Abstract. The role of prostanoids in the regulation of chick myoblast differentiation has been investigated. At 3 x 10 -6 M, indomethacin and chloroquine specifically inhibit cell fusion. They do not affect cell proliferation, alignment, or the expression of two muscle-specific proteins, namely, the acetylcholine receptor and the muscle-specific form of creatine phosphokinase. The results demonstrate that it is indomethacin's activity as an inhibitor of prostaglandin synthesis at the cyclyooxygenase step that causes the block of cell fusion, whereas chloroquine probably acts at the earlier step of phospholipase A. Prostaglandin Et (PGE0, but not prostaglandin E2 (PGE2), rapidly reverses the inhibition of fusion imposed by indomethacin or chloroquine. The dose response of the myoblasts to PGE~ is a bell-shaped curve with a 100% reversal of fusion at ,~10 -9 M. Eicosatrienoate and linoleate reverse the inhibition of fusion with similar kinetics, whereas aracl~idonate is completely ineffective. The ability of PGE~ and eicosatrienoate but not PGF~ and arachidonate to restore fusion to control levels implies that fusion is specifically regulated by a prostanoid of the one series. The reversal of the fusion-block by linoleate further suggests that this fatty acid provides the necessary source of eicosatrienoate in the myoblast plasma membrane. At 10 -8 M and above, PGE~ and PGE2 stimulate adenylate cyclase and depress control fusion as does 10 -5 M isoproterenol.The I]-adrenergic blocker propranolol abolishes both isoproterenol's inhibition of myoblast fusion and its activation of adenylate cyclase. The similar depressions imposed on cell fusion by 10-g-10 -~ M prostanoid and 10 -5 M isoproterenol suggest that in both cases the depressive effects are mediated by cyclic AMP. It is concluded that a prostanoid of the one series regulates fusion by a cyclic AMP-independent mechanism. T HE multinucleated skeletal muscle fiber is formed by the fusion of mononucleated precursor myoblasts. Both in vivo and in culture, the bipolar myoblasts initially undergo a phase of proliferation while aligning along their longitudinal axes. Membrane fusion subsequently takes place between the aligned cells. At approximately the same time as the onset of cell-cell fusion, the embryonic muscle cell ceases DNA synthesis and cell division and initiates the elaboration of the specialized proteins associated with skeletal muscle contraction. The relationship between these three events is not yet fully elucidated. However, it is accepted that the expression of muscle-specific proteins can be uncoupled from the fusion event and that once inside a multinucleate cell, the myogenic nuclei are incapable of either DNA synthesis or cell division.Numerous agents have been identified that inhibit cell-cell fusion (41). Most of these inhibitions probably represent a more general block of differentiation or cellular function. An exception to this is the inhibition of cell fusion achieved by lowering external calcium. Unfortunately, reducing external Ca ++ t...

Epinephrine and isoproterenol provoke primary chick myoblasts to initiate precocious cell fusion. Both the rise in intracellular adenosine 3' ,5-monophosphate (cyclic AMP) and cell fusion generated by these effectors are prevented by propranolol, which is a specific blocker of the beta-adrenergic receptor. Propranolol has no effect either on the precocious cell fusion provoked by prostaglandin E or on cell fusion in control cultures. The results support the idea that a rise in cyclic AMP is the critical intracellular change responsible for initiating events that culminate in myoblast differentiation 4 to 5 hours later. They also indicate that the culminate in myoblast differentiation 4 to 5 hours later. They also indicate that the hormone responsible for the positive regulation of myoblast differentiation in vitro is not acting through the beta-adrenergic receptor.

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The responsiveness of adenylyl cyclase during avian myogenesis in vitro has been examined. Measurements of cyclic AMP generation in intact cells revealed that the precursor myoblast is highly responsive to prostaglandin E1 (11-fold maximum stimulation); whereas its response to isoproterenol is much smaller (2-fold). From the onset of terminal differentiation, responsiveness to the beta-adrenergic agonist increases progressively, reaching a 5.5-fold maximum response by 96 hr of culture. In contrast, there is little change in the cell population's responsiveness to prostaglandin E1. The rise in catecholamine responsiveness is consistent with previously reported increases in beta-receptors accompanying differentiation. DL-propranolol blocks the response of myoblasts and myotubes to 10(-6) M isoproterenol with the same half maximal inhibition value of 1 X 10(-8) mol. The results also suggest a change in the adrenergic character of the receptors and/or coupling to adenylyl cyclase as myoblasts differentiate. First the alpha-adrenergic antagonist phentolamine (10(-7)-10(-4) mol) inhibits the myoblast's response but enhances that of the myotube. Second, the potency ratios for the responses to isoproterenol, epinephrine, and norepinephrine shift from 1.1:1.0:1.0 in the myoblast to 3.3:2.1:1.0 in the myotube. The findings are discussed with reference to the role of prostaglandins in the positive control of muscle differentiation and the changes in the catecholamine-responsive adenylyl cyclase system as an aspect of the expression of the muscle phenotype.

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