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Homologous histamine H1 receptor desensitization results in reduction of H1 receptor agonist efficacy
European Journal of Pharmacology, 196 (1991) 319-322 0 1991 Elsevier Science Publishers B.V. OO14-2999/91/$03.50 A DONIS OOl4299991OW2G
319
EJP 20815
Short c~mwni~tion
omologous histamine
sensitization Fe receptor egotist ~~~~~a~
Rob Leurs, Martine J. Smit, Aalt Bast and Hendrik Timmerman Departmen! of Pharmacochemistry. Faculty ojchemisttp, Vr@ Uniuersiteit, De Boelelaan 1081,1083 HV Ansterabn,
The NetherIan&
Received 26 February 1991, accepted 5 Mar& 1991
Prolonged exposure of the guinea-pig intestinal ion~tudinal smooth muscle to histamine caused homologous de~asit~~on of the H, receptor, which led to reduced H, receptor-mediated production of [3H]inositol phosphates as well as to reduced H, agonist-induced contractions. [3H]Mepyramine binding studies showed that desensitization affected neither the agonist affinity nor the number of H, receptors. Combining the data from the binding studies and the contraction measurements it was found that d~ensitization results in a selective reduction of agonist efficacy. Intestine (guinea-pig); Histamine H, receptors; Desensitization; [ ‘H]Mepyramine; [ 3H]Inositol phosphates; Agonist efficacy
1. Introduction Desensitization of receptor responses is frequently observed in experiments on histamine H, receptor responsiveness (Kenakin and Cook, 1981; Leurs et al., 1990). Previously we noticed a very rapid desensitization of H, receptor responses of the longitudinal smooth muscle of guinea-pig jejunum (Leurs et al., 1990). After a few minutes of desensitization the contractions in response to histamine are initially strongly inhibited. These responses recover slowly and do not reach their initial control levels within a few hours (Leurs et al., 1990). The desensitization with histamine does not attenuate methacholine-induced responses and therefore appears to be homologous (Leurs et al., 1990). These data indicate modulation of signal transduction at the level of the H, receptor. Various modifications of the H, receptor might however affect H, receptor activity. Modulation of agonist binding characteristics, effects on receptor-G-protein coupling or a reduction of receptor number can all exclusively modulate H, receptor function, leading to homologous desensitization. Kenakin and Cook (1981) previously used the irreversible antagonist, phenoxybenzamine, to elucidate the mechanism underlying intestinal H, receptor desensitization. The results of their investigations suggested that the desensitization ob-
served was due to a conformational alteration of the H, receptor, leading to changed binding characteristics (Kenakin and Cook, 1981). Phenoxybenzamine is however not very specific for the H, receptor and might not be appropiate for the study of H, agonist activity (Fox and Friedman, 1987; Graig and Clarke, 1990). In the present study on the mechanism of the rapid H, receptor desensitization we combined studies on contraction with histamine and a partial H, receptor agonist with studies on [3H]mepyramine binding and measurements of the H, receptor-mediated breakdown of the phosphoinositides in -the longitudinal smooth muscle of guinea-pig jejunum.
2. Materials and methods
Histamine dihydrochloride was purchased from Sigma Chemical Co. (St. Louis, USA). 2-Pyridylethylamine was obtained from Janssen Chin&a (Beerse, Belgium). VUF 8961 t2-(3-ethox~henyl}~st~ne dihydrochloride) was from laboratory stock. [ 3H]Mepyramine and [3H]myo-inositol were obtained from Amersham (Bu~~n~arns~re, UK). 2.2, Contraction
Correspondence to: R. Leurs, Department of P~armac~hemist~. Faculty of Chemistry. Vrije Universiteit, De Boelelaan 1081, 1083 HV Amslwdam, The Netherlands.
measurements
Male guin~-pigs (350450 g) were killed by a blow on the head. Jejunal Iongitudinal smooth muscle was carefully separated from the circular muscle. Contrac-
t~~~~s we= ~~?eas~r~ as desc~bed pre~fiously (Leurs et After dose-response curves to histamine and were obtained muscle strips were subseensitized for 5 min with 100 PM histamine tand aE!owed to recover for 20 min. Dose-response curves for the twr, agonists were then recorded again. The ~~~tr~ctio~s in response to the partial agonist, VUF 1, were expressed as a percentage of the maximal ~sta~~e contraption.
