Calcium store depletion reduces the sensitivity of KCl-induced contractions to calcium channel antagonists in mouse aorta

Sarah K. Blackman, Alan Gibson & Ian McFadzean. Pharmaceutical Science Research Division, King’s College London, Guy’s Campus, London, SE1 1UL

Contractile responses to agonists in smooth muscle rely on calcium entry through both store and voltage-operated calcium channels (VOCCs). Whilst it is convenient to consider these processes in isolation, this is likely to be an oversimplification. Membrane depolarisation has been shown not only to activate VOCCs but also enhance agonist-mediated IP3 production and hence calcium release from stores (Best et al, 1986; Mahaut-Smith et al, 1999). We considered whether the converse might also be true; that calcium store depletion might alter responses initiated by membrane depolarisation. In an initial attempt to investigate this possibility we studied the effects of the sarcoplasmic reticulum calcium-ATPase inhibitor, thapsigargin (Tg), on contractile responses to raised potassium in mouse aortic smooth muscle.

Male C57BL6 mice were humanely killed, the aortas dissected, cleaned and cut into 3-4mm lengths. Rings were set up for recording isometric tension changes under 0.5-0.7g resting tension in Krebs’ solution (mM; NaCl 118.1, KCl 4.7, MgSO4 1.0 KH 2PO4 1.0, glucose 11.1, NaHCO3 25.0, CaCl2 2.5) maintained at 37 ° C and gassed with 5% CO2/95% O2. N w -Nitro-L-Arginine (50μM) and indomethacin (3μM) were included in the Krebs’ solution to inhibit the relaxant effects of nitric oxide and prostanoids respectively. Tissues were allowed to rest for 1hr, with the resting tension being adjusted as necessary, before being contracted by a 3 min application of KCl (40mM). This was repeated every 30 min until consistent responses to KCl were obtained. Subsequent contractile responses were expressed as a percentage of the KCl response. Experiments were carried out in paired (control or Tg-treated) tissues from the same animal. Results are expressed as mean ± SEM for the number of observations in parentheses. Data were compared using Students t-test, p<0.05 considered significant.

In control tissues, KCl produced a contraction of 0.34 ± 0.02 grams (n=9). This contraction was reduced to 7.18 ± 0.97% of control in the presence of verapamil (3 μM; n=3). The small, but consistent contraction that remained was not abolished by increasing the verapamil concentration to 10 μM (6.34±0.29% control; n=3), or by substituting verapamil with diltiazem (10 μM) and phentolamine (1μM; 9.02±2.06% control; n=3). Tg (4 μM) produced a slow sustained contraction of the mouse aorta (86.6 ± 6.4% KCl response; n=9). This increase in tension was not reduced by a concomitant application of verapamil (3–10 μM; n=6) or diltiazem (10 μM; n=3) . Application of KCl in the presence of both Tg and verapamil (3 μM) produced contractions (33.4 ± 4.5%, n=3) that were significantly greater than those seen in control tissues. Similar results were obtained using 10 μM verapamil (30.1 ± 5.24%; n=3) and diltiazem (55.0 ± 8.2%; n =3).

These results suggest that pre-treatment of tissues with Tg can alter the response to KCl depolarisation in mouse aorta, notably its sensitivity to block by calcium channel antagonists. Possible reasons for these findings are being investigated.

Best,L., et al (1986) Naunyn Schmeidebergs Arch Pharmacol. 333,78-82.
Mahuaut-Smith, M.P. et al (1999) J Physiol 515, 385.

SKB is the recipient of a BHF research studentship