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Published on: November 29, 2011
Sarafotoxin-induced calcium mobilization in cultured dog tracheal smooth muscle cells
1Department of Pharmacology, Chang Gung College of Medicine and Technology, Tao-Yuan, Taiwan, Republic of China.
Abstract:
Sarafotoxin b (S6b)-induced changes in intracellular Ca2+ concentration ([Ca2+]i) were monitored in cultured canine tracheal smooth muscle cells (TSMCs) by a fluorescent Ca2+ indicator fura-2. S6b elicited an initial transient peak followed by a sustained elevation of [Ca2+]i. BQ-123, an endothelin-A (ETA) receptor antagonist, had a high affinity to block the rise in [Ca2+]i response to S6b. In the absence of external Ca2+, only an initial transient peak of [Ca2+]i was seen, the sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+. Ca2+ influx was required for the changes of [Ca2+]i, since the Ca(2+)-channel blockers, diltiazem, verapamil, and Ni2+, decreased both the initial and sustained elevation of [Ca2+]i in response to S6b. TSMCs pretreated with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min attenuated Ca2+ mobilization induced by S6b, which was reversed by staurosporine, a protein kinase C (PKC) inhibitor. The change of [Ca2+]i induced by S6b was attenuated by cholera toxin pretreatment, but not by pertussis toxin. These data demonstrate that the initial detectable increase in [Ca2+]i stimulated by S6b is due to the activation of ETA receptors and subsequent release of Ca2+ from internal stores, whereas the contribution of external Ca2+ follows and partially involves a diltiazem- and verapamil-sensitive process. The inhibition of PMA on S6b-induced Ca2+ mobilization was inversely correlated with membraneous PKC activity.
Insights
Sarafotoxin b triggers calcium release via endothelin-A receptors in canine tracheal cells, requiring calcium influx for sustained elevation. Protein kinase C activity influences this calcium mobilization.
Area of Science:
- Pharmacology
- Cell Physiology
- Smooth Muscle Biology
Background:
- Sarafotoxin b (S6b) is a peptide toxin known to affect cellular functions.
- Intracellular calcium concentration ([Ca2+]i) plays a critical role in smooth muscle contraction.
- Canine tracheal smooth muscle cells (TSMCs) are a relevant model for studying airway smooth muscle physiology.
Purpose of the Study:
- To investigate the mechanisms underlying Sarafotoxin b (S6b)-induced changes in intracellular calcium concentration ([Ca2+]i) in cultured canine tracheal smooth muscle cells (TSMCs).
- To identify the specific receptors and signaling pathways involved in S6b-mediated calcium mobilization.
Main Methods:
- Monitoring of intracellular Ca2+ concentration ([Ca2+]i) using the fluorescent indicator fura-2 in cultured TSMCs.
- Application of S6b and various pharmacological agents including BQ-123 (ETA receptor antagonist), Ca2+-channel blockers (diltiazem, verapamil, Ni2+), phorbol 12-myristate 13-acetate (PMA), staurosporine (PKC inhibitor), cholera toxin, and pertussis toxin.
- Experiments conducted with and without external Ca2+ to differentiate between internal store release and influx.
Main Results:
- S6b induced a transient peak followed by a sustained elevation of [Ca2+]i.
- The endothelin-A (ETA) receptor antagonist BQ-123 blocked the S6b-induced [Ca2+]i rise, indicating ETA receptor involvement.
- Sustained [Ca2+]i elevation required external Ca2+ influx, which was sensitive to Ca2+-channel blockers.
- Phorbol 12-myristate 13-acetate (PMA) pretreatment attenuated S6b-induced Ca2+ mobilization, an effect reversed by staurosporine, suggesting a role for protein kinase C (PKC).
- Cholera toxin, but not pertussis toxin, attenuated the S6b-induced [Ca2+]i changes, suggesting involvement of a Gs-coupled pathway.
Conclusions:
- The initial increase in [Ca2+]i by S6b is mediated by ETA receptor activation and release of Ca2+ from internal stores.
- Subsequent sustained elevation of [Ca2+]i involves Ca2+ influx through diltiazem- and verapamil-sensitive channels.
- Protein kinase C (PKC) activity modulates S6b-induced calcium signaling in TSMCs.
