Biochemical SocietyTransactions ( 1 992) 20
Myofibrillar bound cyclic nucleotide phosphodiesterase in heart and skeletal muscle.
129s
Table 1 . Inhibition of cardiac rnvofibrillar PDEs.
ANGELA WORBY, LUCY M. MENSAH and KENNETH J. MURRAY SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Herts AL6 9AR It is generally accepted that there are differences between species in their positive inotropic response to the new generation of cardiotonics which act via inhibition of cGMP-inhibited phosphodiesterase (cGI-PDE, also known as PDE Ill; see (11 for nomenclature). Most notable is the lack of effect of these agents on rat cardiac preparations [2,3] and it has been proposed that this is due to either differences in the subcellular location [2] or the ratio of cGIPDE to PDE IV (CAMP specific, rolipram inhibited) in this species [3]. We have investigated the PDE content of purified cardiac myofibrils from rat and a number of other species to see if this can provide an explanation for these observations. Myofibrils were prepared from heart and skeletal muscle by homogenisation (PoIytron,lO-l5s on setting 4) of tissue in 8 vols. of 20mM Tris, 5mM 2-mercaptoethanol, 50pM PMSF, lpg/ml PAL (buffer A) containing 300mM sucrose (pH 7.0). Following centrifugation (15,OOOxg, lomins), the pellet was rehomogenised in 8 vols. of buffer A containing 60mM KCI, 2mM MgCI,, 2mM EGTA (pH 7.0) (buffer B) and recentrifuged (5,OOOxg, 5mins). The pellet was then subjected to 2 washes in buffer B containing 1Yo Triton-X100, followed by a further 4 washes in buffer A alone. The myofibrils were stored at -2OC in buffer A containing 50% glycerol. PDE activity was assayed using [3H]-cAMP and [3H]-cGMP as previously described [4]. Assay of the purified myofibrils from both cardiac and skeletal muscle showed the presence of PDE activity in all species studied (i.e. rat, guinea-pig, cow, rabbit, cat, dog, chicken and pig). Skeletal muscle myofibrils from all the species studied had PDE activity with CAMP, but not with cGMP as a substrate. This substrate specificity suggested that the activity involved was PDE IV. This was confirmed by the use of selective PDE inhibitors; the selective PDE Ill inhibitor siguazodan had no effect whereas the selective PDE IV inhibitors rolipram, denbufylline and Ro 20-1724 caused a complete and potent inhibition. A more complex pattern of activity was found with the cardiac myofibrils. Table 1 shows a comparison of the effects of selective PDE inhibitors on rat and guinea-pig cardiac myofibrils. The results show that rat cardiac myofibril PDE activity was not inhibited by siguazodan but was completely inhibited by rolipram and denbufylline. Therefore, as with the skeletal muscle myofibrils. rat heart myofibrils contain exclusively PDE IV. In contrast, the PDE activity associated with guinea-pig cardiac myofibrils was inhibited by siguazodan and rolipram indicating that these myofibrils c o n t a i n e d b o t h PDE I l l a n d P D E I V . A combination of 1 OOpM siguazodan and 100pM roliprarn completely inhibited all PDE activity, implying that no other PDE isoenzymes were present.
PDE, cyclic nucleotide phosphodiesterase; CAMP, adenosine-3’:5’-cyclic monophosphate; cGMP, guanosine3’:5’-cyclic monophosphate; PAL, pepstatin, antipain, leupeptin; PMSF, phenylmethylsulphonyl flouride.
Siguazodan lmazodan Rolipram Denbufylline Milrinone
>lo0 >lo0 >lo0 >lo0 3.7 3.2 1.3 1.6 27 21
3% 12% 91% 93% 77%
2.4 >lo0 52% 29 45 49% 1.3 2100 50% 0.9 z l 0 0 43% 8.8 22 73%
ls0,the concentration of inhibitor required to inhibit 50% of the activity inhibitable by this inhibitor. 21C50, t h e concentration of inhibitor required to inhibit 50% of the total activity. Yo inhibited at 100pM inhibitor. PDE was assayed using 1pM [3H]-cAMP.
