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M1 mAChR does not regulate L-type calcium current when carbachol increases ventricular myocyte contractions
Vol. 64, Nos. 617, 1999
57-l
Abstracts
46 M 1 mAChR DOES NOT REGULATE L-TYPE CALCIUM CURRENT INCREASES VENTRICULAR MYOCYTE CONTRACTIONS J.-B. Shen, B. Jiang and A.J. Pappano, Department Health Center, Farmington, CT, USA
of Pharmacology,
WHEN CARBACHOL
University
of Connecticut
Activation of Ml muscarinic receptors (mAChR) is reported to increase L-type Ca2+ current (ICY) in guinea pig ventricular myocytes and increase intracellular Ca*+ transients in mammalian ventricular myocytes (Gallo, et al., J. Physiol. 471:41, 1993; Sharma, et al., Circ. Res. 79:86, 1996). We tested this hypothesis in a single ventricular myocytes by measuring ICY under voltage clamp conditions and contractions in externally-stimulated cells. Basal It,(L), measured under conditions with Cs+ present in pipette (120mM) and bath (10mM) solutions, was unaffected by 100 mM McN-A-343 (Ml-selective), oxotremorine (0X0, M2selective) or carbachol (CCh, non-selective). When 1~ L) was increased by 3-10 nM isoproterenol (ISO), CCh and OX0 inhibited by 87+6. ‘/ % (n=8) and 50f7.5% (n=4), respectively. Unlike CCh and 0X0, McN-A-343 had no atropine-sensitive inhibitory effect on ISO-stimulated IQ(L). When ICY was increased by intrapipette cyclic AMP, none of the muscarinic agonists had any effect. Contractions of single ventricular myocytes were increased in 100 n-M CCh by 19+6% (from 4.1f0.72 to 5.OkO.84 mm) at a stimulus frequency of 0.2 Hz (n=19) and by 25?10.8% (from 2.8kO.32 to 3.5kO.50 mm) at 1 Hz (n=lO). McN-A-343 had no significant effect at either 0.2 Hz (from 3.6kO.51 to 3.5f0.53 mm, n=26) or at 1 Hz (from 3.5kO.36 to 3.4f0.34 mm, n=17). We conclude that: 1) there is a negligible role of MI mAChR in either positive or negative regulation of Ic,(L) or contractions, 2) M2 mAChR are implicated in anti-adrenergic action and 3) maximal elevation of intracellular cyclic AMP suppressed muscarinic inhibition of Ica(L).
47 MULTIPHASIC
CALCIUM CURRENTS STIMULATED
BY THE HMI RECEPTORS
K. Lin, J. M. Quillan, J. Lameh and W. SadQ Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-0446. We developed a rapid and sensitive assay of intracellular calcium ions in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells from 96-well plates using a fluorometer equipped with on-line injectors. Compared to the conventional single point measurement with UV-excitable dyes such as Fura-2, this assay was developed in high throughput format with visible light-excitable fluorescent Ca*+dyes Calcium Green-l and Oregon Green 488 BAPTA-1. Both dyes produce a robust and stable signal upon stimulation of intracellular Ca” in HEK293 cells. In CHO cells, the fluorescence signal is stable only with Oregon Green 488 BAPTA- 1 at room temperature in the presence of an organic anion transporter inhibitor. Changes of cytoplasmic calcium concentrations occur either through release from intracellular calcium stores or by the opening of channels in the membrane. To dissect the possible existence of distinct calcium pools and regulation of calcium influx, we measured calcium responses in endogenous and stably transfected hml receptors, in comparison with stably transfected mu-opioid receptors, which also elicit multiphasic Ca*’ currents by stimulating pertussis toxin-sensitive G proteins. Our results suggest that mu-opioid and hml receptors may activate different intracellular Ca*+ stores. Further, we observed at least two calcium influx phases upon agonist stimulation through unidentified Ca’+ channels, suggesting the presence of both receptor-operated and capacitative calcium entry. Supported by NII-I grants GM43102 and PHS grant DA04166.
