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Studies on the muscarinic receptor subtype in chick ileum
Vol. 60, Nos. 13/14,1997
1191
Abstracts
58 STUDIES ON THE MUSCARINIC
RECEPTOR
SUBTYPE IN CHICK ILEUM.
S. Darroch, H. Irving and F.Mitchelson. Department of Pharmaceutical Biology and Pharmacology, Victorian College of Pharmacy (Monash University), Melbourne, Australia The muscarinic receptor initiating contraction in the chick ileum was previously suggested to be a fimctional correlate of the m5 gene product (Darroch & Mitchelson 1995). Functional studies using carbachol as agonist revealed antagonist affinity profiles with a high correlation to those reported for the cloned m5 receptor. Evaluation of the receptor subtype present in the ileum has been extended to binding and molecular analysis studies. Homogenates of whole ileum were prepared in 5OmM Na phosphate buffer (pH 7.4) for binding studies at 37’C. Binding of [3H]-QNB revealed a Kn value of 0.158 * 0.063 nM with a B ,,,= of 419.4 f 39.7 fmol/mg of protein. Pirenzepine and himbacine were found to displace [3H]-QNB binding from a single site with pKr values of 6.85 and 7.24 respectively. p-Fluorohexahydrosiladifenidol also displaced from a single site with a pKr value of 6.46. These profiles support functional studies that suggested the receptor has a different affinity profile to mammalian MI, Ma, M3 or M receptors. Complementary molecular studies are being undertaken. Darroch S and Mitchelson F, (1995) Life Sci, 56, abstract 78
59 INVOLVEMENT OF MUSCARINIC ACETYLCHOLINE SMOOTH MUSCLE OF THE TAENIA CAECI
Mz AND
MS RECEPTORS
A. Shen and F. Mitchelson, Dept. of Pharmaceutical Biology and Pharmacology, of Pharmacy (Monash University), Melbourne, Australia 3052
IN
Victorian College
In the guinea-pig taenia caeci, as in the ileum, there is a preponderance of muscarinic acetylcholine M2 receptors which are coupled to inhibition of adenylyl cyclase, although Ms receptor activation produces the contractile response. While Mj receptors are coupled to the phosphatidylinositol (PI) pathway, there is also evidence for tyrosine kinase involvement. Genistein (20 pM), an inhibitor of tyrosine kinase, caused ca 30-fold rightwards shift of the concentration-response curve to acetylcholine. Histamine curves were also shifted by genistein ca lo-fold, but curves for K’ or Ba*’ were only shifted 2 to 3-fold, suggesting a selective action of genistein on acetylcholine and histamine. Studies were also conducted to evaluate the role of M2 receptors in the taenia which differs from the iteum in possessing a high level of intrinsic tone. M3 receptor inactivation by 4DAMP mustard in the presence of methoctramine to protect MI receptors, was performed on the taenia. Treatment of the taenia with 4-DAMP mustard (60 nM) plus methoctramine (1 @I) for 1 hour led to a 20-fold shift in the concentration-response curve for acetylcholine. Following repeated washing, the tissue was either treated with isoprenaline or isoprenaline plus histamine. Methoctramine (0.1 pM) produced negligible shift of the concentration-response curve to acetylcholine in the presence of isoprenaline alone. However, methoctramine in the presence of histamine plus isoprenaline, shifted the curve for the neurotransmitter with an apparent pKa of 7.3, suggesting histamine was integral to the involvement of Mz receptors in the contractile response after M, receptor inactivation.
1191
Abstracts
58 STUDIES ON THE MUSCARINIC
RECEPTOR
SUBTYPE IN CHICK ILEUM.
S. Darroch, H. Irving and F.Mitchelson. Department of Pharmaceutical Biology and Pharmacology, Victorian College of Pharmacy (Monash University), Melbourne, Australia The muscarinic receptor initiating contraction in the chick ileum was previously suggested to be a fimctional correlate of the m5 gene product (Darroch & Mitchelson 1995). Functional studies using carbachol as agonist revealed antagonist affinity profiles with a high correlation to those reported for the cloned m5 receptor. Evaluation of the receptor subtype present in the ileum has been extended to binding and molecular analysis studies. Homogenates of whole ileum were prepared in 5OmM Na phosphate buffer (pH 7.4) for binding studies at 37’C. Binding of [3H]-QNB revealed a Kn value of 0.158 * 0.063 nM with a B ,,,= of 419.4 f 39.7 fmol/mg of protein. Pirenzepine and himbacine were found to displace [3H]-QNB binding from a single site with pKr values of 6.85 and 7.24 respectively. p-Fluorohexahydrosiladifenidol also displaced from a single site with a pKr value of 6.46. These profiles support functional studies that suggested the receptor has a different affinity profile to mammalian MI, Ma, M3 or M receptors. Complementary molecular studies are being undertaken. Darroch S and Mitchelson F, (1995) Life Sci, 56, abstract 78
59 INVOLVEMENT OF MUSCARINIC ACETYLCHOLINE SMOOTH MUSCLE OF THE TAENIA CAECI
Mz AND
MS RECEPTORS
A. Shen and F. Mitchelson, Dept. of Pharmaceutical Biology and Pharmacology, of Pharmacy (Monash University), Melbourne, Australia 3052
IN
Victorian College
In the guinea-pig taenia caeci, as in the ileum, there is a preponderance of muscarinic acetylcholine M2 receptors which are coupled to inhibition of adenylyl cyclase, although Ms receptor activation produces the contractile response. While Mj receptors are coupled to the phosphatidylinositol (PI) pathway, there is also evidence for tyrosine kinase involvement. Genistein (20 pM), an inhibitor of tyrosine kinase, caused ca 30-fold rightwards shift of the concentration-response curve to acetylcholine. Histamine curves were also shifted by genistein ca lo-fold, but curves for K’ or Ba*’ were only shifted 2 to 3-fold, suggesting a selective action of genistein on acetylcholine and histamine. Studies were also conducted to evaluate the role of M2 receptors in the taenia which differs from the iteum in possessing a high level of intrinsic tone. M3 receptor inactivation by 4DAMP mustard in the presence of methoctramine to protect MI receptors, was performed on the taenia. Treatment of the taenia with 4-DAMP mustard (60 nM) plus methoctramine (1 @I) for 1 hour led to a 20-fold shift in the concentration-response curve for acetylcholine. Following repeated washing, the tissue was either treated with isoprenaline or isoprenaline plus histamine. Methoctramine (0.1 pM) produced negligible shift of the concentration-response curve to acetylcholine in the presence of isoprenaline alone. However, methoctramine in the presence of histamine plus isoprenaline, shifted the curve for the neurotransmitter with an apparent pKa of 7.3, suggesting histamine was integral to the involvement of Mz receptors in the contractile response after M, receptor inactivation.