Results similar to those of the guinea-pig were obtained with the cardiac myofibrils from the other species. Therefore, at present, rat heart myofibrils are unique in that they contain solely PDE IV, whereas those from other species contain both PDE Ill and PDE IV. It is possible that the lack of myofibrillar bound PDE Ill is the reason that PDE Ill inhibitors are not inotropes in the rat although, in this respect, it is important to note that the PDE associated with the myofibril represents only a small amount ( c lo/o) of the total activity. Therefore, further experiments are necessary to determine the physiological role, if any, of the bound PDEs. Similar results were obtained when rat and guinea-pig heart myofibrils were prepared by a different method [5];this and the extensive washing procedure u s e d suggests that the PDEs are tightly and specifically associated with the myofibrils. However, as shown by the 150 values in Table 1, the bound PDEs are inhibited with similar potency t o their cytosolic counterparts. This indicates that cytosolic and myofibril bound PDEs contain similar catalytic sites. The complete inhibition of guinea-pig bound PDEs by milrinone and imodazodan may indicate the decreased selectivity of these agents when compared with siguazodan. In conclusion, most species have two PDE activities tightly associated with heart myofibrils, cGI-PDE (PDE 111) and CAMP-specific, rolipram-inhibited PDE (PDE IV). In contrast, rat cardiac rnyofibrils contained only PDE IV and the absence of myofibril bound PDE Ill in the rat may explain why PDE Ill inhibitors do not show inotropic activity in this species. Skeletal muscle myofibrils of all the species studied, including rat, contained solely PDE IV.
1. Beavo, J . A . & Reifsnyder, D.H. (1990) Trends. Pharmacol. Sci. 1 1 , 150-155 2. Weishaar, R.E., Kobylarz-Singer, D.C., Stefan, R.P. & Kaplan, H.R. (1987) Circ. Res. 61, 539-547 3. S h a h i d , M. & N i c h o l s o n C . D . ( 1 9 9 0 ) N a u n y n Schmiedeberg Arch. Pharmacol. 342, 698-705 4. Reeves, M.L., Leigh B.K. & England P.J. (1987) Biochem. J. 241, 535-541. 5. Knight, P.J. & Trinick, J.A. (1982) Method. Enzymol. 85, 9-13
Myofibrillar bound cyclic nucleotide phosphodiesterase in heart and skeletal muscle.
129s
Table 1 . Inhibition of cardiac rnvofibrillar PDEs.
ANGELA WORBY, LUCY M. MENSAH and KENNETH J. MURRAY SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Herts AL6 9AR It is generally accepted that there are differences between species in their positive inotropic response to the new generation of cardiotonics which act via inhibition of cGMP-inhibited phosphodiesterase (cGI-PDE, also known as PDE Ill; see (11 for nomenclature). Most notable is the lack of effect of these agents on rat cardiac preparations [2,3] and it has been proposed that this is due to either differences in the subcellular location [2] or the ratio of cGIPDE to PDE IV (CAMP specific, rolipram inhibited) in this species [3]. We have investigated the PDE content of purified cardiac myofibrils from rat and a number of other species to see if this can provide an explanation for these observations. Myofibrils were prepared from heart and skeletal muscle by homogenisation (PoIytron,lO-l5s on setting 4) of tissue in 8 vols. of 20mM Tris, 5mM 2-mercaptoethanol, 50pM PMSF, lpg/ml PAL (buffer A) containing 300mM sucrose (pH 7.0). Following centrifugation (15,OOOxg, lomins), the pellet was rehomogenised in 8 vols. of buffer A containing 60mM KCI, 2mM MgCI,, 2mM EGTA (pH 7.0) (buffer B) and recentrifuged (5,OOOxg, 5mins). The pellet was then subjected to 2 washes in buffer B containing 1Yo Triton-X100, followed by a further 4 washes in buffer A alone. The myofibrils were stored at -2OC in buffer A containing 50% glycerol. PDE activity was assayed using [3H]-cAMP and [3H]-cGMP as previously described [4]. Assay of the purified myofibrils from both cardiac and skeletal muscle showed the presence of PDE activity in all species studied (i.e. rat, guinea-pig, cow, rabbit, cat, dog, chicken and pig). Skeletal muscle myofibrils from all the species studied had PDE activity with CAMP, but not with cGMP as a substrate. This substrate specificity suggested that the activity involved was PDE IV. This was confirmed by the use of selective PDE inhibitors; the selective PDE Ill inhibitor siguazodan had no effect whereas the selective PDE IV inhibitors rolipram, denbufylline and Ro 20-1724 caused a complete and potent inhibition. A more complex pattern of activity was found with the cardiac myofibrils. Table 1 shows a comparison of the effects of selective PDE inhibitors on rat and guinea-pig cardiac myofibrils. The results show that rat cardiac myofibril PDE activity was not inhibited by siguazodan but was completely inhibited by rolipram and denbufylline. Therefore, as with the skeletal muscle myofibrils. rat heart myofibrils contain exclusively PDE IV. In contrast, the PDE activity associated with guinea-pig cardiac myofibrils was inhibited by siguazodan and rolipram indicating that these myofibrils c o n t a i n e d b o t h PDE I l l a n d P D E I V . A combination of 1 OOpM siguazodan and 100pM roliprarn completely inhibited all PDE activity, implying that no other PDE isoenzymes were present.