57-l
Abstracts
46 M 1 mAChR DOES NOT REGULATE L-TYPE CALCIUM CURRENT INCREASES VENTRICULAR MYOCYTE CONTRACTIONS J.-B. Shen, B. Jiang and A.J. Pappano, Department Health Center, Farmington, CT, USA
of Pharmacology,
WHEN CARBACHOL
University
of Connecticut
Activation of Ml muscarinic receptors (mAChR) is reported to increase L-type Ca2+ current (ICY) in guinea pig ventricular myocytes and increase intracellular Ca*+ transients in mammalian ventricular myocytes (Gallo, et al., J. Physiol. 471:41, 1993; Sharma, et al., Circ. Res. 79:86, 1996). We tested this hypothesis in a single ventricular myocytes by measuring ICY under voltage clamp conditions and contractions in externally-stimulated cells. Basal It,(L), measured under conditions with Cs+ present in pipette (120mM) and bath (10mM) solutions, was unaffected by 100 mM McN-A-343 (Ml-selective), oxotremorine (0X0, M2selective) or carbachol (CCh, non-selective). When 1~ L) was increased by 3-10 nM isoproterenol (ISO), CCh and OX0 inhibited by 87+6. ‘/ % (n=8) and 50f7.5% (n=4), respectively. Unlike CCh and 0X0, McN-A-343 had no atropine-sensitive inhibitory effect on ISO-stimulated IQ(L). When ICY was increased by intrapipette cyclic AMP, none of the muscarinic agonists had any effect. Contractions of single ventricular myocytes were increased in 100 n-M CCh by 19+6% (from 4.1f0.72 to 5.OkO.84 mm) at a stimulus frequency of 0.2 Hz (n=19) and by 25?10.8% (from 2.8kO.32 to 3.5kO.50 mm) at 1 Hz (n=lO). McN-A-343 had no significant effect at either 0.2 Hz (from 3.6kO.51 to 3.5f0.53 mm, n=26) or at 1 Hz (from 3.5kO.36 to 3.4f0.34 mm, n=17). We conclude that: 1) there is a negligible role of MI mAChR in either positive or negative regulation of Ic,(L) or contractions, 2) M2 mAChR are implicated in anti-adrenergic action and 3) maximal elevation of intracellular cyclic AMP suppressed muscarinic inhibition of Ica(L).
47 MULTIPHASIC
CALCIUM CURRENTS STIMULATED
BY THE HMI RECEPTORS
K. Lin, J. M. Quillan, J. Lameh and W. SadQ Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-0446. We developed a rapid and sensitive assay of intracellular calcium ions in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells from 96-well plates using a fluorometer equipped with on-line injectors. Compared to the conventional single point measurement with UV-excitable dyes such as Fura-2, this assay was developed in high throughput format with visible light-excitable fluorescent Ca*+dyes Calcium Green-l and Oregon Green 488 BAPTA-1. Both dyes produce a robust and stable signal upon stimulation of intracellular Ca” in HEK293 cells. In CHO cells, the fluorescence signal is stable only with Oregon Green 488 BAPTA- 1 at room temperature in the presence of an organic anion transporter inhibitor. Changes of cytoplasmic calcium concentrations occur either through release from intracellular calcium stores or by the opening of channels in the membrane. To dissect the possible existence of distinct calcium pools and regulation of calcium influx, we measured calcium responses in endogenous and stably transfected hml receptors, in comparison with stably transfected mu-opioid receptors, which also elicit multiphasic Ca*’ currents by stimulating pertussis toxin-sensitive G proteins. Our results suggest that mu-opioid and hml receptors may activate different intracellular Ca*+ stores. Further, we observed at least two calcium influx phases upon agonist stimulation through unidentified Ca’+ channels, suggesting the presence of both receptor-operated and capacitative calcium entry. Supported by NII-I grants GM43102 and PHS grant DA04166.