Data in this study show the effects of isoproterenol (ISO), a [bêta]-adrenergic agonist, on the total calcium content of the sarcoplasmic reticulum (SR), denoted [Ca[subscript T]][subscript SR]. Tetramethyl murexide (TMX), a membrane permeable, low affinity Ca[superscript 2+] indicator was used to monitor SR Ca[superscript 2+] content. The fraction of calcium-bound form of TMX in the SR ([f]Ca) is approximately proportional to the concentration of the free Ca[superscript 2+] in the SR ([Ca[superscript 2+]][subscript SR]) which in turn can be used to give [Ca[subscript T]][subscript SR]. Results show that 10 [mu]M ISO increases [Ca[superscript 2+]][subscript SR] by 112% in 30 min. in resting frog skeletal muscle. No significant increase was observed with no Ca[superscript 2+] present in the external solution indicating that ISO caused a Ca[superscript 2+] influx across the surface/T-system membrane. This increase in [Ca[superscript 2+]][subscript SR] occurred with an exponential delay ([tau] = 7.0 min.) and failed to reverse after washing out ISO. These results suggest that ISO stimulates the expression of a channel -permeable to calcium during resting potentials- which would remain active or open even after washing out ISO. These results argue against the involvement of most of voltage-dependent Ca[superscript 2+] channels that are gated by depolarization. Furthermore, we showed that hyperpolarization in ISO further increases Ca[superscript 2+] influx. The increase in rate of influx came on with an exponential delay ([tau] = 1.6 min.), and couldn't be explained simply by the increased driving force for Ca[superscript 2+] or by inward rectification. This delay also argues against the involvement of hyperpolarization-activated channels. The fact that the rate of Ca[superscript 2+] flux did not decrease during hyperpolarization further supports the idea that the Ca[superscript 2+] influx is not due to depolarization-activated Ca[superscript 2+] channel. In the absence of ISO, the level of [f]Ca was the same with and without Ca[superscript 2+] in the external solution, indicative of a lack of Ca[superscript 2+] influx under resting physiological conditions. When the level of [f]Ca was reduced to 30% of the physiological level with 20 mM EGTA, the average value of [f]Ca was the same with or without external Ca[superscript 2+]. These results thereby arguing against the involvement of store operated mechanisms in the regulation of SR Ca[superscript 2+] content in the physiological range. The data show two well distinguished effects when ISO was removed. One is a reversible effect which showed a sudden, transient decrease in SR calcium content (termed"dip"). The"dip" can be described with a single exponential and corresponds with the rate of Ca[superscript 2+] release observed in response to a depolarization to -70 mV. The"dip" appears to require the reversal of the SR calcium pump (SERCA). After the"dip", SR calcium content rose again and reached the same rate as was observed during ISO. This steady rise in SR calcium content appeared to be the other, irreversible effect of ISO. In addition, during the course of ISO stimulation, we observed an increase in myoplasmic pH. One possible explanation could involve the activation of [alpha]-adrenergic receptors by ISO. The receptors activate the PLC-IP3 pathway which, in turn, enhances the Na/H exchanger and thus the removal of H+ ions from the myoplasm. In summary, the data indicate that adrenergic stimulation increases [Ca[subscript T]][subscript SR] in resting fibers by activating Ca[superscript 2+] influx across the surface/T-system membrane and is consistent with the expression of an unknown Ca[superscript 2+] channel. Our results also raise doubts about whether, store-operated mechanisms are involved in fibers depleted to 30% of their normal [Ca[superscript 2+]][subscript SR]. The data also suggest that SERCA is directly enhanced by ISO.
| Identifer | oai:union.ndltd.org:usherbrooke.ca/oai:savoirs.usherbrooke.ca:11143/4283 |
| Date | January 2009 |
| Creators | Gyurkovics, Gabor |
| Contributors | Pape, Paul |
| Publisher | Université de Sherbrooke |
| Source Sets | Université de Sherbrooke |
| Language | English |
| Detected Language | English |
| Type | Thèse |
| Rights | © Gabor Gyurkovics |
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