PDE, cyclic nucleotide phosphodiesterase; CAMP, adenosine-3’:5’-cyclic monophosphate; cGMP, guanosine3’:5’-cyclic monophosphate; PAL, pepstatin, antipain, leupeptin; PMSF, phenylmethylsulphonyl flouride.
Siguazodan lmazodan Rolipram Denbufylline Milrinone
>lo0 >lo0 >lo0 >lo0 3.7 3.2 1.3 1.6 27 21
3% 12% 91% 93% 77%
2.4 >lo0 52% 29 45 49% 1.3 2100 50% 0.9 z l 0 0 43% 8.8 22 73%
ls0,the concentration of inhibitor required to inhibit 50% of the activity inhibitable by this inhibitor. 21C50, t h e concentration of inhibitor required to inhibit 50% of the total activity. Yo inhibited at 100pM inhibitor. PDE was assayed using 1pM [3H]-cAMP.
Results similar to those of the guinea-pig were obtained with the cardiac myofibrils from the other species. Therefore, at present, rat heart myofibrils are unique in that they contain solely PDE IV, whereas those from other species contain both PDE Ill and PDE IV. It is possible that the lack of myofibrillar bound PDE Ill is the reason that PDE Ill inhibitors are not inotropes in the rat although, in this respect, it is important to note that the PDE associated with the myofibril represents only a small amount ( c lo/o) of the total activity. Therefore, further experiments are necessary to determine the physiological role, if any, of the bound PDEs. Similar results were obtained when rat and guinea-pig heart myofibrils were prepared by a different method [5];this and the extensive washing procedure u s e d suggests that the PDEs are tightly and specifically associated with the myofibrils. However, as shown by the 150 values in Table 1, the bound PDEs are inhibited with similar potency t o their cytosolic counterparts. This indicates that cytosolic and myofibril bound PDEs contain similar catalytic sites. The complete inhibition of guinea-pig bound PDEs by milrinone and imodazodan may indicate the decreased selectivity of these agents when compared with siguazodan. In conclusion, most species have two PDE activities tightly associated with heart myofibrils, cGI-PDE (PDE 111) and CAMP-specific, rolipram-inhibited PDE (PDE IV). In contrast, rat cardiac rnyofibrils contained only PDE IV and the absence of myofibril bound PDE Ill in the rat may explain why PDE Ill inhibitors do not show inotropic activity in this species. Skeletal muscle myofibrils of all the species studied, including rat, contained solely PDE IV.
1. Beavo, J . A . & Reifsnyder, D.H. (1990) Trends. Pharmacol. Sci. 1 1 , 150-155 2. Weishaar, R.E., Kobylarz-Singer, D.C., Stefan, R.P. & Kaplan, H.R. (1987) Circ. Res. 61, 539-547 3. S h a h i d , M. & N i c h o l s o n C . D . ( 1 9 9 0 ) N a u n y n Schmiedeberg Arch. Pharmacol. 342, 698-705 4. Reeves, M.L., Leigh B.K. & England P.J. (1987) Biochem. J. 241, 535-541. 5. Knight, P.J. & Trinick, J.A. (1982) Method. Enzymol. 85, 9-13
