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1.
Nat Rev Mol Cell Biol ; 2(12): 898-907, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11733769

ABSTRACT

The ability to visualize protein dynamics and biological processes by in vivo microscopy is revolutionizing many areas of biology. These methods generate large, kinetically complex data sets, which often cannot be intuitively interpreted. The combination of dynamic imaging and computational modelling is emerging as a powerful tool for the quantitation of biophysical properties of molecules and processes. The new discipline of computational cell biology will be essential in uncovering the pathways, mechanisms and controls of biological processes and systems as they occur in vivo.


Subject(s)
Microscopy, Fluorescence/methods , Models, Biological , Proteins/metabolism , Animals , Biophysical Phenomena , Biophysics , Computational Biology , Cyclic AMP/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Humans , Kinetics , Photochemistry , Protein Binding , Proteins/chemistry
2.
J Cell Biol ; 153(3): 529-41, 2001 Apr 30.
Article in English | MEDLINE | ID: mdl-11331304

ABSTRACT

The endocytic itineraries of lipid raft markers, such as glycosyl phosphatidylinositol (GPI)-anchored proteins and glycosphingolipids, are incompletely understood. Here we show that different GPI-anchored proteins have different intracellular distributions; some (such as the folate receptor) accumulate in transferrin-containing compartments, others (such as CD59 and GPI-linked green fluorescent protein [GFP]) accumulate in the Golgi apparatus. Selective photobleaching shows that the Golgi pool of both GPI-GFP and CD59-GFP constantly and rapidly exchanges with the pool of these proteins found on the plasma membrane (PM). We visualized intermediates carrying GPI-GFP from the Golgi apparatus to the PM and separate structures delivering GPI-GFP to the Golgi apparatus.GPI-GFP does not accumulate within endocytic compartments containing transferrin, although it is detected in intracellular structures which are endosomes by the criteria of accessibility to a fluid phase marker and to cholera and shiga toxin B subunits (CTxB and STxB, which are also found in rafts). GPI-GFP and a proportion of the total CTxB and STxB taken up into cells are endocytosed independently of clathrin-associated machinery and are delivered to the Golgi complex via indistinguishable mechanisms. Hence, they enter the Golgi complex in the same intermediates, get there independently of both clathrin and rab5 function, and are excluded from it at 20 degrees C and under conditions of cholesterol sequestration. The PM-Golgi cycling pathway followed by GPI-GFP could serve to regulate lipid raft distribution and function within cells.


Subject(s)
Cell Membrane/metabolism , Golgi Apparatus/metabolism , Membrane Microdomains/metabolism , Biological Transport , CD59 Antigens/metabolism , Cell Compartmentation , Cholera Toxin/metabolism , Cholesterol , Clathrin/metabolism , Exocytosis , Glycosylphosphatidylinositols/metabolism , Green Fluorescent Proteins , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Photomicrography , Shiga Toxins/metabolism , Transferrin/metabolism
4.
Nature ; 404(6778): 604-9, 2000 Apr 06.
Article in English | MEDLINE | ID: mdl-10766243

ABSTRACT

The mammalian cell nucleus contains numerous sub-compartments, which have been implicated in essential processes such as transcription and splicing. The mechanisms by which nuclear compartments are formed and maintained are unclear. More fundamentally, it is not known how proteins move within the cell nucleus. We have measured the kinetic properties of proteins in the nucleus of living cells using photobleaching techniques. Here we show that proteins involved in diverse nuclear processes move rapidly throughout the entire nucleus. Protein movement is independent of energy, which indicates that proteins may use a passive mechanism of movement. Proteins rapidly associate and dissociate with nuclear compartments. Using kinetic modelling, we determined residence times and steady-state fluxes of molecules in two main nuclear compartments. These data show that many nuclear proteins roam the cell nucleus in vivo and that nuclear compartments are the reflection of the steady-state association/dissociation of its 'residents' with the nucleoplasmic space. Our observations have conceptual implications for understanding nuclear architecture and how nuclear processes are organized in vivo.