~~osito~ pb~phate production was measured as described by Watson et al. (1988). Smooth muscle slices were treated with 1 mM 2-pyridylethylamine for 15 . washed for 10 min and incubated with the indicated agents for 30 min at 37OC. The reaction was tenanted by the addition of 1 ml chloroform/meth~o~/~~~ (~~/~~/~. v/v). and equal volumes of water and chloroform were then added. The total inositol phospbat~ were obtained from the aqeous layer in a 10 ml fraction of 1 M HCI after sequential anion exchange chromatography with 10 ml water and 10 ml 0.025 M ammonium formate. f “Hjlnositol incorporation in the phospbolipids was measured in samples from the c~orofo~ layer. This inco~oration was also used as a measure of the amount of intestinal tissue in each sample, as previously suggested by Watson et al. (19S8).
Desensitization of isolated longitudinal smooth muscle of guinea-pig jejunum with 100 CM ~st~ne for 5 min resulted in a marked desensitization of the H, receptor-mediated responses after 20 min of recovery (fig. 1A). This desensitization was reflected by a rightward shift of the dose-response curve for the full agonist, ~sta~ne, from 6.7 rt 0.1 to 6.1 + 0.1 (n = 8, P c 0.05). The maximal response of the smooth muscle strips to histamine was however not altered by this desensitization protocol. Treatment of the muscle strips with 100 CM histamine also affected the response to the partial agonist, VUF 8961. Under control conditions this age:tt contracted the longitudinal smooth muscle with a pD, vaiue of 4.7 + 0.1 (n = 7) and reached 52 f 5% of the maximal histamine contraction. After recovery for 20 min there was a loss of sensitivity to VUF 8961, resulting in a pD* value of 4.4 jr 0.1 (n = 7) and also in attenuation of the maximal response to VUF 8961 (fig. 1A). The maximal response of the desensitized tissues to VUF 8961 was reduced approximately 20% to 43 f 3% (n = 7, P < 0.05 compared to the control).
24. Receptor birzditzg studies Longitudinal smooth muscle slices were incubated for X5 tin with 100 FM histamine in Krebs buffer. After extensive washing of the tissue with ice-cold 50 mM ~a/K-phosphate buffer (pH = 7.4). the tissue was homogenized in the same buffer. [ “H]Mepyramine binding in membrane preparations was determined according to Donaldson and Hill (1985), with minor modifications as suggested by DeBlasi et al. (1989), and analyzed by non-linear regression.
Combining the results of both functional studies and radioligand binding studies provides a direct method for calculating the efficacy (e) of a full agonist. According to Stephenson (1956) agonist efficacy is defined as e = ([&,I + K,)/[EC-,I.
The data shown are means &-SE of several independent experiments. A P value of 0.05 (t-test) was regarded as the limit to indicate a significant differer?ce.
-8.5 -7.5 -6.5 -5.5 -4.5 -3.5 -2.5 loglagonistj
basal
PEA
NaF
Fig. 1. (A) Dose-response curves for histamine (square} and VUF 8961 (circle) in control (open) or desensitized (closed, 100 PM histamine for 5 min) smooth muscle sitips of guinea-pig jejunum. The data shown are means+S.E. of seven or eight experiments. (B) Total inositol phosphate production in smooth mu&e slices from guinea-pig jejunum under control (open columns) or desensitized (hatched columns. 1 mM PEA for 15 min) conditionsSlices were stimulated with 100 pM 2-pyridylethylamine (PEA) or 10 mM NaF for 30 min. The data shown are the means f S.E. of six experiments. The basal accumulation of inositd phosphates was 204227 dpm/lO~ dpm in the lipid fraction. The asterisk indjcato., a significant difference from control conditions (P < 0.05).
