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Desensitisation off histamine H1-receptor mediated cyclic-GMP production in guinea-pig lung slices
2167 P.fr.057 I
Desensitisation of histamine H ~-receptor mediated cyclic-GMP production in guinea-pig hmg slices Leurs, R., Jansen, W., Bast, A. and T i m m e r m a n , H. Department of Pharmacochemistry. Fa~-~dtyof Chemist,-y, Vrije Universiteit. De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
Histamine is one of the many mediators, that is released from lung tissue after application of allergic stimuli. Histamine can contract airway smooth muscle, but also causes an elevation of the cyclic guanosine-3',5'-monophosphate (c-GMP) content of non-muscle cells in guinea-pig lung tissue (Sertl et al., 1987). In the present study we examined the desensitisation of the histamine-induced c-GMP formation in gninea-pig lung slices. Histamine stimulates the c-GMP production in lung slices in a concentration- and time dependent fashion. The ECso-Value for histamine-induced c-GMP production was 26.8/~M. Already 2 min after stimulation with ~30 ttM histamine maximal responses could be measured (9 ± 1 fold increase above basal levels, mean ± s.e.m., n -- 25). After continued incubation with histamine c-GMP tissue levels drop rather rapidly and reach control levels after 20 min stimulation. Using the two stereoisomers of the Hi-antagonist chlorpheniramine (0.1/tM) a distinct stereoselectivity in favor of the d-isomer was encountered. These data indicate the involvement of the Hi-receptor in the obse, Ted stimulation of the c-GMP production. The Hi-receptor mediated c-GMP production desensitised very rapidly. After one l~l~ndesensifisation with 300/~M histamine and subsequent washing of the lung slices for 20 rain, responses to 100/tM histamine were reduced to 39 + 5~ of the control value (mean ± s.e.m., n - - 4 ) , whereas after 10 min desensitisation response~ to l ~ /tM histamine were reduced to 19 ± 2~ of the control value. Desensitisation of the Ht-receptor mediated responses appeared to be homologous. After desensitisation with 300 /tM histamine for 5 or 20 min, increases in c-GMP tissue levels, induced by 100 jtM metacholine or 100 /~M nitroprusside were not significantly altered. These data indicate that the observed desensitisation is not the result of activation of c-GMP-phophodiesterases, but imply a rather specific action in the signal transfer mechansim of the H~-receptor. Although the histamine H~-receptor is considered to be a so-called calcium-mobilising receptor, desensitisation of the histamine-induced c-GMP production was not dependent upon the presence of extracellular calcium. After a 30 rain preincubation in calcium-free Krebs-buffer, supplemented with 2 mM EGTA, lung slices were desensitised for 5 rain with 300 /~M histamine in the same calcium-free buffer. Thereafter lung slices were washed in normal Krebs-buffer and stimulated for 2 rain with 100 ttM histamine. After desensitisation in calcium-free buffer the response to 100/tM histamine was reduced to 19 ± 3~ (mean ± s.e.m, n -- 5) of the control value. This inhibition did not differ from desensitisation in normal, calcium-containing buffer. Protein kinase C is often involved in a negative feedback loop of various receptor systems. Therefore, we examined the effects of the protein kinase C activator phorbol-12,13-dibutyrate (PDB) on the Ht-receptor mediated c-GMP production in guinea-pig lung slices. After 1 hour preincubation of lung slices with 1 ~M PDB subsequent responses to 100/tM histamine were inhibited to 47 ± 3~; (mean ± s.e.m., n -- 8) of the control value, whereas 1 hour preincubation with 1/~M 4a-phorbol, a compound that does not activate protein kinase C and is used as chemical control, did not significantly attenuate the histamine Hi-receptor response. Both compounds did not attenuate basal c-GMP levels in the lung slices. Due to the remarkable difference in desensitisation rate after protein kinase C stimulation or excessive Ht-receptor activation we do not con~der protein kinase C as an important regulatory component in the observed desensitisafion of the Hl-r~.eptor mediated c-GMP production in guinea-pig l:ang slices. References Sertl, K., Casale, T.B., Wcscott, S.L. and Kaliner, M.A., 1987, Am. Rev. Resp. Dis., 135, 456.