Subject(s)
Cell Nucleus/metabolism , Nuclear Proteins/metabolism , Biological Transport , Cell Line , Chromosomal Proteins, Non-Histone/metabolism , Fluorescence , HeLa Cells , High Mobility Group Proteins/metabolism , Humans , Kinetics , RNA-Binding Proteins , Recombinant Fusion Proteins/metabolism , Serine-Arginine Splicing Factors
5.
Cell ; 99(6): 589-601, 1999 Dec 10.
Article in English | MEDLINE | ID: mdl-10612395

ABSTRACT

Quantitative imaging and photobleaching were used to measure ER/Golgi recycling of GFP-tagged Golgi proteins in interphase cells and to monitor the dissolution and reformation of the Golgi during mitosis. In interphase, recycling occurred every 1.5 hr, and blocking ER egress trapped cycling Golgi enzymes in the ER with loss of Golgi structure. In mitosis, when ER export stops, Golgi proteins redistributed into the ER as shown by quantitative imaging in vivo and immuno-EM. Comparison of the mobilities of Golgi proteins and lipids ruled out the persistence of a separate mitotic Golgi vesicle population and supported the idea that all Golgi components are absorbed into the ER. Moreover, reassembly of the Golgi complex after mitosis failed to occur when ER export was blocked. These results demonstrate that in mitosis the Golgi disperses and reforms through the intermediary of the ER, exploiting constitutive recycling pathways. They thus define a novel paradigm for Golgi genesis and inheritance.


Subject(s)
Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Mitosis/physiology , Saccharomyces cerevisiae Proteins , Animals , Cell Line , Cytokines/metabolism , Endoplasmic Reticulum/ultrastructure , Fluorescent Antibody Technique , Galactosyltransferases/genetics , Golgi Apparatus/ultrastructure , Green Fluorescent Proteins , Humans , Interphase/physiology , Intracellular Membranes/metabolism , Luminescent Proteins/genetics , Metaphase/physiology , Microscopy, Electron , Monomeric GTP-Binding Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Vesicular Transport Proteins
6.
J Cell Biol ; 143(6): 1485-503, 1998 Dec 14.
Article in English | MEDLINE | ID: mdl-9852146

ABSTRACT

Quantitative time-lapse imaging data of single cells expressing the transmembrane protein, vesicular stomatitis virus ts045 G protein fused to green fluorescent protein (VSVG-GFP), were used for kinetic modeling of protein traffic through the various compartments of the secretory pathway. A series of first order rate laws was sufficient to accurately describe VSVG-GFP transport, and provided compartment residence times and rate constants for transport into and out of the Golgi complex and delivery to the plasma membrane. For ER to Golgi transport the mean rate constant (i.e., the fraction of VSVG-GFP moved per unit of time) was 2.8% per min, for Golgi to plasma membrane transport it was 3.0% per min, and for transport from the plasma membrane to a degradative site it was 0.25% per min. Because these rate constants did not change as the concentration of VSVG-GFP in different compartments went from high (early in the experiment) to low (late in the experiment), secretory transport machinery was never saturated during the experiments. The processes of budding, translocation, and fusion of post-Golgi transport intermediates carrying VSVG- GFP to the plasma membrane were also analyzed using quantitative imaging techniques. Large pleiomorphic tubular structures, rather than small vesicles, were found to be the primary vehicles for Golgi to plasma membrane transport of VSVG-GFP. These structures budded as entire domains from the Golgi complex and underwent dynamic shape changes as they moved along microtubule tracks to the cell periphery. They carried up to 10,000 VSVG-GFP molecules and had a mean life time in COS cells of 3.8 min. In addition, they fused with the plasma membrane without intersecting other membrane transport pathways in the cell. These properties suggest that the post-Golgi intermediates represent a unique transport organelle for conveying large quantities of protein cargo from the Golgi complex directly to the plasma membrane.