321
The full H, receptor agonist, Z-pyridylethyla~ne, was used instead of histamine for the measurement of inositol phosphate production, because it has been reported that the inositol phosphate response to histamine in the intestine is obscured by a non-H, receptor component, whereas the stimulation of inositol phosphate production by 2-pyridylethyla~ne has been shown to reflect H, receptor activation of inositol phosphate production (Donaldson and Hill, 1986). Desensitization of muscle strips with 1 mM of 2-pyridylethylamine for 15 min also resulted in a rightward shift of the dose response curve for histamine. After 20 min washing the pD, value for histamine was reduced from 6.8 + 0.1 (control, n = 4) to 6.2 4 0.1 (desensitized, n = 4). As can be seen in fig. 1B desensitization of the jejunal slices with 1 mM of Zpyridylethylamine for 15 min did not affect the basal accumulation of inositol phoshates. H, receptor responsiveness was however markedly reduced after desensitization (fig. 1B). Under control conditions, 100 PM 2-pyridylethylamine increased inositol phosphate production to 206 rfi 18% (n = 6) of the basal value. After desensitization with 2-pyridylethylamine the response to 100 pM 2-py~dylethyla~~e was only 131 f 9% (n = 6) of the basal level. Breakdown of the phosphoinositides could also be stimulated by NaF, a modulator of G-proteins. At a concentration of 10 mM NaF the inositol phosphate levels are stimulated to 314 + 38% (n = 6) of the basal accumulation. Desensitization of the tissue with 2-py~dylethylamine did not reduce the response to NaF (fig. 1B). Finally, [ 3H]mepyramine binding in membrane preparations of the longitudinal smooth muscle of guineapig jejunum was determined. Desensitization of the smooth muscle slices with 100 FM histamine for 15 min did not attenuate the binding characteristics for mepyramine (table 1). Desensitization affected neither the number of H, receptors, nor the affinity of the H, antagonist (table 1). Using non-linear regression the various experiments on control and desensitized longitudinal smooth muscle could be fitted simultaneously, resulting in an affinity of 7 f 1 nM (n = 8) for mepyramine. The binding characteristics for the agonist, TABLE
1
Binding characteristics of histamine and mepyramine for the H, receptor of the longitudinal smooth muscle of guinea-pig jejunum under control conditions or after desensitisation with IOG FM histamine for 15 min.
~e~yrumjfle K, @Ml B (fmol/mg H~s%nine Ki (PM) Efficacy a P e 0.05
protein)
Contnb!
Desensitized
n
8.2* 2.2 373 *51
5.6+ 1.8 411 *39
4 4
55 291 compared to control,
f 5 f44
50 6-l
rt 6 f9”
4 4
~sta~ne, were also not attenuated after deserrsitization (table 1). The K, values for histamine and the EC, values from the contraction studies were used for a direct calculation of the efficacy of histamine (see Materials and methods). Based on these results it appears that the efficacy of histamine was si~fi~tiy reduced after the desensitization with 100 FM histamine (table 1).
4. Discussion Homologous desensitization of histamine H, receptor responses is observed in several experimental models, including the guinea-pig intestine (Kenakin and Cook, 1981; Leurs et al., 1990). Such an exclusive modulation of the receptor response indicates a modification of the H, receptor as the basic mechanism for the loss of receptor responsiveness. Reduction of agonist affinity, reduced capacity of the agonist to stimulate the occupied receptor or loss of H, receptors are all possible explanations for the development of homologous desensit~ation of H, receptor function_ Desensitization of longitudinal smooth muscle strips for 5 min with 100 FM histamine clearly attenuated subsequent contractions due to the full agonist, histamine, and to the partial agonist, VUF 8961. Not only muscle contraction but the H, receptor-mediated production of inositol phosphates in guinea-pig small intestine is also inhibited by previous desensitization. Under control conditions stimulation of the H, receptor with a submaximal concentration of 2-pyridylethylamine leads to a 106% increase above basal values. However H, receptor stimulation only leads to a 31% increase above basal levels after desensitization. Inositol phosphate production via G-protein activation with NaF is not attenuated after prolonged stimulation of the H, receptor. These data are completely compatible with the occurrence of homologous desensitization and suggest