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Homeostatic plasticity of excitatory synapses plays an important role in stabilizing neuronal activity, but the mechanism of this form of plasticity is incompletely understood. In particular, whether the locus of expression is presynaptic or postsynaptic has been controversial. Here we show that the expression locus depends on the time neurons have spent in vitro. In visual cortical cultures < or =14 days in vitro (DIV), 2 days of TTX treatment induced an increase in miniature excitatory postsynaptic current (mEPSC) amplitude onto pyramidal neurons, without affecting mEPSC frequency. However, in cultures > or =18 DIV, the same TTX treatment induced a large increase in mEPSC frequency, whereas the amplitude effect was reduced. The increased mEPSC frequency was associated with an increased density of excitatory synapses and increased presynaptic vesicle release in response to electrical stimulation. This indicates a shift from a predominantly postsynaptic response to TTX in < or =14 DIV cultures, to a coordinated pre- and postsynaptic response in > or =18 DIV cultures. This shift was not specific for cortical cultures because a similar shift was observed in cultured hippocampal neurons. Culturing neurons from older animals showed that the timing of the switch depends on the time the neurons have spent in vitro, rather than their postnatal age. This temporal switch in expression locus can largely reconcile the contradictory literature on the expression locus of homeostatic excitatory synaptic plasticity in central neurons. Furthermore, our results raise the intriguing possibility that the expression mechanism of homeostatic plasticity can be tailored to the needs of the network during different stages of development or in response to different challenges to network function.

Mucosal healing requires migration and proliferation. Most studies of focal adhesion kinase (FAK), a protein that regulates motility, proliferation, and apoptosis, have focused on rapid phosphorylation. We reported lower FAK protein levels in motile Caco-2 colon cancer cells and postulated that this reduction in FAK available for activation might impact cell migration and mucosal healing. Therefore, total and active FAK (FAK(397)) immunoreactivity was assessed at the migrating fronts of human Caco-2 and rat IEC-6 intestinal epithelial cells. Caco-2 and IEC-6 motility, quantitated as migration into linear or circular wounds, was examined following FAK protein inhibition by small interfering RNA (siRNA). FAK protein stability and mRNA expression were ascertained by cycloheximide decay, RT-PCR, and in situ hybridization in static and migrating Caco-2 cells. Cells at the migrating front of Caco-2 and IEC-6 monolayers exhibited lower immunostaining for both total and activated FAK than cells immediately behind the front. Western blot analysis also demonstrated diminished FAK protein levels in motile cells by >/=30% in both the differential density seeding and multiple scrape models. siRNA FAK protein inhibition enhanced motility in both the linear scrape (20% in Caco-2) and circular wound (16% in Caco-2 and 19% in IEC-6 cells) models. FAK protein degradation did not differ in motile and static Caco-2 cells and was unaffected by FAK(397) phosphorylation, but FAK mRNA was lower in migrating Caco-2 cells. Thus FAK protein abundance appears regulated at the mRNA level during gut epithelial cell motility and may influence epithelial cell migration coordinately with signals that modify FAK phosphorylation.

Obesity plays a pivotal role in the pathophysiology of metabolic and cardiovascular diseases. Resistance to insulin is commonly seen in metabolic disorders such as obesity and diabetes. Insulin-like growth factor-I (IGF-I) mimics insulin in many tissues and has been shown to enhance cardiac contractile function and growth. Because IGF-I resistance often accompanies resistance to insulin, we sought to determine whether IGF-I-induced myocardial contractile was elevated and whether heart and kidney size were enlarged in obese compared with lean rats. The myocyte contraction profile in the obese rats showed a decreased peak shortening associated with prolonged relengthening and normal shortening duration, a pattern similar to that observed in diabetes. IGF-I (1-500 ng/ml) caused a dose-dependent increase in peak shortening in lean but not obese animals, but it did not alter the duration of shortening and relengthening. Consistent with contractile data, IGF-I induced a dose-dependent increase in Ca(2+) transients only in myocytes of lean rats. IGF-I receptor mRNA levels were significantly reduced in obese rat hearts. These results suggest that the IGF-I-induced cardiac contractile responses are attenuated in the Zucker model of obesity. The mechanisms underlying this alteration may be related to the decreased receptor number and/or changes in intracellular Ca(2+) handling in these animals.

Normally, insulin-like growth factor I (IGF-I) exerts positive effects on cardiac growth and myocardial contractility, but resistance to its action has been reported in diabetes. This study was designed to determine whether IGF-I-induced myocardial contractile action is altered in diabetes as a result of an intrinsic alteration of contractile properties at the cellular level. Contractile responses to IGF-I were examined in left ventricular papillary muscles and ventricular myocytes from normal and short-term (5-7 days) streptozotocin-induced diabetic rats. Mechanical properties of muscles and myocytes were evaluated using a force transducer and an edge detector, respectively. Preparations were electrically stimulated at 0.5 Hz, and contractile properties analyzed include peak tension development (PTD) or peak twitch amplitude (PTA), time to peak contraction/shortening, and time to 90% relaxation/relengthening. Intracellular Ca2+ transients were measured as fura 2 fluorescence intensity changes. IGF-I (1-500 ng/ml) caused a dose-dependent increase in PTD and PTA in preparations from normal but not diabetic animals. IGF-I did not alter time to peak contraction/shortening or time to 90% relaxation/relengthening. Pretreatment with the NO synthase inhibitor Nomega-nitro-L-arginine methyl ester (100 microM) attenuated IGF-I-induced increases in PTD in normal myocardium but unmasked a positive inotropic action in diabetic animals. Pretreatment with Nomega-nitro-L-arginine methyl ester blocked IGF-I-induced increases in PTA in single myocytes. Consistent with its inotropic actions on muscles and myocytes, IGF-I induced a dose-dependent increase in Ca2+ transients in normal but not diabetic myocytes. These results suggest that the IGF-I-induced inotropic response is depressed in diabetes because of an intrinsic alteration at the myocyte level. Mechanisms underlying this alteration in IGF-I-induced myocardial response may be related to changes in intracellular Ca2+ and/or NO production in diabetes.

Insulin-like growth factor I (IGF-I) is vasodilatory and mitogenic for vascular smooth muscle cells (VSMC). Alteration in VSMC Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity is hypothesized to underlie abnormal vascular tone and growth in hypertension and diabetes. Therefore, we investigated effects of IGF-I on Na(+)-K(+)-ATPase activity in rat aortic VSMC. IGF-I increases pump activity in a dose- and time-dependent manner: the minimal dose required was 10(-10) M, and the minimal time required was 20 min (at 10(-8) M) to increase activity. Similar effects persisted through 12 h. In Na(+)-loaded cells, IGF-I does not further stimulate activity. Blockade of Na+/H+ exchange attenuates IGF-I-induced increases in activity after 30 min but has no effect after 12 h. Northern blot analyses reveal that expression of the alpha 1- and the alpha 2-subunits of the pump were unaffected by IGF-I. Plasma membrane alpha 1- and alpha 2-protein were also unaffected, suggesting translocation of preformed pools was not responsible for the increases. Inhibitors revealed that neither tyrosine kinase activity, RNA transcription, protein synthesis, nitric oxide synthase activity, or protein kinase C activity mediated this IGF-I effect. Therefore, IGF-I regulates Na pump activity in the short term by an Na+/H+ exchange-dependent but transcription/translocation-independent mechanism. These data suggest that IGF-I, known to be produced by VSMC, may regulate tone and growth responses abnormal in disease states such as hypertension and diabetes.