Subject(s)
Cell Membrane/metabolism , Golgi Apparatus/metabolism , Membrane Glycoproteins , Viral Envelope Proteins/metabolism , Aluminum Compounds/pharmacology , Animals , Biological Transport , COS Cells , Cell Membrane/drug effects , Cell Membrane/ultrastructure , Cytochalasin B/pharmacology , Fluorides/pharmacology , Golgi Apparatus/drug effects , Golgi Apparatus/ultrastructure , Green Fluorescent Proteins , Kinetics , Luminescent Proteins/metabolism , Models, Biological , Nocodazole/pharmacology , Recombinant Fusion Proteins/metabolism , Time Factors , Transfection , Vesicular stomatitis Indiana virus/genetics , Viral Envelope Proteins/genetics
7.
J Lipid Res ; 38(12): 2422-35, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9458266

ABSTRACT

Niemann-Pick C disease (NP-C) is a rare inborn error of metabolism with hepatic involvement and neurological sequelae that usually manifest in childhood. Although in vitro studies have shown that the lysosomal distribution of LDL-derived cholesterol is defective in cultured cells of NP-C subjects, no unusual characteristics mark the plasma lipoprotein profiles. We set out to determine whether anomalies exist in vivo in the cellular distribution of newly synthesized, HDL-derived or LDL-derived cholesterol under physiologic conditions in NP-C subjects. Three affected and three normal male subjects were administered [14C]mevalonate as a tracer of newly synthesized cholesterol and [3H]cholesteryl linoleate in either HDL or LDL to trace the distribution of lipoprotein-derived free cholesterol. The rate of appearance of free [14C]- and free [3H]cholesterol in the plasma membrane was detected indirectly by monitoring their appearance in plasma and bile. The plasma disappearance of [3H]cholesteryl linoleate was slightly faster in NP-C subjects regardless of its lipoprotein origin. Appearance of free [14C] cholesterol ill the plasma (and in bile) was essentially identical in normal and affected individuals as was the initial appearance of free [3H]cholesterol derived from HDL, observed before extensive exchange occurred of the [3H]cholesteryl linoleate among lipoproteins. In contrast, the rate of appearance of LDL-derived free [3H]cholesterol in the plasma membrane of NP-C subjects, as detected in plasma and bile, was retarded to a similar extent that LDL cholesterol metabolism was defective in cultured fibroblasts of these affected subjects. These findings show that intracellular distribution of both newly synthesized and HDL-derived cholesterol are essentially unperturbed by the NP-C mutation, and therefore occur by lysosomal-independent paths. In contrast, in NP-C there is defective trafficking of LDL-derived cholesterol to the plasma membrane in vivo as well as in vitro. The in vivo assay of intracellular cholesterol distribution developed herein should prove useful to quickly evaluate therapeutic interventions for NP-C.


Subject(s)
Cholesterol Esters/metabolism , Cholesterol/metabolism , Lipoproteins, HDL/metabolism , Lipoproteins, LDL/metabolism , Niemann-Pick Diseases/genetics , Niemann-Pick Diseases/metabolism , Adult , Bile/chemistry , Bile/metabolism , Cholesterol/blood , Cholesterol Esters/blood , Fibroblasts , Histocytochemistry , Humans , Male , Mevalonic Acid/administration & dosage , Mevalonic Acid/metabolism , Mutation
8.
Metabolism ; 46(12): 1489-95, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9439549

ABSTRACT

Increasingly, successful research on metabolic systems relies on teams of specialists. Because of the enormous complexity of these systems, many experimental groups have sought collaborations with theoreticians for data analysis and modeling. Predictably, cultural differences in scientific approach, methodology, assumptions, and language have led to some persistent difficulties in communication across the experiment-theory frontier. This report attempts to diagnose some of these difficulties from the perspective of 30 years' experience in both experimental and theoretical biology, and to suggest guidelines for effective collaboration between experimentalists and theorists. As these collaborations move to the level of cellular and molecular biology, effective communication will become all the more important because the simple linear rate laws of radiotracer and stable-isotope kinetics will no longer suffice. This is because every form of regulation and control, hallmarks of metabolic systems, results in nonlinear kinetics. To advance this transition to nonlinear cellular and molecular metabolic models and to facilitate communication between experimental and theoretical collaborators, a general procedure for incorporating control mechanisms in metabolic rate laws is developed based on the familiar rapid-equilibrium assumption of classical enzyme kinetics.