modulation at the H, receptor after prolonged agonist stimulation. Since there is a vast surplus of spare receptors for histamine in the guinea-pig intestine (Kenakin and Cook, 1981; Donaldson and Hill, 1985) both a reduction of rhe number of H, receptors or a reduction of agonist affinity might be reasonable explanations for the obsewed loss of agonist responsiveness. Using a partial aponist it was attempted to distinguish between these two possibilities for the desensitization of the H, receptor-mediated contractions. However, contractions in response to VUF 8961 are influenced in two ways. Both redu:*tion of agonist sensitivity and in~bition of the maximal response were found after desensitization with histardne. These findings might indicate that the I-I, receptor desensitization is the result of both a reduction of receptor number, leading to a reduction @f the
~~~~~~a~ response to VUF 8961, and a loss of agonist a~~~n~t~,resulting in a shift of the dose-response curve. Nevertheless. Haenen et al. (1990). who previously applied the receptor theory of Stephenson (1956) to similar problems. reported that dual modulation of the d-response curve of a partial agonist might also be related to a reduction of the ability of the agonist to stimtdate the receptor after receptor occupation {agonist efficacy). [~~~~ep~~ne b‘md mg studies were performed to try and distinguish between the various explanations. The [“H~mepyramine binding characteristics appeared not to be altered after H, receptor desensitization. Both the affinity and the maximal binding capacity for mepyramine were unchanged. Reduction of H, receptor number was therefore not responsible for the observed loss of responsiveness. Moreover, the affinity of histamine for the H, receptor also was not affected by the desensitization. The Ki values observed correspond with previously reported data (Donaldson and Hill, 1985). Efficacy values for ~st~ne also can be calculated based OII these Ki vahzes and on EC,, values from the contraction experiments. It was now shown that agonist efficacy is markedly reduced as a consequence of homologous H, receptor desensitization. In conclusion, combining the results of both functianaf and radioligand binding studies, the rapid loss of H, receptor responsiveness in the guinea-pig small intestine was shown to lead to reduction of agonist efficacy. The observed findings can be explained by modu&ion of the receptor protein. It is not yet known how the HI receptor is modulated or by which mech~sm the receptor protein is modulated after homologous
desensitization, although a role for protein kinase C has already been reported to be unlikely (Leurs et al., 1990).
References DeBlasi. A., K. G’Reilly and H.J. Motulsky, 1989, Calculating receptor number from binding experiments using the same compound as mdioligand and competitor, Trends Pharmacol. Sci. 10.227. Donaldson, J. and S.J. Hill, 1985, Hista~5induc~ inositol phospholipids breakdown in the lon~tudinal smooth muscle of guineapig ileum, Br. J. Pharmacol. 85, 499. Donaldson, J. and S.J. Hill, 1986, Histamine-induced hydrolysis of polyphosphoinositides in guinea-pig ileum and brain, European J. Phartnacol. 124, 255. Fox, A.W. and P.A. Friedman, 1987, Evidence for spare a,-adrenoceptors for the accumulation of inositoi phosphates in smooth muscle, J. Pharrn. Pharmacol. 39, 68. Graig, D.A. and D.E. Clarke, 1994 Pharmacological characterization of a neuronal receptor for 5-hydroxytryptamine in guinea-pig ileum with properties similar to the 5-hydroxytryptamine, receptor, J. Pharmacol. Exp. Ther. 252,1378. Haenen, G.R.M.M., M. Veerman and A. Bast, 1990, Reduction of &adrenoceptor function by oxidative stress in the heart, Free Rad. Biol. Med. 9, 279. Kenakin, T.P. and D.A. Cook, 1981. The effect of desensitization on the antagonism of the histamine response by phenoxybenzatnine, Mol. Pharrnacol. 17, 309. Leurs, R., M.J. Stnit, A. Bast and H. Timmennan, 1990, Different profiles of desensitization dynamics in guinea-pig jejunal longitudinal smooth muscle after stimulation with histamine and metacholine, Br. J. Pharmacol. 101, 881. Stephenson, R.P., 1956, A modification of receptor theory, Br. J. Pharmacol. 11, 379. Watson, S.P., A.F. Stanley and T. Sasaguri, 1988, Does the hyd~ly~s of inositol pho~hofipi~ lead to the opening of voftage operated Ca2+ channels in guinea-pig ileum? Studies with Ruoride ions and caffeine, B&hem. Biophys. Res. Connnun. 153, 14.