Desensitisation of histamine H ~-receptor mediated cyclic-GMP production in guinea-pig hmg slices Leurs, R., Jansen, W., Bast, A. and T i m m e r m a n , H. Department of Pharmacochemistry. Fa~-~dtyof Chemist,-y, Vrije Universiteit. De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
Histamine is one of the many mediators, that is released from lung tissue after application of allergic stimuli. Histamine can contract airway smooth muscle, but also causes an elevation of the cyclic guanosine-3',5'-monophosphate (c-GMP) content of non-muscle cells in guinea-pig lung tissue (Sertl et al., 1987). In the present study we examined the desensitisation of the histamine-induced c-GMP formation in gninea-pig lung slices. Histamine stimulates the c-GMP production in lung slices in a concentration- and time dependent fashion. The ECso-Value for histamine-induced c-GMP production was 26.8/~M. Already 2 min after stimulation with ~30 ttM histamine maximal responses could be measured (9 ± 1 fold increase above basal levels, mean ± s.e.m., n -- 25). After continued incubation with histamine c-GMP tissue levels drop rather rapidly and reach control levels after 20 min stimulation. Using the two stereoisomers of the Hi-antagonist chlorpheniramine (0.1/tM) a distinct stereoselectivity in favor of the d-isomer was encountered. These data indicate the involvement of the Hi-receptor in the obse, Ted stimulation of the c-GMP production. The Hi-receptor mediated c-GMP production desensitised very rapidly. After one l~l~ndesensifisation with 300/~M histamine and subsequent washing of the lung slices for 20 rain, responses to 100/tM histamine were reduced to 39 + 5~ of the control value (mean ± s.e.m., n - - 4 ) , whereas after 10 min desensitisation response~ to l ~ /tM histamine were reduced to 19 ± 2~ of the control value. Desensitisation of the Ht-receptor mediated responses appeared to be homologous. After desensitisation with 300 /tM histamine for 5 or 20 min, increases in c-GMP tissue levels, induced by 100 jtM metacholine or 100 /~M nitroprusside were not significantly altered. These data indicate that the observed desensitisation is not the result of activation of c-GMP-phophodiesterases, but imply a rather specific action in the signal transfer mechansim of the H~-receptor. Although the histamine H~-receptor is considered to be a so-called calcium-mobilising receptor, desensitisation of the histamine-induced c-GMP production was not dependent upon the presence of extracellular calcium. After a 30 rain preincubation in calcium-free Krebs-buffer, supplemented with 2 mM EGTA, lung slices were desensitised for 5 rain with 300 /~M histamine in the same calcium-free buffer. Thereafter lung slices were washed in normal Krebs-buffer and stimulated for 2 rain with 100 ttM histamine. After desensitisation in calcium-free buffer the response to 100/tM histamine was reduced to 19 ± 3~ (mean ± s.e.m, n -- 5) of the control value. This inhibition did not differ from desensitisation in normal, calcium-containing buffer. Protein kinase C is often involved in a negative feedback loop of various receptor systems. Therefore, we examined the effects of the protein kinase C activator phorbol-12,13-dibutyrate (PDB) on the Ht-receptor mediated c-GMP production in guinea-pig lung slices. After 1 hour preincubation of lung slices with 1 ~M PDB subsequent responses to 100/tM histamine were inhibited to 47 ± 3~; (mean ± s.e.m., n -- 8) of the control value, whereas 1 hour preincubation with 1/~M 4a-phorbol, a compound that does not activate protein kinase C and is used as chemical control, did not significantly attenuate the histamine Hi-receptor response. Both compounds did not attenuate basal c-GMP levels in the lung slices. Due to the remarkable difference in desensitisation rate after protein kinase C stimulation or excessive Ht-receptor activation we do not con~der protein kinase C as an important regulatory component in the observed desensitisafion of the Hl-r~.eptor mediated c-GMP production in guinea-pig l:ang slices. References Sertl, K., Casale, T.B., Wcscott, S.L. and Kaliner, M.A., 1987, Am. Rev. Resp. Dis., 135, 456.