Subject(s)
Models, Biological , Molecular Biology/methods , Animals , Computer Simulation , Endocrine System/cytology , Guidelines as Topic , Humans , Linear Models , Models, Theoretical
9.
Am J Physiol ; 270(6 Pt 2): H1972-8, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8764246

ABSTRACT

Steady-state cytosolic calcium (Ca2+i) concentration in a vascular smooth muscle cell is determined by Ca2+ influx and Ca2+ extrusion across the plasma membrane, yet no means for determining the absolute magnitude of these transmembrane Ca2+ fluxes in the basal state of the resting cell has been devised. We now report a method that combines fluorescence measurement of Ca2+i, 45Ca kinetics, and computer modeling to yield the basal plasma membrane Ca2+ flux in A7r5 vascular smooth muscle cells. Kinetic analysis of basal Ca2+i and Ca2+i transients following chelation of extracellular Ca2+ yields a unique value for the ratio of the rate constant governing Ca2+ pumping into the sarcoplasmic reticulum (SR) to that for plasma membrane Ca2+ extrusion (1.12 +/- 0.06). When this ratio was used to constrain the least-squares fitting of 45Ca efflux data from A7r5 cells, it was possible to determine unique values for the unidirectional, steady-state Ca2+ fluxes across both SR and plasma membranes. The basal unidirectional plasma membrane Ca2+ flux was 0.062 +/- 0.018 fmol . min-1 . cell, and the basal SR Ca2+ flux was 0.069 +/- 0.019 fmol . min-1 . cell-1. These results demonstrate, within the limitations of measuring the absolute value of Ca2+i, the feasibility of measuring previously unresolvable subpicoamp basal Ca2+ fluxes in intact cells under normal physiological conditions.


Subject(s)
Calcium/metabolism , Cell Membrane/metabolism , Muscle, Smooth, Vascular/metabolism , Cell Line , Cytosol/metabolism , Electronic Data Processing , Fluorescent Dyes , Fura-2 , Kinetics , Microscopy, Fluorescence , Models, Cardiovascular , Muscle, Smooth, Vascular/cytology
10.
Cell Calcium ; 19(2): 167-84, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8689674

ABSTRACT

Established cell lines are now widely used in experiments concerning vascular smooth muscle (VSM) function; however, considerable evidence suggests that cultured VSM cells are functionally different from VSM cells in intact blood vessels. In order to test the hypothesis that calcium signaling mechanisms are comparable in these two preparations, we developed a new method for high resolution 45Ca efflux studies in A7r5 cells. Briefly, this method involves plating cells in the lumen of a tubular glass efflux chamber and, after loading the cells with 45Ca, perfusing the chamber with a physiological saline solution and collecting the effluent. Using this method we found that the plasma membrane in cultured cells is not rate limiting for calcium efflux, since the efflux curves from both permeabilized and intact cells are kinetically the same. We also found the plasma membrane is not rate limiting in whole aortic segments by using a depolarizing solution followed by dihydropyridine solution. Thus, we demonstrated that the data obtained from cells or tissues with intact membranes reveal information about the intracellular stores (sarcoplasmic reticulum and mitochondria). Combining efflux data with a detailed kinetic model of cellular Ca transport allows least-squares estimation of the rate constants for release and uptake of Ca2+ by intracellular stores with a high degree of confidence (CV < 25%) as well as the Ca2+ contents and transmembrane fluxes associated with these stores. Quantitative comparison of results obtained from A7r5 cells with those we previously obtained for rabbit aortic segments reveals marked similarities and suggests that A7r5 cells serve as excellent model experiments for VSM cell Ca2+ homeostasis.


Subject(s)
Calcium/metabolism , Muscle, Smooth, Vascular/metabolism , Animals , Aorta/metabolism , Calcium/pharmacokinetics , Calcium Radioisotopes/metabolism , Calcium-Transporting ATPases/antagonists & inhibitors , Cell Compartmentation/physiology , Cell Membrane/metabolism , In Vitro Techniques , Models, Molecular , Muscle, Smooth, Vascular/cytology , Platelet-Derived Growth Factor/pharmacology , Rabbits , Rats , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/metabolism , Signal Transduction , Terpenes/pharmacology , Thapsigargin , Time Factors
11.
Arterioscler Thromb Vasc Biol ; 15(1): 44-51, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7749815