319
EJP 20815
Short c~mwni~tion
omologous histamine
sensitization Fe receptor egotist ~~~~~a~
Rob Leurs, Martine J. Smit, Aalt Bast and Hendrik Timmerman Departmen! of Pharmacochemistry. Faculty ojchemisttp, Vr@ Uniuersiteit, De Boelelaan 1081,1083 HV Ansterabn,
The NetherIan&
Received 26 February 1991, accepted 5 Mar& 1991
Prolonged exposure of the guinea-pig intestinal ion~tudinal smooth muscle to histamine caused homologous de~asit~~on of the H, receptor, which led to reduced H, receptor-mediated production of [3H]inositol phosphates as well as to reduced H, agonist-induced contractions. [3H]Mepyramine binding studies showed that desensitization affected neither the agonist affinity nor the number of H, receptors. Combining the data from the binding studies and the contraction measurements it was found that d~ensitization results in a selective reduction of agonist efficacy. Intestine (guinea-pig); Histamine H, receptors; Desensitization; [ ‘H]Mepyramine; [ 3H]Inositol phosphates; Agonist efficacy
1. Introduction Desensitization of receptor responses is frequently observed in experiments on histamine H, receptor responsiveness (Kenakin and Cook, 1981; Leurs et al., 1990). Previously we noticed a very rapid desensitization of H, receptor responses of the longitudinal smooth muscle of guinea-pig jejunum (Leurs et al., 1990). After a few minutes of desensitization the contractions in response to histamine are initially strongly inhibited. These responses recover slowly and do not reach their initial control levels within a few hours (Leurs et al., 1990). The desensitization with histamine does not attenuate methacholine-induced responses and therefore appears to be homologous (Leurs et al., 1990). These data indicate modulation of signal transduction at the level of the H, receptor. Various modifications of the H, receptor might however affect H, receptor activity. Modulation of agonist binding characteristics, effects on receptor-G-protein coupling or a reduction of receptor number can all exclusively modulate H, receptor function, leading to homologous desensitization. Kenakin and Cook (1981) previously used the irreversible antagonist, phenoxybenzamine, to elucidate the mechanism underlying intestinal H, receptor desensitization. The results of their investigations suggested that the desensitization ob-
served was due to a conformational alteration of the H, receptor, leading to changed binding characteristics (Kenakin and Cook, 1981). Phenoxybenzamine is however not very specific for the H, receptor and might not be appropiate for the study of H, agonist activity (Fox and Friedman, 1987; Graig and Clarke, 1990). In the present study on the mechanism of the rapid H, receptor desensitization we combined studies on contraction with histamine and a partial H, receptor agonist with studies on [3H]mepyramine binding and measurements of the H, receptor-mediated breakdown of the phosphoinositides in -the longitudinal smooth muscle of guinea-pig jejunum.
2. Materials and methods
Histamine dihydrochloride was purchased from Sigma Chemical Co. (St. Louis, USA). 2-Pyridylethylamine was obtained from Janssen Chin&a (Beerse, Belgium). VUF 8961 t2-(3-ethox~henyl}~st~ne dihydrochloride) was from laboratory stock. [ 3H]Mepyramine and [3H]myo-inositol were obtained from Amersham (Bu~~n~arns~re, UK). 2.2, Contraction
Correspondence to: R. Leurs, Department of P~armac~hemist~. Faculty of Chemistry. Vrije Universiteit, De Boelelaan 1081, 1083 HV Amslwdam, The Netherlands.
measurements
Male guin~-pigs (350450 g) were killed by a blow on the head. Jejunal Iongitudinal smooth muscle was carefully separated from the circular muscle. Contrac-
t~~~~s we= ~~?eas~r~ as desc~bed pre~fiously (Leurs et After dose-response curves to histamine and were obtained muscle strips were subseensitized for 5 min with 100 PM histamine tand aE!owed to recover for 20 min. Dose-response curves for the twr, agonists were then recorded again. The ~~~tr~ctio~s in response to the partial agonist, VUF 1, were expressed as a percentage of the maximal ~sta~~e contraption.
