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1.
Circulation ; 103(18): 2289-95, 2001 May 08.
Article in English | MEDLINE | ID: mdl-11342479

ABSTRACT

BACKGROUND: Despite limiting elastic recoil and late vascular remodeling after angioplasty, coronary stents remain vulnerable to restenosis, caused primarily by neointimal hyperplasia. Paclitaxel, a microtubule-stabilizing drug, has been shown to inhibit vascular smooth muscle cell migration and proliferation contributing to neointimal hyperplasia. We tested whether paclitaxel-coated coronary stents are effective at preventing neointimal proliferation in a porcine model of restenosis. METHODS AND RESULTS: Palmaz-Schatz stents were dip-coated with paclitaxel (0, 0.2, 15, or 187 microgram/stent) by immersion in ethanolic paclitaxel and evaporation of the solvent. Stents were deployed with mild oversizing in the left anterior descending coronary artery (LAD) of 41 minipigs. The treatment effect was assessed 4 weeks after stent implantation. The angiographic late loss index (mean luminal diameter) decreased with increasing paclitaxel dose (P<0.0028 by ANOVA), declining by 84.3% (from 0.352 to 0.055, P<0.05) at the highest level tested (187 microgram/stent versus control). Accompanying this change, the neointimal area decreased (by 39.5%, high-dose versus control; P<0.05) with increasing dose (P<0.040 by ANOVA), whereas the luminal area increased (by 90.4%, high-dose versus control; P<0.05) with escalating dose (P<0.0004 by ANOVA). Inflammatory cells were seen infrequently, and there were no cases of aneurysm or thrombosis. CONCLUSIONS: Paclitaxel-coated coronary stents produced a significant dose-dependent inhibition of neointimal hyperplasia and luminal encroachment in the pig LAD 28 days after implantation; later effects require further study. These results demonstrate the potential therapeutic benefit of paclitaxel-coated coronary stents in the prevention and treatment of human coronary restenosis.


Subject(s)
Coronary Vessels/drug effects , Graft Occlusion, Vascular/prevention & control , Paclitaxel/administration & dosage , Stents , Tunica Intima/drug effects , Animals , Coronary Angiography , Coronary Vessels/chemistry , Coronary Vessels/surgery , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Graft Occlusion, Vascular/pathology , Hyperplasia/pathology , Hyperplasia/prevention & control , Infusion Pumps, Implantable , Male , Paclitaxel/analysis , Surface Properties , Swine, Miniature , Tunica Intima/pathology , Tunica Intima/surgery
2.
Biochem Cell Biol ; 76(5): 743-9, 1998.
Article in English | MEDLINE | ID: mdl-10353707

ABSTRACT

The Na+/H+ exchangers (NHE1-6) are integral plasma membrane proteins that catalyze the exchange of extracellular Na+ for intracellular H+. In addition to Na+ and H+ transport sites, NHE has an intracellular allosteric H+ modifier site that increases exchange activity when occupied by H+. NHE activity is also subject to control by a variety of extrinsic factors including hormones, growth factors, cytokines, and pharmacological agents. Many of these factors, working through second messenger pathways acting directly or indirectly on NHE, regulate NHE activity by shifting the apparent affinity of the H+ modifier site to more alkaline or more acid pH. The underlying molecular mechanisms involved in the activation of NHE by the H+ modifier site are poorly understood at this time, but likely involve slow protein conformational changes within a NHE oligomer. In this paper, we present initial experiments measuring intracellular pH-dependent transition rates between active and inactive oligomeric conformations and describe how these transition rates may be important for overall regulation of NHE activity.


Subject(s)
Sodium-Hydrogen Exchangers/chemistry , Sodium-Hydrogen Exchangers/metabolism , Animals , Hydrogen-Ion Concentration , Models, Biological , Sodium-Hydrogen Exchanger 3 , Temperature , Time Factors
3.
Am J Physiol ; 272(1 Pt 2): H244-55, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9038944

ABSTRACT

beta-Adrenergic agonists induce the relaxation of vascular smooth muscle by a mechanism that activates the extrusion of Na+ and Ca2+ from the cell. A primary source of contractile Ca2+ resides in the sarcoplasmic reticulum (SR), which releases Ca2+ in response to vasoactive agents through inositol trisphosphate-mediated channels. To determine if smooth muscle relaxation induced by beta 2-adrenergic agonists involves the redistribution of intracellular Ca2+, we studied the effects of isoproterenol (Iso) on freshly isolated, single rat tail artery smooth muscle cells loaded with fura 2, using digital ratiometric fluorescence imaging. Stimulation with 1 microM phenylephrine (PE) or norepinephrine produced phasic and tonic increases in cytoplasmic intracellular Ca2+ concentration ([Ca2+]i) associated associated with cell shortening. Exposure to caffeine and to Ca2(+)-free solutions eliminated the phasic and tonic components, respectively, from the Ca2+ signal. Intermittent superfusion with PE or caffeine was used to evaluate SR Ca2+ stores after stimulation by Iso. Exposure to 1 microM Iso induced a time-dependent decrease in PE-activated peak and tonic [Ca2+]i without any change in resting [Ca2+]i. Intermittent stimulation with 10 mM caffeine revealed a similar decline in peak [Ca2+]i, indicating Iso-dependent depletion of SR Ca2+ stores. The Ca2+ that remained in the SR after prolonged exposure to Iso (30% of the pre-Iso level by 80 min at 22 degrees C) failed to elicit a contractile response. The cells, perfused with a Na(+)- and Ca2(+)-free medium to block Na+/ Ca2+ exchange, prevented depletion of the SR Ca2+ stores by Iso. We propose that Iso inhibits agonist-mediated Ca2+ influx through sarcolemmal Ca2+ channels and activates Ca2+ redistribution from storage sites in the SR to the extracellular compartment by a mechanism that involves Na+/Ca2+ exchange. These combined effects of Iso facilitate smooth muscle relaxation (and reduce vascular tonus) by reducing the increase in cytoplasmic Ca2+ evoked by vasoconstrictors.


