The mechanism of Ca2' release from sarcoplasmic reticulum, which triggers contraction in skeletal muscle, remains the key unresolved problem in excitation-contraction coupling. Recently, we have described the isolation of purified fractions referable to terminal and longitudinal cisternae of sarcoplasmic reticulum. Junctional terminal cisternae are distinct in that they have a low net energized Ca2+ loading, which can be enhanced 5-fold or more by addition of ruthenium red. The loading rate, normalized for calcium pump protein content, then approaches that of longitudinal cisternae of sarcoplasmic reticulum. We now find that the ruthenium red-enhaniced Ca2+ loading rate can be blocked by the previous addition of ryanodine. The inhibition constant is in the nanomolar range (20-180 nM). Ryanodine and ruthenium red have no effect on the Ca21 loading rate of longitudinal cisternae. Direct binding studies with [3HJryano-dine localized the receptors to the terminal cisternae and not to longitudinal cisternae. Scatchard analysis of the binding data gives a dissociation constant for ryanodine in the range of the drug action on the terminal cisternae ("100 nM range) with approximately 4 to 20 pmol bound per mg of protein.Ryanodine is known to be toxic in animals, leading to irreversible muscle contractures. These studies provide evidence on the mode of action of ryanodine and its localization to the terminal cisternae. The low concentration at which the drug is effective appears to account for its toxicity. Ryanodine locks the Ca2+ release channels in the "open state," so that Ca2+ is not reaccumulated and the muscle fiber cannot relax.

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Stimulation of hepatocyte proliferation by epidermal growth factor (EGF) and insulin is inhibited by transforming growth factor ␤ (TGF-␤) and by glucagon. It is also suppressed by inhibitors of various protein kinases, including rapamycin, which blocks activation of p70 S6 kinase (p70 S6k ), PD98059, which inhibits the activation of extracellular-regulated kinase (ERK), and SB 203580, an inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK). In this study, we investigated whether the inhibition of proliferation by TGF-␤ involves these protein kinase cascades. Culture of hepatocytes with TGF-␤ for 16 hours decreased the stimulation by EGF of ERK2 and p70 S6k (by 50% and 35%, respectively), but did not affect the stimulation of either p38 MAPK, c-jun NH 2 -terminal kinase (JNK), or protein kinase B (PKB). Culture of hepatocytes with glucagon for 16 hours also inhibited the stimulation by EGF of activation of ERK2 and p70 S6k (by E50%). The inhibitory effects of glucagon were observed when the hormone was added either 10 minutes or 60 minutes before EGF addition, whereas no effects of TGF-␤ were observed after 10-minute or 60-minute incubation. These results suggest that the inhibition of hepatocyte proliferation by TGF-␤ may be in part mediated by inhibition of ERK2 and p70 S6k , but does not involve PKB, JNK, or p38 MAPK. Unlike glucagon, the effects of TGF-␤ are not elicited in response to short-term treatment. (HEPATOLOGY 1999;29:1418-1424 Hepatocyte proliferation following injury or partial hepatectomy is regulated by the action of both stimulatory (mitogenic) and inhibitory growth factors and hormones (reviewed in Michalopoulos 1 and Fausto 2 ). Studies in primary hepatocyte cultures have identified several complete mitogens capable of inducing DNA synthesis in serum-free medium,

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