ABSTRACT

Since the platelet-derived growth factor (PDGF)-induced increase in cellular inositol 1,4,5-trisphosphate (InsP3) has been found to decay to basal levels soon after the onset of PDGF exposure, it has been argued that activation of Ca2+ release from intracellular stores must be similarly transient. The possibility remains, however, that PDGF-induced release of stored Ca2+ is initiated and sustained by other second-messenger systems. To test the hypothesis that PDGF-BB initiates sustained Ca2+ release from cellular stores, we performed 4-hour 45Ca effluxes on monolayers of A7r5 vascular smooth muscle cells in small, continuously perfused chambers. Isoform PDGF-BB (5 ng/mL for 30 minutes or 30 ng/mL for 15 minutes) was added to the perfusate beginning at 30 minutes of efflux. A dose-related increase in 45Ca release was sustained as long as PDGF-BB was present. Detailed kinetic analysis and nonlinear least-squares fitting of the experimental data revealed that (1) PDGF-BB induced sustained increases of 2.86-fold (5 ng/mL) and 6.50-fold (30 ng/mL) in the rate constant governing Ca2+ release from intracellular stores, (2) the apparent Km for this effect was 13.4 +/- 1.31 ng PDGF-BB/mL, and (3) the entire agonist-releasable Ca2+ store (presumably sarcoplasmic reticulum) is sensitive to PDGF-BB. These data indicate that PDGF-BB causes a sustained depletion of intracellular Ca2+ stores by means of sustained activation of Ca2+ release and suggest that intraorganellar Ca2+ may be one of the signals that mediates long-term smooth muscle responses to PDGF.


Subject(s)
Calcium/metabolism , Muscle, Smooth, Vascular/metabolism , Platelet-Derived Growth Factor/pharmacology , Animals , Becaplermin , Caffeine/pharmacology , Cell Line , Inositol Phosphates/metabolism , Ion Transport/drug effects , Kinetics , Models, Biological , Proto-Oncogene Proteins c-sis , Second Messenger Systems , Signal Transduction
12.
J Lipid Res ; 32(7): 1133-41, 1991 Jul.
Article in English | MEDLINE | ID: mdl-1940637

ABSTRACT

Studies were carried out in three normolipidemic non-obese men with insulin-dependent diabetes mellitus (IDDM) and three normal men, to assess whether the clearance of postprandial Sf 100-400 lipoproteins is decreased in IDDM. Sf greater than 100 lipoproteins isolated from plasma 4.5 h after fat ingestion were labeled with 125I and injected into the same subject intravenously. ApoB radioactivity was measured over time in Sf greater than 400, Sf 100-400, and Sf 20-100 lipoproteins isolated from plasma and analyzed using a kinetic model that included both fast and slow delipidation cascades, where lipolysis and uptake of particles by the liver and other tissues were represented. Fractional catabolic rates of Sf 100-400 lipoproteins (min-1) were decreased in diabetic versus control subjects: fast = 0.170 +/- 0.126 versus 0.680 +/- 0.242 (mean +/- SD) (P less than 0.05, two-tailed) and slow = 0.011 +/- 0.006 versus 0.031 +/- 0.015 (P less than 0.05, one-tailed). Kinetic analysis showed that the data were consistent with decreased uptake by the tissues for the fast cascade (diabetic, 0.084 +/- 0.082, vs. control, 0.617 +/- 0.328, P less than 0.05, one-tailed). A similar trend was observed for the slow cascade. There were no significant differences between the two groups in the intraplasma lipolysis rates of Sf 100-400 particles. Analysis of the composition of the injected particles showed that they were total cholesterol (TC)- versus triglyceride (TG)-enriched (P less than 0.001, log-ratio analysis of composition) in IDDM subjects.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Diabetes Mellitus, Type 1/blood , Dietary Fats/pharmacokinetics , Lipoproteins/pharmacokinetics , Triglycerides/blood , Adult , Dietary Fats/blood , Humans , Lipoproteins/blood , Lipoproteins/isolation & purification , Male , Metabolic Clearance Rate , Models, Molecular
13.
Am J Physiol ; 257(3 Pt 1): C413-8, 1989 Sep.
Article in English | MEDLINE | ID: mdl-2782384

