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Acetylcholine receptor (ACHR) metabolic stability in myotubes is epsilon and gamma subunits indipendent
CROSS
TALK
BETWEEN
M2-MUSCARINIC
AND
BETA-2-
ACETYLCHOLINE RECEPTOR (ACHR) METABOLIC STABILITY IN MYOTUBES IS EPSILON AND GAMMA SUBUNITS INDIPENDENT Carlo Sala and *Guido Fumagalli j Department of Medical Pharmacology, University of Milano and *Institute of Pharmacology, University of Verona, Italy.
ADRENERGIC RECEPTORS IN HEL 299 CELLS. J. Rousell, E-B. Haddad, B. L. J. Webb, M. A, Giembycz J. C. W, Mak and P. J. Barnes. Department of Thoracic Medicine, National Heart and Lung institute, London SW3 6L Y.
In cultured muscle cells, two types of AChR can be distinguished by their degradation rates (O'Malley et at., Exp Cell Res 208, 44-47, 1993): most of the receptor degrades rapidly (called Rr, tl/2=l d) and approximately 10% of the molecules degrade slowly (Rs,tl/2=~3 d). R s degradation rate is reversibly modulated between 3 and 10 d by cAMP. A similar situation exists at neuromuscular junctions (nmj): Rs is predominant in innervated nmj and Rr appears after denervation and replaces Rs. The coexistence at a same endplate and the different sensitivity to modulatory events suggest that Rr and R s are two distinct molecular entities. Muscle AChRs contain either 7 or ~ subunits: the second is present in innervated nmj, the first appears after denervation. With this study we are testing the hypothesis that the differences in degradation rates are due to the presence of e subunit in the Rs molecule. After labelling superficial AChR molecules of new-bum rat myetubes in culture with 125I-o~-bungarotoxin (125I-BGT),' E subunit was detectable by immunoprecipimtion with subunit specific mabs starting from day 5 of culture and reached a maximum level of -10% of total superficial AChR at day 9. To measure the contribution of the ~ subunit-containing AChR to the Rs population, myotubes were labelled at day 9 of culture with 125I-BGT. Degradation rates were then calculated from the release of radioactivity in the medium; using ~ subunit-specific mabs and polyclenal anti-AChR antibodies, we calculated the fraction of e subunit-containing AChR remaining on myotubes surface at various time points after labelling. T1/2 were ld in most and 3.5 d in 10.5+_0.8% of AChRs. On the other hand, E subunit-containing receptors were -10% of total AChR at all the time point tested (0, 4 and 6 days after labelling with 125I-BGT) indicating that this subunit is present in both R s and Rr. Incubation of cells with 1 mM dbcAMP shifted Rs degradation rate to 5,5 d but did not change the e/total AChR ratio measured 0 and 4 d after toxin labelling. These results and the late appearance of e subunit strongly suggest that this subunit is not responsible for the metabolic differences between Rr and R s.
Cross talk was investigated between ~2-adrenergic ([52AR) and muscarinic M2 receptors. Stimulation of ~2AR receptors with procaterol (5 ~M) caused a biphasic decrease in M2-muscarinic receptor binding (40 and 50% after 0.5 and 24 hrs respectively) and could be mimicked with the cAMP analogue 8-Br-cAMP (5 mM) and forskolin (50 ~M). Recovery to control levels after 2 hrs procaterol treatment did not require de novo protein synthesis as cycloheximide had no effect on this process. Procaterol treatments also resulted in a transient increase in m2 mRNA levels. While 24 hr procaterol and 8-Br-cAMP treatments had no effect on m2-mRNA levels a 50% reduction was seen following forskolin stimulation. Procaterol-induced down-regulation was blocked by the PKC inhibitor GF 109203X but not PKA or tyrosine kinase inhibitors. Accumulation of cAMP with procaterol could be significantly inhibited (50-60%) by carbachol (15 rain-2 hrs) but was reduced following 24 hr stimulations with procaterol to 15% indicating uncoupling of the receptor. Therefore, cross talk between M2muscadnic and ~2-AR receptors can be demonstrated in HEL 299 cells, This work was funded by the Medical Research Council, UK.
