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Tissue peptidase activity as a possible determinant of relative activity of tachykinins and other peptides
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TISSUE PEPTIDASE ACTIVITY AS A POSSIBLE ACTIVITY OF TACHYKININS AND OTHER PEPTIDES
DETERMINANT
OF
RELATIVE
Alyson J. FOX, Judith M. HALL and Ian K. M. MORTON Department of Pharmacology, King's College London, Strand, London WC2R 2LS In the continuing absence of selective receptor antagonists, the characterisation of peptide receptors has relied heavily on the determination of relative activities of series of agonists. However, differences in structure between peptides renders them differentially susceptible to degradation by tissue peptidases which may in turn vary between tissues (1,2). Consequently, enzymic degradation may distort effective biophase concentrations of agonists to an extent that results in erroneous relative activity determinations. We therefore thought it important to redetermine the relative activities of some tachykinins and bombesin-like peptides in a number of peripheral preparations which we have previously studied (3,4) in order to assess the influence of peptidase activity on such estimates. Agonlsts were selected to include relatively stable analogues and those known to be substrates for certain peptidases. Test systems included potentiation of the contractile response of the guinea-pig vas deferens to nerve stimulation and contraction of the guinea-pig taenia caeci and rat urinary bladder. Either two point or full dose-response curves were determined in the presence (test) or absence (concurrent control) of a peptidase inhibitor 'cocktail' containing phosphoramidon (l~H), captopril (I~M) and bestatin (100~M) to inhibit endopeptidase 24.11, ACE and some amlnopeptidases. Concentrations chosen were those already shown to be effective in the trachea preparation (5) In no case was a significant difference seen in peak responses to tachyklnins including SP, NKA, NKB and physalaemin or BB-like peptides including BB, NMB, GRPI8_27 and litorin, between control and test preparations. However, interestingly enough, the duration of action of the tachykinins after bath washout became prolonged for test tissues in the guinea-pig vas deferens series, now resembling the more prolonged kinetics of the BB-like peptides. Thus, somewhat surprisingly, there was no evidence in these preparations that agonist breakdown had seriously distorted the effective biophase concentrations of the peptides tested. However, it seems likely that access to the receptors is relatively unimpaired in these preparations where maximal responses are achieved in approximately 20s. Thus, even when bioassay has shown slow depletion of bath peptide the initial biophase concentration adequately reflects the nominal bath concentration, with only the kinetics of the slower processes (such as offset) becoming peptidase sensitive. In contrast, it would be expected that in thick tissues showing slow responses to cumulative dosing regimes these slow kinetics would become all important, so dramatic effects of peptidase inhibition might not be surprising. This has recently been described for airways smooth muscle and peptidase inhibition had marked effects on doseresponse relationships of certain tachykinins where slopes, relative position, maximal effects and rank order were all influenced (5,6). The findings in these and similar studies is significant in relation to the design of experiments where the biophase concentration of peptide agonists, and for that matter antagonists, is of critical importance. We thank the MRC and SERC for support of this work. i. Nau R., Schafer G.. Deacon C.F., Cole T., Agoston D.V., Conlon J.M. (1986) J Neurochem 47:856-864 2. Sooper N.M., Kerry A.J., Tu~er A.J. (1985) Biochem J 231:357-361 3. Hall J.M., Fox A.J., Morton I.K.M. (lgSF) Hr J ~ a ~ @1:475P 4. Fox A.J., Hall J.M., Morton I.K.M. (lg88) Br J ~ a ~ 93:283P 5. Stephens-SmithM.L, Irel~d S.J., Jordan C.C., (1988) Sis meeting 6. Seklzawa K. et al (1987) J ~ a ~ Exp ~er 243:1211-121F
TISSUE PEPTIDASE ACTIVITY AS A POSSIBLE ACTIVITY OF TACHYKININS AND OTHER PEPTIDES
DETERMINANT
OF
RELATIVE
Alyson J. FOX, Judith M. HALL and Ian K. M. MORTON Department of Pharmacology, King's College London, Strand, London WC2R 2LS In the continuing absence of selective receptor antagonists, the characterisation of peptide receptors has relied heavily on the determination of relative activities of series of agonists. However, differences in structure between peptides renders them differentially susceptible to degradation by tissue peptidases which may in turn vary between tissues (1,2). Consequently, enzymic degradation may distort effective biophase concentrations of agonists to an extent that results in erroneous relative activity determinations. We therefore thought it important to redetermine the relative activities of some tachykinins and bombesin-like peptides in a number of peripheral preparations which we have previously studied (3,4) in order to assess the influence of peptidase activity on such estimates. Agonlsts were selected to include relatively stable analogues and those known to be substrates for certain peptidases. Test systems included potentiation of the contractile response of the guinea-pig vas deferens to nerve stimulation and contraction of the guinea-pig taenia caeci and rat urinary bladder. Either two point or full dose-response curves were determined in the presence (test) or absence (concurrent control) of a peptidase inhibitor 'cocktail' containing phosphoramidon (l~H), captopril (I~M) and bestatin (100~M) to inhibit endopeptidase 24.11, ACE and some amlnopeptidases. Concentrations chosen were those already shown to be effective in the trachea preparation (5) In no case was a significant difference seen in peak responses to tachyklnins including SP, NKA, NKB and physalaemin or BB-like peptides including BB, NMB, GRPI8_27 and litorin, between control and test preparations. However, interestingly enough, the duration of action of the tachykinins after bath washout became prolonged for test tissues in the guinea-pig vas deferens series, now resembling the more prolonged kinetics of the BB-like peptides. Thus, somewhat surprisingly, there was no evidence in these preparations that agonist breakdown had seriously distorted the effective biophase concentrations of the peptides tested. However, it seems likely that access to the receptors is relatively unimpaired in these preparations where maximal responses are achieved in approximately 20s. Thus, even when bioassay has shown slow depletion of bath peptide the initial biophase concentration adequately reflects the nominal bath concentration, with only the kinetics of the slower processes (such as offset) becoming peptidase sensitive. In contrast, it would be expected that in thick tissues showing slow responses to cumulative dosing regimes these slow kinetics would become all important, so dramatic effects of peptidase inhibition might not be surprising. This has recently been described for airways smooth muscle and peptidase inhibition had marked effects on doseresponse relationships of certain tachykinins where slopes, relative position, maximal effects and rank order were all influenced (5,6). The findings in these and similar studies is significant in relation to the design of experiments where the biophase concentration of peptide agonists, and for that matter antagonists, is of critical importance. We thank the MRC and SERC for support of this work. i. Nau R., Schafer G.. Deacon C.F., Cole T., Agoston D.V., Conlon J.M. (1986) J Neurochem 47:856-864 2. Sooper N.M., Kerry A.J., Tu~er A.J. (1985) Biochem J 231:357-361 3. Hall J.M., Fox A.J., Morton I.K.M. (lgSF) Hr J ~ a ~ @1:475P 4. Fox A.J., Hall J.M., Morton I.K.M. (lg88) Br J ~ a ~ 93:283P 5. Stephens-SmithM.L, Irel~d S.J., Jordan C.C., (1988) Sis meeting 6. Seklzawa K. et al (1987) J ~ a ~ Exp ~er 243:1211-121F