~~osito~ pb~phate production was measured as described by Watson et al. (1988). Smooth muscle slices were treated with 1 mM 2-pyridylethylamine for 15 . washed for 10 min and incubated with the indicated agents for 30 min at 37OC. The reaction was tenanted by the addition of 1 ml chloroform/meth~o~/~~~ (~~/~~/~. v/v). and equal volumes of water and chloroform were then added. The total inositol phospbat~ were obtained from the aqeous layer in a 10 ml fraction of 1 M HCI after sequential anion exchange chromatography with 10 ml water and 10 ml 0.025 M ammonium formate. f “Hjlnositol incorporation in the phospbolipids was measured in samples from the c~orofo~ layer. This inco~oration was also used as a measure of the amount of intestinal tissue in each sample, as previously suggested by Watson et al. (19S8).
Desensitization of isolated longitudinal smooth muscle of guinea-pig jejunum with 100 CM ~st~ne for 5 min resulted in a marked desensitization of the H, receptor-mediated responses after 20 min of recovery (fig. 1A). This desensitization was reflected by a rightward shift of the dose-response curve for the full agonist, ~sta~ne, from 6.7 rt 0.1 to 6.1 + 0.1 (n = 8, P c 0.05). The maximal response of the smooth muscle strips to histamine was however not altered by this desensitization protocol. Treatment of the muscle strips with 100 CM histamine also affected the response to the partial agonist, VUF 8961. Under control conditions this age:tt contracted the longitudinal smooth muscle with a pD, vaiue of 4.7 + 0.1 (n = 7) and reached 52 f 5% of the maximal histamine contraction. After recovery for 20 min there was a loss of sensitivity to VUF 8961, resulting in a pD* value of 4.4 jr 0.1 (n = 7) and also in attenuation of the maximal response to VUF 8961 (fig. 1A). The maximal response of the desensitized tissues to VUF 8961 was reduced approximately 20% to 43 f 3% (n = 7, P < 0.05 compared to the control).
24. Receptor birzditzg studies Longitudinal smooth muscle slices were incubated for X5 tin with 100 FM histamine in Krebs buffer. After extensive washing of the tissue with ice-cold 50 mM ~a/K-phosphate buffer (pH = 7.4). the tissue was homogenized in the same buffer. [ “H]Mepyramine binding in membrane preparations was determined according to Donaldson and Hill (1985), with minor modifications as suggested by DeBlasi et al. (1989), and analyzed by non-linear regression.
Combining the results of both functional studies and radioligand binding studies provides a direct method for calculating the efficacy (e) of a full agonist. According to Stephenson (1956) agonist efficacy is defined as e = ([&,I + K,)/[EC-,I.
The data shown are means &-SE of several independent experiments. A P value of 0.05 (t-test) was regarded as the limit to indicate a significant differer?ce.
-8.5 -7.5 -6.5 -5.5 -4.5 -3.5 -2.5 loglagonistj
basal
PEA
NaF
Fig. 1. (A) Dose-response curves for histamine (square} and VUF 8961 (circle) in control (open) or desensitized (closed, 100 PM histamine for 5 min) smooth muscle sitips of guinea-pig jejunum. The data shown are means+S.E. of seven or eight experiments. (B) Total inositol phosphate production in smooth mu&e slices from guinea-pig jejunum under control (open columns) or desensitized (hatched columns. 1 mM PEA for 15 min) conditionsSlices were stimulated with 100 pM 2-pyridylethylamine (PEA) or 10 mM NaF for 30 min. The data shown are the means f S.E. of six experiments. The basal accumulation of inositd phosphates was 204227 dpm/lO~ dpm in the lipid fraction. The asterisk indjcato., a significant difference from control conditions (P < 0.05).
