We have presented evidence that only muscle FBPase is detect

We have presented evidence that only muscle FBPase is detectable within HL-1 cells. This is consistent with past findings of the Eschrich's group, which showed that in rat heart ESI-09 expression of FBPase is almost exclusively limited to the muscle isozyme [3]. Results of our preliminary experiments reveal that, in contrast to the liver isozyme, which plays the main role in gluconeogenesis regulation [2], [16], neither elevation nor subsequent withdrawal of glucose have an effect on the nuclear localization of muscle FBPase (data not shown). This might suggest that the cellular function of the enzyme in muscle tissue goes far beyond participation in glycogen synthesis from carbohydrate precursors. Subcellular localization of the muscle FBPase isozyme seems to change, depending on the differentiation stage of the cell [14]. One of the factors critical to maintaining a differentiated phenotype of HL-1 cell line is norepinephrine [17], [18]. To identify the signals involved in the nuclear targeting of muscle FBPase, we examined the effect of NE withdrawal on FBPase localization in HL-1 cardiomyocytes. In the presence of NE (that is, in standard culture medium for HL-1), approximately 85% of the HL-1 cells contained high levels of FBPase in the nucleus, in addition to the cytoplasm. This distribution was expected, and it is consistent with our previous experiments on vertebrate heart muscle tissue, showing that FBPase is a nucleo-cytoplasmic enzyme [7], [12]. When the HL-1 cells were examined following a shift to NE-free medium, it appeared that the amount of nuclear FBPase decreased in a time-dependent manner, and after 48 h nuclear FBPase was undetectable with indirect immunofluorescence in a vast majority of the cardiomyocytes. This withdrawal of FBPase was then effectively blocked by a specific inhibitor of nuclear export — Leptomycin B, suggesting that FBPase translocation to cytoplasm is an active, NES-dependent process. β-ARs family play a critical role in regulating cardiac function in response to catecholamines, and β1-AR is the predominant subtype within HL-1 cells [21]. Therefore, it appeared plausible that FBPase enters the nucleus following the activation of β1-ARs. This hypothesis was confirmed through our experiments with ISO (a nonselective β-AR agonist) and with MTP (a selective antagonist of β1-AR). ISO mimicked the stimulatory effect of NE on nuclear accumulation of FBPase, and this effect was, in turn, suppressed by MTP. Searching for further validation of our findings, we discovered that in the absence of NE, the activation of adenylyl cyclase by forskolin is sufficient to stimulate FBPase targeting to the nucleus. On the other hand, inhibition of cAMP-dependent PKA effectively abolishes the NE-activated nuclear transport of FBPase. These findings suggest a role of PKA-dependent phosphorylation in the nucleo-cytoplasmic shuttling of the enzyme. However, although we have demonstrated that muscle FBPase might be a substrate for PKA [27], the relatively slow rate of FBPase translocation might suggest that an additional modification of FBPase is necessary for the nuclear localization of the enzyme. NE-stimulated PKA activation is an important step in the process of cellular growth and differentiation of cardiac myocytes [28]. However, many lines of evidence suggest that it is β2-ARs (rather than β1-ARs) which are responsible for the mitogenic effect of catecholamines (for example see [29]). Our results demonstrate that the NE signaling accelerates the rate of HL-1 proliferation significantly (about twofold). The same effect is achieved by substituting NE with a β-AR agonist, while a selective β1-AR antagonist does not slow the proliferation rate. Consequently, it seems that a β1-AR-dependent nuclear localization of FBPase is not directly connected with the accelerating effect of NE on cell proliferation. However, a disruption of β1-AR signaling increases the mortality of HL-1 cells about threefold.