Involvement of different Ca2+ pools during the canine bronchial sustained contraction in Ca2+-free medium:: lack of effect of PKC inhibition

We evaluated the role of protein kinase C (PKC) in the sustained bronchial contraction (SBC) induced by carbachol (Cch) or histamine in a Ca2+-free medium and the possibility that each agonist uses a different Ca2+ store for this response. We studied third-order bronchi and airway smooth muscle (ASM) from first-order bronchi dissected free of cartilage and epithelium. Bronchial and ASM responsiveness to Cch or histamine were evaluated in Krebs solution (2.5 mM Ca2+) and in Ca2+-free medium. Cch and histamine induced an SEC in bronchial tissues in Ca2+-free medium. In ASM each agonist produced a transient contraction, but the response to histamine was much smaller. Cch induced a concentration-dependent accumulation of inositol phosphates (IPs) in both bronchi and ASM; however, histamine did not induce significant accumulation of IPs. Repeated exposure to histamine in bronchial rings abolished contractile responses in Ca2+-free media, but Cch added afterwards still produced a sustained contraction. This response was blocked when bronchial tissues were preincubated with 10 mu M cyclopiazonic acid (CPA). Brief incubation of these preparations with a high EGTA concentration (1 mM) abolished the histamine-induced SEC. The SEC induced by Cch or histamine in Ca2+-free medium was not affected by the preincubation of the tissues with calphostin C, chelerythrine or staurosporine. We concluded that Cch mobilizes Ca2+ from two different sources during the SEC in Ca2+-free medium: from a CPA-sensitive one from sarcoplasmic reticulum (SR) and from a putative extracellular membrane Ca2+ pool sensitive to 1 mM EGTA, and neither process involved PKC activation. Histamine appeared to utilize the extracellular membrane pool only.