Effect of nitrendipine on i n v i v o - induced desensitization of ~]-adrenoceptors in rat lung P. P. Vassiley, N. Danchev*, D. Staneva-Stoytcheva Institute of Physiology, BulgarianAcademy of Sciences, Acad. G. Bonchev Str., bl. 23, 1113 Sofia * Faculty of Pharmacy, Medical Academy, Sofia
MECHANISM OF ESTROGEN RECEPTOR DOWNR E G U L A T I O N IN SK-ER3 N E U R O B L A S T O M A C E L L S . S. Santagati, G. Pellio, Z.Q Ma and A. Maggi Milano Molecular Pharmacology Lab, Institute o f Pharmacological Sciences, University o f Milan, Via Balzaretti 9, 20133 Milan, Italy Ligand-indueed down regulation o f membrane as well as intraeellular receptors has an important role in hormone action. However, the mechanisms involved in this process are still far to be c0mpletely clarified. The present study is aimed at the comprehension o f estrogen receptor down-regulation and stems from two separate Observations: a. down regulation o f the ER was observed in cells not physiologically expressing ER, but stably transfected with ER eDNA b. oligonucleotides representing selected regions o f the ER eDNA determined up regulation o f the ER (as shown by western analysis). On the basis o f these observations, we hypothesized that the ER itself, once linked to its cognate hormone, could interact with,the ER gene or mRNA and interfere with its own synthesis. This hypothesis was further supported by subsequent studies where we proved that ER binds with high affinity the oligos determining its up-regulation. This binding, as assessed by gel shift, occurs at high affinity with the double strand DNA, at lower affinity with single strand elinueleotides supporting the view o f ER binding to the DNA and not to the mRNA. Studies are in progress to better define the kinetics and specificity o f this binding.
The effect of nitrendipine on desensitization of ~adrenoceptors induced by isoprenaline was investigated using rat lungs. Rats were divided into four groups: (1) the control group, (2) the isoprenaline-treated group, (3) the nitrendipine-treated group, and (4) isoprenaline plus nitrendipine-treated group. After 7 days treatment the tracheas were isolated and dissociation constant (Kb) for the propranolol- ~-receptor complex was determined. The lung parenchymal t~ssue was used to study the binding characteristics of ~-adrenoceptors. The Kb value for the propranolol- ~-receptor complex was find to be up to 6,4-fold higher in the tracheas isolated from isoprenaline-treated rats (3.2x10 8 -" 5.4x10 9) than in tracheas isolated from control rats (4.9 *- 1.2x10-9). The K b in tracheas from isoprenaline plus nitrendipine-treated rats w a s 7.0-'1.2x10 -9. A 31% reduction in the number of ~-adrenoceptors as compared to those of the control group was observed in the isoprenaline treated group. The number of the ~-adrenoceptors in the isoprenaline plus nitrendipine-treated group was significantly increased as compared to that of the isoprenaline-treated group.
Acknowledgements This work was supported by the CNR, P.S. Antisense eligonucleotides ( A . M )
- -
203
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TALK
BETWEEN
M2-MUSCARINIC
AND
BETA-2-
ACETYLCHOLINE RECEPTOR (ACHR) METABOLIC STABILITY IN MYOTUBES IS EPSILON AND GAMMA SUBUNITS INDIPENDENT Carlo Sala and *Guido Fumagalli j Department of Medical Pharmacology, University of Milano and *Institute of Pharmacology, University of Verona, Italy.
ADRENERGIC RECEPTORS IN HEL 299 CELLS. J. Rousell, E-B. Haddad, B. L. J. Webb, M. A, Giembycz J. C. W, Mak and P. J. Barnes. Department of Thoracic Medicine, National Heart and Lung institute, London SW3 6L Y.
In cultured muscle cells, two types of AChR can be distinguished by their degradation rates (O'Malley et at., Exp Cell Res 208, 44-47, 1993): most of the receptor degrades rapidly (called Rr, tl/2=l d) and approximately 10% of the molecules degrade slowly (Rs,tl/2=~3 d). R s degradation rate is reversibly modulated between 3 and 10 d by cAMP. A similar situation exists at neuromuscular junctions (nmj): Rs is predominant in innervated nmj and Rr appears after denervation and replaces Rs. The coexistence at a same endplate and the different sensitivity to modulatory events suggest that Rr and R s are two distinct molecular entities. Muscle AChRs contain either 7 or ~ subunits: the second is present in innervated nmj, the first appears after denervation. With this study we are testing the hypothesis that the differences in degradation rates are due to the presence of e subunit in the Rs molecule. After labelling superficial AChR molecules of new-bum rat myetubes in culture with 125I-o~-bungarotoxin (125I-BGT),' E subunit was detectable by immunoprecipimtion with subunit specific mabs starting from day 5 of culture and reached a maximum level of -10% of total superficial AChR at day 9. To measure the contribution of the ~ subunit-containing AChR to the Rs population, myotubes were labelled at day 9 of culture with 125I-BGT. Degradation rates were then calculated from the release of radioactivity in the medium; using ~ subunit-specific mabs and polyclenal anti-AChR antibodies, we calculated the fraction of e subunit-containing AChR remaining on myotubes surface at various time points after labelling. T1/2 were ld in most and 3.5 d in 10.5+_0.8% of AChRs. On the other hand, E subunit-containing receptors were -10% of total AChR at all the time point tested (0, 4 and 6 days after labelling with 125I-BGT) indicating that this subunit is present in both R s and Rr. Incubation of cells with 1 mM dbcAMP shifted Rs degradation rate to 5,5 d but did not change the e/total AChR ratio measured 0 and 4 d after toxin labelling. These results and the late appearance of e subunit strongly suggest that this subunit is not responsible for the metabolic differences between Rr and R s.