321
The full H, receptor agonist, Z-pyridylethyla~ne, was used instead of histamine for the measurement of inositol phosphate production, because it has been reported that the inositol phosphate response to histamine in the intestine is obscured by a non-H, receptor component, whereas the stimulation of inositol phosphate production by 2-pyridylethyla~ne has been shown to reflect H, receptor activation of inositol phosphate production (Donaldson and Hill, 1986). Desensitization of muscle strips with 1 mM of 2-pyridylethylamine for 15 min also resulted in a rightward shift of the dose response curve for histamine. After 20 min washing the pD, value for histamine was reduced from 6.8 + 0.1 (control, n = 4) to 6.2 4 0.1 (desensitized, n = 4). As can be seen in fig. 1B desensitization of the jejunal slices with 1 mM of Zpyridylethylamine for 15 min did not affect the basal accumulation of inositol phoshates. H, receptor responsiveness was however markedly reduced after desensitization (fig. 1B). Under control conditions, 100 PM 2-pyridylethylamine increased inositol phosphate production to 206 rfi 18% (n = 6) of the basal value. After desensitization with 2-pyridylethylamine the response to 100 pM 2-py~dylethyla~~e was only 131 f 9% (n = 6) of the basal level. Breakdown of the phosphoinositides could also be stimulated by NaF, a modulator of G-proteins. At a concentration of 10 mM NaF the inositol phosphate levels are stimulated to 314 + 38% (n = 6) of the basal accumulation. Desensitization of the tissue with 2-py~dylethylamine did not reduce the response to NaF (fig. 1B). Finally, [ 3H]mepyramine binding in membrane preparations of the longitudinal smooth muscle of guineapig jejunum was determined. Desensitization of the smooth muscle slices with 100 FM histamine for 15 min did not attenuate the binding characteristics for mepyramine (table 1). Desensitization affected neither the number of H, receptors, nor the affinity of the H, antagonist (table 1). Using non-linear regression the various experiments on control and desensitized longitudinal smooth muscle could be fitted simultaneously, resulting in an affinity of 7 f 1 nM (n = 8) for mepyramine. The binding characteristics for the agonist, TABLE
1
Binding characteristics of histamine and mepyramine for the H, receptor of the longitudinal smooth muscle of guinea-pig jejunum under control conditions or after desensitisation with IOG FM histamine for 15 min.
~e~yrumjfle K, @Ml B (fmol/mg H~s%nine Ki (PM) Efficacy a P e 0.05
protein)
Contnb!
Desensitized
n
8.2* 2.2 373 *51
5.6+ 1.8 411 *39
4 4
55 291 compared to control,
f 5 f44
50 6-l
rt 6 f9”
4 4
~sta~ne, were also not attenuated after deserrsitization (table 1). The K, values for histamine and the EC, values from the contraction studies were used for a direct calculation of the efficacy of histamine (see Materials and methods). Based on these results it appears that the efficacy of histamine was si~fi~tiy reduced after the desensitization with 100 FM histamine (table 1).
4. Discussion Homologous desensitization of histamine H, receptor responses is observed in several experimental models, including the guinea-pig intestine (Kenakin and Cook, 1981; Leurs et al., 1990). Such an exclusive modulation of the receptor response indicates a modification of the H, receptor as the basic mechanism for the loss of receptor responsiveness. Reduction of agonist affinity, reduced capacity of the agonist to stimulate the occupied receptor or loss of H, receptors are all possible explanations for the development of homologous desensit~ation of H, receptor function_ Desensitization of longitudinal smooth muscle strips for 5 min with 100 FM histamine clearly attenuated subsequent contractions due to the full agonist, histamine, and to the partial agonist, VUF 8961. Not only muscle contraction but the H, receptor-mediated production of inositol phosphates in guinea-pig small intestine is also inhibited by previous desensitization. Under control conditions stimulation of the H, receptor with a submaximal concentration of 2-pyridylethylamine leads to a 106% increase above basal values. However H, receptor stimulation only leads to a 31% increase above basal levels after desensitization. Inositol phosphate production via G-protein activation with NaF is not attenuated after prolonged stimulation of the H, receptor. These data are completely compatible with the occurrence of homologous desensitization and suggest