ABSTRACT

We combined techniques of 45Ca efflux and computer-assisted kinetic analysis to investigate the effects of forskolin and caffeine on intracellular Ca2+ metabolism in intact rabbit aortic segments. When either 1 microM forskolin or 10 mM caffeine was present during the 45Ca load and efflux, the amount of 45Ca released from the tissue was reduced during the interval between 50 and 250 min of efflux. In contrast caffeine but not forskolin induced an acute increase in 45Ca efflux when added to the perfusion medium after 1 h of efflux in physiological salt solution. Preincubation with caffeine abolished phenylephrine-stimulated 45Ca release, but preincubation with forskolin had no effect. Kinetic analysis of these data indicated that caffeine reduced the Ca2+ content of the same intracellular compartment depleted by phenylephrine, whereas forskolin depleted a different intracellular Ca2+ store. Forskolin-induced depletion of an intracellular Ca2+ store was surprising in light of current evidence suggesting adenosine 3',5'-cyclic monophosphate stimulation of sarcoplasmic reticular Ca2+ uptake, but hypotheses that included only this mechanism are inconsistent with our findings.


Subject(s)
Aorta/metabolism , Caffeine/pharmacology , Calcium/metabolism , Colforsin/pharmacology , Animals , Aorta/cytology , Biological Transport/drug effects , Calcium/pharmacokinetics , Electronic Data Processing , Female , Male , Rabbits
14.
Cell Calcium ; 9(5-6): 275-84, 1988 Dec.
Article in English | MEDLINE | ID: mdl-3066492

ABSTRACT

Evidence for and against the theory that cell calcium is causally involved in the pathogenesis of atherosclerosis is presented and evaluated. In particular, it is argued that: (1) arterial calcium is increased in atherosclerosis; (2) this increase in tissue calcium content is largely intracellular; (3) this increased intracellular calcium content is caused by increased plasma membrane calcium permeability; (4) the increased calcium content is causally related to atherogenesis; (5) many of the cell physiological, cell biological, biochemical, and molecular biological processes, known to function abnormally in atherosclerosis, are also known to be calcium regulated; and (6) these processes are activated or inactivated in atherosclerosis in a manner consistent with increased cell calcium. It is concluded that the calcium-atherogenesis hypothesis has the potential to unify macroscopic clinical risk factors in terms of intracellular mechanisms that are controlled by cell calcium, and that this hypothesis deserves further experimental tests.


Subject(s)
Arteriosclerosis/etiology , Calcium/physiology , Animals , Arteries/analysis , Arteries/pathology , Arteriosclerosis/blood , Calcium/analysis , Calcium/blood , Cell Membrane Permeability , Humans
15.
Circ Res ; 62(1): 75-80, 1988 Jan.
Article in English | MEDLINE | ID: mdl-3335059

ABSTRACT

Several lines of evidence, including the reported ability of calcium channel blockers to prevent atherogenesis in cholesterol-fed rabbits, suggest that calcium mediates one or more of the pathologic changes in atherosclerosis. Moreover, it has long been known that calcium accumulates in atherosclerotic blood vessels. To test the hypothesis that a substantial fraction of this accumulated calcium is intracellular and to identify possible causes of this accumulation, calcium fluxes and contents were determined in aortic segments from cholesterol-fed rabbits and age-matched controls. A new method, based on 45Ca efflux experiments and computer-assisted kinetic analysis, was used to measure intracellular and extracellular calcium contents (nmol calcium/g wet wt tissue) and fluxes. Total intracellular calcium increased from 269 +/- 11.6 to 1,300 +/- 352 nmol/g in cholesterol-fed animals compared with controls (p less than 0.01). This change was sufficient to account for the observed increase in total tissue calcium from 4,190 +/- 211 to 5,240 +/- 477 nmol/g (p less than 0.05). Thus, the fraction of tissue calcium that is intracellular increased significantly from 0.065 +/- 0.006 to 0.223 +/- 0.048 (p less than 0.01) in experimental atherosclerosis. In addition, the data were quantitatively consistent with the hypothesis that these changes are brought about by a 4.8-fold increase in the plasma membrane calcium permeability of aortic smooth muscle cells. These results provide evidence that increased intracellular calcium is a possible mediator of cholesterol-induced atherogenesis.