Cross talk was investigated between ~2-adrenergic ([52AR) and muscarinic M2 receptors. Stimulation of ~2AR receptors with procaterol (5 ~M) caused a biphasic decrease in M2-muscarinic receptor binding (40 and 50% after 0.5 and 24 hrs respectively) and could be mimicked with the cAMP analogue 8-Br-cAMP (5 mM) and forskolin (50 ~M). Recovery to control levels after 2 hrs procaterol treatment did not require de novo protein synthesis as cycloheximide had no effect on this process. Procaterol treatments also resulted in a transient increase in m2 mRNA levels. While 24 hr procaterol and 8-Br-cAMP treatments had no effect on m2-mRNA levels a 50% reduction was seen following forskolin stimulation. Procaterol-induced down-regulation was blocked by the PKC inhibitor GF 109203X but not PKA or tyrosine kinase inhibitors. Accumulation of cAMP with procaterol could be significantly inhibited (50-60%) by carbachol (15 rain-2 hrs) but was reduced following 24 hr stimulations with procaterol to 15% indicating uncoupling of the receptor. Therefore, cross talk between M2muscadnic and ~2-AR receptors can be demonstrated in HEL 299 cells, This work was funded by the Medical Research Council, UK.
Effect of nitrendipine on i n v i v o - induced desensitization of ~]-adrenoceptors in rat lung P. P. Vassiley, N. Danchev*, D. Staneva-Stoytcheva Institute of Physiology, BulgarianAcademy of Sciences, Acad. G. Bonchev Str., bl. 23, 1113 Sofia * Faculty of Pharmacy, Medical Academy, Sofia
MECHANISM OF ESTROGEN RECEPTOR DOWNR E G U L A T I O N IN SK-ER3 N E U R O B L A S T O M A C E L L S . S. Santagati, G. Pellio, Z.Q Ma and A. Maggi Milano Molecular Pharmacology Lab, Institute o f Pharmacological Sciences, University o f Milan, Via Balzaretti 9, 20133 Milan, Italy Ligand-indueed down regulation o f membrane as well as intraeellular receptors has an important role in hormone action. However, the mechanisms involved in this process are still far to be c0mpletely clarified. The present study is aimed at the comprehension o f estrogen receptor down-regulation and stems from two separate Observations: a. down regulation o f the ER was observed in cells not physiologically expressing ER, but stably transfected with ER eDNA b. oligonucleotides representing selected regions o f the ER eDNA determined up regulation o f the ER (as shown by western analysis). On the basis o f these observations, we hypothesized that the ER itself, once linked to its cognate hormone, could interact with,the ER gene or mRNA and interfere with its own synthesis. This hypothesis was further supported by subsequent studies where we proved that ER binds with high affinity the oligos determining its up-regulation. This binding, as assessed by gel shift, occurs at high affinity with the double strand DNA, at lower affinity with single strand elinueleotides supporting the view o f ER binding to the DNA and not to the mRNA. Studies are in progress to better define the kinetics and specificity o f this binding.
The effect of nitrendipine on desensitization of ~adrenoceptors induced by isoprenaline was investigated using rat lungs. Rats were divided into four groups: (1) the control group, (2) the isoprenaline-treated group, (3) the nitrendipine-treated group, and (4) isoprenaline plus nitrendipine-treated group. After 7 days treatment the tracheas were isolated and dissociation constant (Kb) for the propranolol- ~-receptor complex was determined. The lung parenchymal t~ssue was used to study the binding characteristics of ~-adrenoceptors. The Kb value for the propranolol- ~-receptor complex was find to be up to 6,4-fold higher in the tracheas isolated from isoprenaline-treated rats (3.2x10 8 -" 5.4x10 9) than in tracheas isolated from control rats (4.9 *- 1.2x10-9). The K b in tracheas from isoprenaline plus nitrendipine-treated rats w a s 7.0-'1.2x10 -9. A 31% reduction in the number of ~-adrenoceptors as compared to those of the control group was observed in the isoprenaline treated group. The number of the ~-adrenoceptors in the isoprenaline plus nitrendipine-treated group was significantly increased as compared to that of the isoprenaline-treated group.
Acknowledgements This work was supported by the CNR, P.S. Antisense eligonucleotides ( A . M )
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203
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