American Journal of Physiology最新文献

Cardiac hypothermia alters contractility and intracellular Ca2+ concentration ([Ca2+]i) homeostasis. We examined how left ventricular pressure (LVP) is altered as a function of cytosolic [Ca2+]i over a range of extracellular CaCl2 concentration ([CaCl2]e) during perfusion of isolated, paced guinea pig hearts at 37 degrees C, 27 degrees C, and 17 degrees C. Transmural LV phasic [Ca2+] was measured using the Ca2+ indicator indo 1 and calibrated (in nM) after correction was made for autofluorescence, temperature, and noncytosolic Ca2+. Noncytosolic [Ca2+]i, cytosolic diastolic and systolic [Ca2+]i, phasic [Ca2+]i, and systolic Ca2+ released per beat (area Ca2+) were plotted as a function of 0.3-4.5 mM [CaCl2]e, and indexes of contractility [LVP, maximal rates of LVP development (+dLVP/dt) and relaxation (-dLVP/dt), and the integral of the LVP curve per beat (LVParea)] were plotted as a function of [Ca2+]i. Hypothermia increased systolic [Ca2+]i and slightly changed systolic LVP but increased diastolic LVP and [Ca2+]i. The relationship of diastolic and noncytosolic [Ca2+] to [CaCl2]e was shifted upward at 17 degrees C and 27 degrees C, whereas that of phasic [Ca2+]) to [CaCl2]e was shifted upward at 17 degrees C but not at 27 degrees C. The relationships of phasic [Ca2+]i to developed LVP, +dLVP/dt, and LVP(area) were progressively reduced by hypothermia so that maximal Ca2+-activated LVP decreased and hearts were desensitized to Ca2+. Thus mild hypothermia modestly increases diastolic and noncytosolic Ca2+ with little effect on systolic Ca2+ or released (area) Ca2+, whereas moderate hypothermia markedly increases diastolic, noncytosolic, peak systolic, and released Ca2+ and results in reduced maximal Ca2+-activated LVP and myocardial sensitivity to systolic Ca2+.

The actin cytoskeleton is an important contributor to the modulation of the cell function. However, little is known about the regulatory role of this supermolecular structure in the membrane events that take place in the heart. In this report, the regulation of cardiac myocyte function by actin filament organization was investigated in neonatal mouse cardiac myocytes (NMCM) from both wild-type mice and mice genetically devoid of the actin filament severing protein gelsolin (Gsn-/-). Cardiac L-type calcium channel currents (I(Ca)) were assessed using the whole cell voltage-clamp technique. Addition of the actin filament stabilizer phalloidin to wild-type NMCM increased I(Ca) by 227% over control conditions. The basal I(Ca) of Gsn-/- NMCM was 300% higher than wild-type controls. This increase was completely reversed by intracellular perfusion of the Gsn-/- NMCM with exogenous gelsolin. Further, cytoskeletal disruption of either Gsn-/- or phalloidin-dialyzed wild-type NMCM with cytochalasin D (CD) decreased the enhanced I(Ca) by 84% and 87%, respectively. The data indicate that actin filament stabilization by either a lack of gelsolin or intracellular dialysis with phalloidin increase I(Ca), whereas actin filament disruption with CD or dialysis of Gsn-/- NMCM with gelsolin decrease I(Ca). We conclude that cardiac L-type calcium channel regulation is tightly controlled by actin filament organization. Actin filament rearrangement mediated by gelsolin may contribute to calcium channel inactivation.

We tested the hypothesis that a fish oil (FO) diet promotes positive inotropy of ouabain without increased toxicity. For 2 mo, two groups of adult male rats were fed 1) a regular food diet supplemented with dietary long-chain polyunsaturated fatty acid from FO or 2) a regular food diet (control). The responsiveness to ouabain was evaluated for the two groups in Langendorff-perfused hearts, by (31)P nuclear magnetic resonance spectroscopy, and on purified membrane-bound Na-K-ATPase. The maximum positive inotropy achieved with ouabain was nearly two times higher in the FO than in the control group and was not associated with significant changes in energetics. Alteration of function and energetic metabolism and inhibition of Na-K-ATPase in response to 3 x 10(-4) M ouabain were delayed in the FO group. This study demonstrates that dietary FO, by a cardiac membrane incorporation of n-3 polyunsaturated fatty acid, promotes positive inotropy of ouabain without toxicity and changes in cardiac metabolism.