Subject(s)
Aorta/metabolism , Arteriosclerosis/metabolism , Calcium/metabolism , Animals , Biological Transport, Active , Calcium Channel Blockers/pharmacology , Cell Membrane/metabolism , Cholesterol, Dietary , Kinetics , Male , Rabbits
16.
Circ Res ; 59(1): 74-84, 1986 Jul.
Article in English | MEDLINE | ID: mdl-2426004

ABSTRACT

A new method, based on computer-assisted kinetic analysis of 45Ca efflux data, was used to measure calcium contents and fluxes for extracellular and intracellular compartments in intact segments of rabbit aorta. After a 1-hour loading period, efflux data were collected for 8 hours using a flow-through tissue chamber. These long-term effluxes were necessary because information on intracellular calcium metabolism was concentrated in the slow components of the efflux curves while earlier components appeared to be dominated by washout of extracellular calcium. Intracellular compartments were identified as those whose calcium contents were altered by 10 microM phenylephrine. This method complements previous approaches by providing simultaneous estimates of compartmental calcium contents and fluxes without requiring the assumption of isotopic equilibrium and without recourse to standard wash techniques for removal of extracellular calcium. In normal, calcium-containing, bicarbonate-buffered physiological salt solution these compartments contained a total of approximately 300 nmol Ca/g wet aorta. Of this total, 55 nmol/g were associated with the slowest resolvable compartment whose turnover time was 170 minutes and whose exchange flux was 0.32 nmol min-1g-1. Two other intracellular compartments had turnover times of 30 minutes. One of these was phenylephrine releasable and contained 145 nmol/g; it exchanged calcium at 4.9 nmol min-1g-1. In normal physiological salt solution the plasma membrane was, surprisingly, not rate limiting for Ca efflux; and in 10 microM phenylephrine the membrane Ca flux was even greater, increasing 3.5-fold compared to control.


Subject(s)
Aorta, Thoracic/metabolism , Calcium/metabolism , Ion Channels/metabolism , Muscle, Smooth, Vascular/metabolism , Animals , Calcium Radioisotopes , Cell Membrane Permeability , Female , Ion Channels/drug effects , Kinetics , Male , Muscle, Smooth, Vascular/drug effects , Phenylephrine/pharmacology , Rabbits , Time Factors
17.
Circ Res ; 59(1): 85-92, 1986 Jul.
Article in English | MEDLINE | ID: mdl-2426005

ABSTRACT

Previous work from this laboratory revealed the presence of at least three distinct intracellular calcium compartments in intact segments of rabbit aorta. In this study one of these intracellular compartments is shown to be sensitive to dinitrophenol and to increased extracellular phosphate. Intact aortic segments were loaded with 45Ca in bicarbonate-buffered physiologic salt solution for 1 hour, and then transferred to a flow-through chamber perfused with physiologic salt solution. Effluent from the chamber was collected for 8 hours, and 45Ca efflux curves were analyzed using compartmental analysis. When aortic segments were loaded and washed out in dinitrophenol, the slowest component of the efflux curve was less prominent; in high phosphate it was more prominent. The rate constant changes required to account for these data were primarily in the exchange between the cytosolic and slowest intracellular calcium compartment, suggesting that the slowest calcium compartment resolved during the 8-hour washout was mitochondrial. This compartment contained 5.4 +/- 3.2 nmol calcium/g wet wt. tissue. The calcium flux across its membranes was 0.32 +/- 0.04 nmol min-1g-1. Because this flux is much smaller than the plasma-membrane calcium flux, we suggest that, in normal physiological circumstances, plasma-membrane extrusion is more important for the removal of Ca from the smooth muscle cytosol than is uptake into this slow intracellular compartment.


Subject(s)
Aorta, Thoracic/metabolism , Calcium/metabolism , Dinitrophenols/pharmacology , Ion Channels/metabolism , Muscle, Smooth, Vascular/metabolism , Phosphates/pharmacology , Animals , Calcium Radioisotopes , Cell Membrane Permeability , Cytosol/metabolism , Extracellular Space/metabolism , Female , Ion Channels/drug effects , Kinetics , Male , Rabbits , Time Factors
18.
Diabete Metab ; 10(2): 71-7, 1984 May.
Article in English | MEDLINE | ID: mdl-6430728