modulation at the H, receptor after prolonged agonist stimulation. Since there is a vast surplus of spare receptors for histamine in the guinea-pig intestine (Kenakin and Cook, 1981; Donaldson and Hill, 1985) both a reduction of rhe number of H, receptors or a reduction of agonist affinity might be reasonable explanations for the obsewed loss of agonist responsiveness. Using a partial aponist it was attempted to distinguish between these two possibilities for the desensitization of the H, receptor-mediated contractions. However, contractions in response to VUF 8961 are influenced in two ways. Both redu:*tion of agonist sensitivity and in~bition of the maximal response were found after desensitization with histardne. These findings might indicate that the I-I, receptor desensitization is the result of both a reduction of receptor number, leading to a reduction @f the
~~~~~~a~ response to VUF 8961, and a loss of agonist a~~~n~t~,resulting in a shift of the dose-response curve. Nevertheless. Haenen et al. (1990). who previously applied the receptor theory of Stephenson (1956) to similar problems. reported that dual modulation of the d-response curve of a partial agonist might also be related to a reduction of the ability of the agonist to stimtdate the receptor after receptor occupation {agonist efficacy). [~~~~ep~~ne b‘md mg studies were performed to try and distinguish between the various explanations. The [“H~mepyramine binding characteristics appeared not to be altered after H, receptor desensitization. Both the affinity and the maximal binding capacity for mepyramine were unchanged. Reduction of H, receptor number was therefore not responsible for the observed loss of responsiveness. Moreover, the affinity of histamine for the H, receptor also was not affected by the desensitization. The Ki values observed correspond with previously reported data (Donaldson and Hill, 1985). Efficacy values for ~st~ne also can be calculated based OII these Ki vahzes and on EC,, values from the contraction experiments. It was now shown that agonist efficacy is markedly reduced as a consequence of homologous H, receptor desensitization. In conclusion, combining the results of both functianaf and radioligand binding studies, the rapid loss of H, receptor responsiveness in the guinea-pig small intestine was shown to lead to reduction of agonist efficacy. The observed findings can be explained by modu&ion of the receptor protein. It is not yet known how the HI receptor is modulated or by which mech~sm the receptor protein is modulated after homologous
desensitization, although a role for protein kinase C has already been reported to be unlikely (Leurs et al., 1990).
References DeBlasi. A., K. G’Reilly and H.J. Motulsky, 1989, Calculating receptor number from binding experiments using the same compound as mdioligand and competitor, Trends Pharmacol. Sci. 10.227. Donaldson, J. and S.J. Hill, 1985, Hista~5induc~ inositol phospholipids breakdown in the lon~tudinal smooth muscle of guineapig ileum, Br. J. Pharmacol. 85, 499. Donaldson, J. and S.J. Hill, 1986, Histamine-induced hydrolysis of polyphosphoinositides in guinea-pig ileum and brain, European J. Phartnacol. 124, 255. Fox, A.W. and P.A. Friedman, 1987, Evidence for spare a,-adrenoceptors for the accumulation of inositoi phosphates in smooth muscle, J. Pharrn. Pharmacol. 39, 68. Graig, D.A. and D.E. Clarke, 1994 Pharmacological characterization of a neuronal receptor for 5-hydroxytryptamine in guinea-pig ileum with properties similar to the 5-hydroxytryptamine, receptor, J. Pharmacol. Exp. Ther. 252,1378. Haenen, G.R.M.M., M. Veerman and A. Bast, 1990, Reduction of &adrenoceptor function by oxidative stress in the heart, Free Rad. Biol. Med. 9, 279. Kenakin, T.P. and D.A. Cook, 1981. The effect of desensitization on the antagonism of the histamine response by phenoxybenzatnine, Mol. Pharrnacol. 17, 309. Leurs, R., M.J. Stnit, A. Bast and H. Timmennan, 1990, Different profiles of desensitization dynamics in guinea-pig jejunal longitudinal smooth muscle after stimulation with histamine and metacholine, Br. J. Pharmacol. 101, 881. Stephenson, R.P., 1956, A modification of receptor theory, Br. J. Pharmacol. 11, 379. Watson, S.P., A.F. Stanley and T. Sasaguri, 1988, Does the hyd~ly~s of inositol pho~hofipi~ lead to the opening of voftage operated Ca2+ channels in guinea-pig ileum? Studies with Ruoride ions and caffeine, B&hem. Biophys. Res. Connnun. 153, 14.