Acetylcholine and tachykinins involvement in the caffeine‐induced biphasic change in intracellular Ca2+ in bovine airway smooth muscle

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Abstract
1. Caffeine has been widely used as a pharmacological tool to evaluate Ca(2+) release from the sarcoplasmic reticulum in isolated smooth muscle cells. However, in nervous tissue this drug also causes neurotransmitters release, which might cause additional effects when smooth muscle strips are evaluated. To assess this last possibility, simultaneous measurements of contraction and cytosolic Ca(2+) concentration (using Fura-2/AM) were carried out in bovine airway smooth muscle strips during caffeine stimulation. 2. A first stimulation (S1, n=11) with caffeine (10 mM) induced a biphasic change in cytosolic Ca(2+), which consisted of a transient Ca(2+) peak (254+/-40 nM, X+/-SEM) followed by a plateau (92+/-13 nM), and a transient contraction (204.72+/-31.56 mg tension mg tissue(-1)). A second caffeine stimulation (S2) produced a similar response but these parameters had a different magnitude. The S2/S1 ratios for these parameters were 0.69+/-0.02, 0.83+/-0.06 and 1.01+/-0.03, respectively. Addition of omega-conotoxin GVIA (1 micro M) and tetrodotoxin (3.1 micro M) before S2 significantly diminished these S2/S1 ratios (0.26+/-0.05, 0.26+/-0.09 and 0.64+/-0.11, respectively, n=5, P<0.05), implicating the neurotransmitters release involvement in the response to caffeine. A similar effect (P<0.01) was observed with atropine (1 micro M, n=4), the fragment 4-11 of substance P (SP) (an SP receptor antagonist, 10 micro M, n=5), and with both substances (n=4). 3. We discarded a direct effect of omega-conotoxin GVIA (1 micro M) plus tetrodotoxin (3.1 micro M) or of atropine (1 micro M) plus SP fragment 4-11 on smooth muscle cells because they did not modify caffeine responses in isolated tracheal myocytes. 4. We confirmed by HPLC that caffeine increased the release of acetylcholine (from 0.43+/-0.19 to 2.07+/-0.56 nM mg tissue(-1), P<0.02) in bovine airway smooth muscle strips. Detection of substance P by ELISA was not statistically different after caffeine stimulation (geometric means before and after caffeine, 0.69 vs. 1.97 pg ml(-1) mg tissue(-1), respectively, P=0.053). 5. We concluded that acetylcholine and tachykinins release are involved in the caffeine-induced biphasic changes in cytosolic Ca(2+) concentration.

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