Little is known about the development of smooth muscle. Excitation-contraction coupling and relaxation are still not fully understood even in mature smooth muscles. By studying the mechanisms as they develop new insights may emerge. A thorough understanding of the normal development will be necessary to interpret causes of abnormal development and pathological conditions. Much of the activity of smooth muscle cells (contractile or metabolic) is affected by changes in the cytoplasmic free Ca concentration ([Ca++]i). Changes in the [Ca++]i can be indirectly monitored by changes in tension in mature muscle. In embryonic cells, contractions are difficult or impossible to monitor. The recent introduction of a nondisruptive method to directly measure changes in [Ca++]i in small cells now makes this study feasible. This well-designed method uses a permeant ester (Quin-2/AM) of a fluorescent chelator (Quin-2) with a high affinity and selectivity for calcium (Kd=115 nM). Cellular enzymes de-esterify Quin-2/AM thereby freeing the carboxyl groups which bind Ca and make the molecule too hydrophilic to escape. The binding of Ca affects the fluorescence intensity of the molecule. The development of receptors for neurotransmitters, neuropeptides and hormones that initiate signals for contraction or relaxation of visceral or vascular smooth muscles and Ca transport mechanisms will be studied. This proposal focuses on the development of cholinergic receptors in embryonic chicken gizzard smooth muscle. This tissue is easily accessible and the best characterized embryonic smooth muscle to date. The developmental process will be studied in ovo and in tissue cultures. Tissue culture conditions favoring appropriate differentiation and suitable for the Quin-2 method will be developed.