ABSTRACT

The involvement of prostaglandins in the effects of arachidonic acid (20:4n-6) on insulin and glucagon release was investigated, using the isolated, perfused rat pancreas model. 20:4n-6, the substrate for dienoic prostaglandins, or 20:3n-3, a fatty acid that cannot be metabolized to prostaglandins were perfused over 55 min. 20: 4n-6 evoked triphasic insulin release: early and late phase during, and "off-response" following the perfusion. With 20:3n-3 the early phase of insulin release was 57% of that with 20:4n-6. 20:4n-6 stimulated only an early phase release of glucagon; 20:3n-3 had no effect. Indomethacin (10 microM, a cyclooxygenase inhibitor) inhibited by 50% the early phase of insulin and glucagon release induced by 20:4n-6, but did not modify insulin release during the early phase with 20:3n-3, or the late phase or off-response with either 20:4n-6 or 20:3n-3. We conclude that 1) the early phase release of insulin and glucagon which occurs with arachidonic acid is due in part to pancreatic biosynthesis of prostaglandins; and 2) in the other phases of insulin release evoked by the fatty acids, alternate "nonspecific" mechanisms may be involved.


Subject(s)
Arachidonic Acids/pharmacology , Glucagon/metabolism , Insulin/metabolism , Pancreas/metabolism , Prostaglandins/physiology , 8,11,14-Eicosatrienoic Acid/pharmacology , Animals , Arachidonic Acid , Indomethacin/pharmacology , Insulin Secretion , Male , Pancreas/drug effects , Perfusion , Rats , Rats, Inbred Strains , Time Factors
19.
Am J Physiol ; 246(4 Pt 2): R587-96, 1984 Apr.
Article in English | MEDLINE | ID: mdl-6326602

ABSTRACT

Adrenal secretory rates of cortisol and arterial concentrations of adrenocorticotropin (ACTH) were measured in conscious trained dogs subjected to intravenous infusion of ACTH. To investigate the causal relation of ACTH to the secretion of cortisol, a mechanistic mathematical model based on current hypotheses of adrenocortical function was constructed and tested. It is widely believed that ACTH stimulates cortisol secretion through adenosine 3',5'-cyclic monophosphate (cAMP), which provides substrate cholesterol by activating cholesterol ester hydrolase and facilitating transport of cholesterol to the side-chain cleavage enzyme. In addition, cholesterol modulates its own synthesis by inhibiting beta-hydroxy-beta-methylglutaryl (HMG)-CoA reductase in the adrenocortical cell. These and other steps in the biosynthetic reaction sequence were described using differential equations subject to the additional constraints imposed by available measurements of intracellular quantities. The resulting model is consistent with many of the known characteristics of the canine adrenal response to ACTH. In this model, steady-state nonlinearities arise from cooperative binding of cAMP to its receptor protein and saturation of mitochondrial pregnenolone transport. The transient response is dominated by a depletable pool of intracellular free cholesterol. Other inferences based on the model are presented, and a quantifiable cellular basis for increased adrenal sensitivity to ACTH is proposed.


Subject(s)
Adrenocorticotropic Hormone/pharmacology , Hydrocortisone/blood , Models, Biological , Animals , Dogs , Enzyme Activation , Female , Kinetics , Male , Mathematics , Pregnenolone/metabolism , Sterol Esterase/metabolism
20.
Am J Physiol ; 237(1): H1-9, 1979 Jul.
Article in English | MEDLINE | ID: mdl-464063

ABSTRACT

If newly formed adenosine is the mediator of active hyperemia in skeletal muscle, tissue adenosine must increase and remain elevated during sustained muscle contraction. We tested this prediction using isolated canine anterior calf muscles. Muscle samples were obtained before and during contraction by punch biopsy and adenosine was measured by spectrophotometric enzyme assay. Tissue adenosine content didnot increase significantly above the precontraction level during either the 2- or 6-Hz contraction. We estimated (by infusing adenosine) that the adenosine concentration required to cause a dilation equal to that observed during 6-Hz contractions was 3.7 X 10(-5) M. We would have detected an increase in tissue adenosine if this concentration were established in as little as 10% of the skeletal muscle interstitium during free flow. These results indicate that adenosine released into the whole interstitial space is not the cause of free-flow exercise hyperemia.


Subject(s)
Adenosine/analysis , Hyperemia/etiology , Muscles/analysis , Adenosine/biosynthesis , Animals , Dogs , Female , Male , Muscle Contraction , Oxygen Consumption
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