A reduction of renal kallikrein has been found in non-insulin-treated diabetic individuals, suggesting that an impaired renal kallikrein-kinin system (KKS) contributes to the development of diabetic nephropathy. We analyzed relevant components of the renal KKS in non-insulin-treated streptozotocin (STZ)-induced diabetic rats. Twelve weeks after a single injection of STZ, rats were normotensive and displayed hyperglycemia, polyuria, proteinuria, and reduced glomerular filtration rate. Blood bradykinin (BK) levels and prekallikrein activity were significantly increased compared with controls. Renal kallikrein activity was reduced by 70%, whereas urinary BK levels were increased up to threefold. Renal kininases were decreased as indicated by a 3-fold reduction in renal angiotensin-converting enzyme activity and a 1.8-fold reduction in renal expression of neutral endopeptidase 24.11. Renal cortical expression of kininogen and B2 receptors was enhanced to 1.4 and 1. 8-fold, respectively. Our data suggest that increased urinary BK levels found in severely hyperglycemic STZ-diabetic rats are related to increased filtration of components of the plasma KKS and/or renal kininogen synthesis in combination with decreased renal kinin-degrading activity. Thus, despite reduced renal kallikrein synthesis, renal KKS is activated in the advanced stage of diabetic nephropathy.

The characteristics and modulation of glucose transport were investigated in skeletal muscles of 5-wk-old Muscovy ducklings (Cairina moschata). Glucose uptake by sarcolemmal vesicles isolated from gastrocnemius muscle followed typical Michaelis-Menten kinetics with a K(m) value (17 mM) similar to that described in equivalent mammalian preparations. Western blot analysis of duckling sarcolemma using antibodies directed against rat GLUT-4 transporter revealed an immunoreactive protein of similar molecular mass (45 kDa) to that present in rats. When ducklings were killed in the postabsorptive state, GLUT-4 homologous protein was located predominantly (80%) in intracellular membranes. Insulin stimulation of a perfused leg muscle preparation in vitro led to the translocation of GLUT-4 homologous proteins from intracellular pools to the sarcolemma, with a subsequent increase in glucose uptake by sarcolemmal vesicles and perfused muscles. Glucose transport was positively controlled by the metabolic needs of skeletal muscle as reflected by the increased glucose uptake of sarcolemmal vesicles isolated from cold-acclimated ducklings. Present results, therefore, demonstrate, for the first time in an avian species, the existence in skeletal muscle of a glucose transporter showing molecular and functional homologies with the mammalian GLUT-4 transporter.

Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(-)]. cAMP stimulation of ABP(-) cells or cells genetically rescued with ABP-280 cDNA [ABP(+)] was without effect on whole cell Cl(-) currents. In ABP(-) cells expressing CFTR, cAMP was also without effect on Cl(-) conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell Cl(-) currents of ABP(+)/CFTR(+) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, however, resulted in a DPC-inhibitable increase in the whole cell currents of ABP(-)/CFTR(+) cells. At the single-channel level, protein kinase A plus ATP activated single Cl(-) channels only in excised patches from ABP(+)/CFTR(+) cells. Furthermore, filamin alone also induced Cl(-) channel activity in excised patches of ABP(-)/CFTR(+) cells. The present data indicate that an organized actin cytoskeleton is required for cAMP-dependent activation of CFTR.

NHE1, NHE2, and NHE3 are well-characterized cloned members of the mammalian Na(+)/H(+) exchanger (NHE) gene family. Given the specialized function and regulation of NHE1, NHE2, and NHE3, we compared basal turnover numbers of NHE1, NHE2, and NHE3 measured in the same cell system: PS120 fibroblasts lacking endogenous NHEs. NHE1, NHE2, and NHE3 were epitope tagged with vesicular stomatitis virus glycoprotein (VSVG). The following characteristics were determined on the same passage of cells transfected with NHE1V, NHE2V, or NHE3V: 1) maximal reaction velocity (V(max)) by (22)Na(+) uptake and fluorometery, 2) total amount of NHE protein by quantitative Western analysis with internal standards of VSVG-tagged maltose-binding protein, and 3) cell surface expression by cell surface biotinylation. Cell surface expression (percentage of total NHE) was 88.8 +/- 3.5, 64.6 +/- 3.3, 20.0 +/- 2.6, and 14.0 +/- 1.3 for NHE1V, 85- and 75-kDa NHE2V, and NHE3V, respectively. Despite these divergent cell surface expression levels, turnover numbers for NHE1, NHE2, and NHE3 were similar (80.3 +/- 9.6, 92.1 +/- 8.6, and 99.2 +/- 9.1 s(-1), when V(max) was determined using (22)Na uptake at 22 degrees C and 742 +/- 47, 459 +/- 16, and 609 +/- 39 s(-1) when V(max) was determined using fluorometry at 37 degrees C). These data indicate that, in the same cell system, intrinsic properties that determine turnover number are conserved among NHE1, NHE2, and NHE3.

