Roles of mechano-sensitive ion channels, cytoskeleton, and contractile activity in stretch-induced immediate-early gene expression and hypertrophy of cardiac myocytes.

Mechanical loading of cardiac and skeletal muscles in vivo and in vitro causes rapid activation of a number of immediate-early (IE) genes and hypertrophy of muscle cells. However, little is known as to how muscle cells sense mechanical load and transduce it into intracellular signals of gene regulation. We examined roles of putative cellular mechanotransducers, mechanosensitive ion channels, the cytoskeleton, and contractile activity in stretch-induced hypertrophy of cardiac myocytes grown on a deformable silicone sheet. Using the patch-clamp technique, we found a single class of stretch-activated cation channel that was completely blocked by gadolinium (Gd3+). Inhibition of this channel by Gd3+ did not affect either the stretch-induced expression of IE genes or the increase in protein synthesis. Neither disruption of microtubules with colchicine nor that of actin microfilaments by cytochalasin D prevented the stretch-induced IE gene expression and increase in protein synthesis. Arresting contractile activity of myocytes by high K+, tetrodotoxin, or Ba2+ did not affect the stretch-induced IE gene expression. Tetrodotoxin-arrested myocytes could increase protein synthesis in response to stretch. These results suggest that Gd(3+)-sensitive ion channels, microtubules, microfilaments, and contractile activity may not be necessary for transduction of mechanical stretch into the IE gene expression and hypertrophy. The stimulus of membrane stretch may be transmitted to the cell nucleus through some mechanisms other than electrical or direct mechanical transduction in cardiac myocytes.     

Isolation and characterization of a homologue of mammalian prolactin-releasing peptide from the tilapia brain and its effect on prolactin release from the tilapia pituitary

In the tilapia (Oreochromis mossambicus), as in many teleosts, prolactin (PRL) plays a major role in osmoregulation in freshwater. Recently, PRL-releasing peptides (PrRPs) have been characterized in mammals. Independently, a novel C-terminal RF (arginine-phenylalanine) amide peptide (Carrasius RF amide; C-RFa), which is structurally related to mammalian PrRPs, has been isolated from the brain of the Japanese crucian carp. The putative PrRP was purified from an acid extract of tilapia brain by affinity chromatography with antibody against synthetic C-RFa and HPLC on a reverse-phase ODS-120 column. The tilapia PrRP cDNA was subsequently cloned by polymerase chain reaction. The cDNA consists of 619 bp encoding a preprohormone of 117 amino acids. Sequence comparison of the isolated peptide and the preprohormone revealed that tilapia PrRP contains 20 amino acids and is identical to C-RFa. Incubation of the tilapia pituitary with synthetic C-RFa (100 nM) significantly stimulated the release of two forms of tilapia PRL (PRL188 and PRL177). However, the effect of C-RFa was less pronounced than the marked increase in PRL release in response to hyposmotic medium. The ability of C-RFa to stimulate PRL release appears to be specific, since C-RFa failed to stimulate growth hormone release from the pituitary in organ culture. In contrast, rat and human PrRPs had no effect on PRL release. C-RFa was equipotent with chicken GnRH in stimulating PRL release in the pituitary preincubated with estradiol 17beta. Circulating levels of PRL were significantly increased 1 h after intraperitoneal injection of 0.1 microg/g of C-RFa in female tilapia in freshwater but not in males. These results suggest that C-RFa is physiologically involved in the control of PRL secretion in tilapia.     

Clinical utility and approach to estimate postprandial hypertriglycemia by a newly designed oral fat-loading test

The objective of this study was to estimate postprandial hypertriglycemia by a newly designed oral fat-loading test. Twenty-three healthy normolipidemic volunteers were orally administered a test meal consisting of a mixture of Telmeal 2.0 and 20 g of salt-free butter after fasting for 12 h. To measure the levels of total cholesterol (T-Cho), triglycerides (TG), high-density lipoprotein-cholesterol (HDL-C), remnant-like particle-cholesterol (RLP-C), lipoprotein (a) [Lp (a)], free fatty acid, apolipoproteins (Apos), plasma glucose (PG), immunoreactive insulin (IRI), and high-sensitivity C-reactive protein (hs-CRP), venous blood samples were collected before the meal and at each hour until 9 h after fat-loading. The levels of both TG and RLP-C were drastically elevated at 2 h after fat-loading and these levels remained high until 4 h (p < 0.01). A significant correlation between TG and RLP-C was also observed at 2, 3 and 4 h, and the values of the correlation coefficients (r) were 0.837, 0.838, and 0.908, respectively. In contrast, the levels of T-Cho, HDL-C, Lp (a), Apos, PG, and hs-CRP did not change. Furthermore, there were no gastrointestinal symptoms during or after the study. These results strongly suggested that this newly designed fat-loading test was very useful for evaluating postprandial hypertriglycemia, including remnant concentrations.     

Growth hormone insensitivity syndrome associated with syringomyelia and type I Chiari malformation

A 49-year-old man with syringomyelia and a Type I Arnold-Chiari malformation (Chiari-I) was diagnosed with growth hormone insensitivity syndrome (GHIS). He was short in stature, had high circulating levels of GH, and low circulating levels of insulin-like growth factor-I (IGF-I) and IGF binding protein-3 (IGFBP-3). His GH responses to the administration of growth hormone-releasing hormone (GHRH) and L-DOPA were normal, but his levels of IGF-I and IGFBP-3 did not increase after the administration of exogenous GH. Direct genomic DNA sequencing revealed neither a mutation nor deletion in this patient's GH receptor (GHR) gene, though one polymorphism was detected, indicating that his GHR gene was normal. This is the first reported case of an association of GHIS with syringomyelia and Chiari-I malformation.     

Fasting induces a large, leptin-dependent increase in the intrinsic action potential frequency of orexigenic arcuate nucleus neuropeptide Y/Agouti-related protein neurons

The neuropeptide Y (NPY)/Agouti-related protein (AgRP) neurons of the hypothalamic arcuate nucleus are thought to promote feeding. Here, we demonstrate that feeding state in vivo, through a leptin-dependent process, induces large and persistent changes in the electrophysiological activity of these neurons as measured extracellularly in vitro. Consistent with an orexigenic role, fasting induced a 4-fold increase in the basal action potential frequency of NPY/AgRP neurons. Leptin, when injected into fasted wild-type mice, induced a dose- and time-dependent decrease in spike frequency, which approached fed levels 2-3 h post treatment. In leptin-deficient (lep(ob)/lep(ob)) and leptin receptor-deficient (lepr(db)/lepr(db)) mice, NPY/AgRP spike frequency was not significantly increased by fasting, and even in mutant mice fed ad libitum, spike frequency was at least as high as in fasted wild-type mice. All recordings included GABA(A) and ionotropic glutamate receptor antagonists, suggesting that expression of this modulation is potentially intrinsic and not synaptically dependent. Recorded neurons were unambiguously identified using NPY-Sapphire transgenic mice. This is a remarkably straightforward example of a very robust in vitro electrophysiogical effect produced by a simple behavioral manipulation, food restriction.