Experimental Procedures. The A₁-AdoR occupancy by p-DITC-ADAC was determined as the percent loss of specific [³H]CPX (5 nM) binding to cell membranes compared with untreated controls. Values are mean ± standard error of three or four separate experiments, each performed in duplicate or triplicate. The control (−)-isoproterenol (5 μM)-stimulated cAMP formation was 50 ± 5 pmol/mg of protein/min, and the control-specific [³H]CPX binding was 195 ± 4 fmol/mg of protein. The maximal inhibition of (−)-isoproterenol-stimulated cAMP accumulation in the presence of 5 μM p-DITC-ADAC was 82 ± 3% (three experiments).

Fig. 7

Time course of the effects of CPA, m-DITC-ADAC, and A_1-AdoR antagonist CPT on SH interval of guinea pig isolated, perfused hearts. Hearts were paced at an atrial cycle length of 300 msec throughout an experiment. A, CPA (50 nM) maximally and reversibly prolonged the SH interval. This effect was rapidly and nearly completely reversed by its washout or by the antagonist CPT (5 μM). Points, averages of SH interval determinations from two hearts. B, Lack of reversibility of m-DITC-ADAC (5 μM, 20 min)-induced SH interval prolongation. m-DITC-ADAC (5 μM) added to the perfusate increased the SH interval and caused second-degree AV block. After a 40-min washout, 1:1 AV conduction resumed, but the SH interval remained prolonged (79 ± 2 msec). A supramaximal concentration of CPT (5 μM) caused only a small (p > 0.05) reversal of the effect of m-DITC-ADAC. Points, mean ± standard error of responses of four hearts.

Fig. 8

Ado-R blockade prevents the negative dromotropic effect of m-DITC-ADAC on the guinea pig isolated, perfused heart. The heart was paced at a constant atrial cycle length of 300 msec. CPT (5 μM) prevented the prolongation by m-DITC-ADAC (5 μM) of the SH interval. After washout of CPT in the presence of m-DITC-ADAC, the SH interval rapidly lengthened and remained prolonged despite washout of m-DITC-ADAC. Identical results were obtained in two additional hearts.

Fig. 9

Rosenthal plot of specific [³H]CPX binding to guinea pig ventricular membranes after pretreatment with m-DITC-ADAC. Membranes were incubated with 1 μM m-DITC-ADAC for 20 min and then washed four times by centrifugation and resuspension. The A₁-AdoR density was then determined by the specific binding of [³H]CPX (0.6–10 nM) as described in Experimental Procedures. Points, mean of four determinations assayed in triplicate. The K_d values are 4.3 ± 0.5 and 1.4 ± 0.1 nM for control and m-DITC-ADAC-treated membranes, respectively.

Fig. 10

Correlation between SH interval prolongation and A₁-AdoR occupancy by m-DITC-ADAC in guinea pig isolated, perfused hearts. Hearts paced at a constant atrial cycle length of 300 msec were perfused with Krebs-Henseleit solution containing 5 μM m-DITC-ADAC for 5, 10, 15, and 30 min. At the end of each perfusion period, the tissue was perfused with 5 μM CPT, and the irreversible component of SH interval prolongation was recorded. After washout for 1 hr, the ventricles were harvested, and membranes were prepared for determination of the number (B_max) and affinity (K_d) of A₁-AdoRs by measuring specific binding of [³H]CPX. The irreversible component of SH interval prolongation caused by m-DITC-ADAC is plotted as a function of percent of total A₁-AdoRs occupied [B_max (fmol/mg of protein of untreated control)/B_max of m-DITC-ADAC-treated hearts × 100). The B_max and K_d values of four untreated hearts were 33 ± 4 fmol/mg of protein and 3.2 ± 0.2 nM, respectively. There was no significant difference between the K_d values of [³H]CPX binding to membranes of untreated and m-DITC-ADAC-treated hearts.