The continuous intragastric enteral feeding protocol in the rat was a major development in alcohol-induced liver injury (ALI) research. Much of what has been learned to date involves inhibitors or nutritional manipulations that may not be specific. Knockout technology avoids these potential problems. Therefore, we used long-term intragastric cannulation in mice to study early ALI. Reactive oxygen species are involved in mechanisms of early ALI; however, their key source remains unclear. Cytochrome P-450 (CYP)2E1 is induced predominantly in hepatocytes by ethanol and could be one source of reactive oxygen species leading to liver injury. We aimed to determine if CYP2E1 was involved in ALI by adapting the enteral alcohol (EA) feeding model to CYP2E1 knockout (-/-) mice. Female CYP2E1 wild-type (+/+) or -/- mice were given a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 wk. All mice gained weight steadily over 4 wk, and there were no significant differences between groups. There were also no differences in ethanol elimination rates between CYP2E1 +/+ and -/- mice after acute ethanol administration to naive mice or mice receiving EA for 4 wk. However, EA stimulated rates 1.4-fold in both groups. EA elevated serum aspartate aminotransferase levels threefold to similar levels over control in both CYP2E1 +/+ and -/- mice. Liver histology was normal in control groups. In contrast, mice given ethanol developed mild steatosis, slight inflammation, and necrosis; however, there were no differences between the CYP2E1 +/+ and -/- groups. Chronic EA induced other CYP families (CYP3A, CYP2A12, CYP1A, and CYP2B) to the same extent in CYP2E1 +/+ and -/- mice. Furthermore, POBN radical adducts were also similar in both groups. Data presented here are consistent with the hypothesis that oxidants from CYP2E1 play only a small role in mechanisms of early ALI in mice. Moreover, this new mouse model illustrates the utility of knockout technology in ALI research.

The ability to localize transplanted hepatocytes in the liver offers exciting new opportunities. Transplanted hepatocytes enter liver plates, form hybrid plasma membrane structures with adjacent hepatocytes, express liver genes correctly, and survive indefinitely. The transplanted cell mass is regulated, such that cell proliferation is limited in the normal adult liver, whereas the liver is repopulated extensively when proliferation rates in transplanted and host hepatocytes become dissociated or host hepatocytes are ablated selectively. Transplanted hepatocytes are susceptible to hepatitis viruses. These aspects of transplanted hepatocyte biology indicate that liver repopulation systems can help address questions concerning pathophysiological mechanisms.

We have developed a simple laboratory exercise that uses an inexpensive dialysis membrane (molecular weight cutoff = 100) to illustrate the generation of membrane potentials (Vm) across plasma membranes of animal cells. A piece of membrane approximately 2.0 cm2 is mounted in an Ussing-like chamber. One chamber half is designated cytosol and the other half external. Chamber sidedness helps students relate their findings to those of real cells. As in real cells, outward directed K+ concentration gradients [high cytosolic K+ concentration ([K+]c) and low extracellular K+ concentration] generate cytosol electrically negative Vm with a slope of approximately -45 mV/decade change in [K+]c. The polarity of Vm reflects the outward flow of potassium ions because flow of the larger counterion, H2PO4-, is restricted to the pores in the membrane. A slope less than Nernstian (<59 mV/decade) suggests that the membrane is slightly permeable to H2PO4-. Importantly, this facilitates teaching the use of the Nernst equation to quantify the relationship between ion concentration ratios across membranes and magnitude of Vm. For example, students use their data and calculate a permeability ratio PK/PH2PO4 that corresponds to a slope of approximately 24% less than Nernstian. This calculation shows that Nernstian slopes are achieved only when permeability to the counterion is zero. Finally, students use the concept of membrane capacitance to calculate the number of ions that cross the membrane. They learn where these ions are located and why the bulk solutions conform to the principle of electroneutrality.