Subject(s)
Adrenergic beta-Agonists/pharmacology , Arteries/metabolism , Calcium/metabolism , Intracellular Membranes/metabolism , Muscle, Smooth/metabolism , Animals , Arteries/cytology , Arteries/drug effects , Fura-2 , Intracellular Membranes/drug effects , Isoproterenol/pharmacology , Male , Muscle, Smooth/cytology , Muscle, Smooth/drug effects , Phenylephrine/pharmacology , Rats , Rats, Inbred F344 , Sarcoplasmic Reticulum/metabolism , Tail/blood supply , Tissue Distribution
4.
J Cell Sci ; 108 ( Pt 12): 3685-94, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8719875

ABSTRACT

We performed differential cDNA hybridization using RNA from endothelial cells cultured for 4 hours on either plastic or basement membrane matrix (Matrigel), and identified early genes induced during the morphological differentiation into capillary-like tubes. The mRNA for one clone, thymosin beta 4, was increased 5-fold. Immunostaining localized thymosin beta 4 in vivo in both growing and mature vessels as well as in other tissues. Endothelial cells transfected with thymosin beta 4 showed an increased rate of attachment and spreading on matrix components, and an accelerated rate of tube formation on Matrigel. An antisense oligo to thymosin beta 4 inhibited tube formation on Matrigel. The results suggest that thymosin beta 4 is induced and likely involved in differentiating endothelial cells. Thymosin beta 4 may play a role in vessel formation in vivo.


Subject(s)
Collagen/pharmacology , Endothelium, Vascular/drug effects , Extracellular Matrix , Gene Expression Regulation/drug effects , Laminin/pharmacology , Microfilament Proteins/genetics , Proteoglycans/pharmacology , Thymosin/genetics , Base Sequence , Capillaries/metabolism , Cell Cycle/physiology , Cell Differentiation/genetics , Cloning, Molecular , DNA, Complementary/analysis , Drug Combinations , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Humans , Molecular Sequence Data , Transfection
5.
Exp Cell Res ; 215(2): 310-8, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7526993

ABSTRACT

Recent studies have suggested that fibroblasts, widely distributed mesenchymal cells, not only function to sustain various organs and tissues as stroma cells but also act directly to regulate adjacent cell behavior including migration, proliferation, and differentiation. Since fibroproliferative diseases and lesions (fibroplasia) are accompanied by new capillary growth (angiogenesis), we hypothesized that fibroblasts may have direct effects on endothelial cell behavior, independent of the elaboration of extracellular matrix, that are relevant to complex process of angiogenesis. To test this hypothesis, bovine aortic endothelial cells were cocultured in collagen gels with human skin fibroblasts. This coculture system caused the endothelial cells to become spindle shaped and to organize into a capillary-like structure within the collagen gels. We found that fibroblast-conditioned medium (FCM) also induced endothelial cells initially to elongate and subsequently to organize into a capillary-like structure within collagen gels. While FCM had no significant effect on endothelial cell DNA synthesis, the soluble factor(s) in FCM increased endothelial cell motility in an in vitro wound assay and in a Boyden chamber assay. The chemoattractant(s) in FCM was alkaline (pH 9.0)--and acid (pH 3.0)--stable, relatively heat stable (stable at 60 degrees for 30 min, unstable at 98 degrees C for 3 min), dithiothreitol (DTT)-sensitive, and bound to an anionic exchange resin (DEAE-cellulose). Another factor(s) stimulated endothelial cell reorganization into capillary-like structure both within a collagen gel and on a reconstituted basement membrane matrix, Matrigel. This factor(s) was alkaline (pH 9.0)- and acid (pH 3.0)--stable, heat (98 degrees C for 3 min)-stable, and DTT-sensitive and bound an anionic exchange resin (DEAE-cellulose). These in vitro results suggest that fibroblasts secrete soluble factors that can influence endothelial cell behaviors relevant to the angiogenesis process with possible implications for vascularization in fibroproliferative conditions.


Subject(s)
Endothelium, Vascular/cytology , Fibroblasts/physiology , Animals , Cattle , Cell Communication/physiology , Cell Differentiation/physiology , Cell Movement/physiology , Cell Size , Culture Media, Conditioned , DNA/biosynthesis , Endothelium, Vascular/metabolism , Humans , Neovascularization, Pathologic/etiology , Solubility
6.
J Cell Physiol ; 161(2): 267-76, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7962111

ABSTRACT

The formation of capillary-like network structures by cultured vascular endothelial cells on reconstituted basement membrane matrix, Matrigel, models endothelial cell differentiation, the final step of angiogenesis (Kubota et al., 1988; Grant et al., 1989). When endothelial cells derived from bovine aorta and brain capillaries were plated on Matrigel, DNA synthesis was suppressed and a network of capillary-like structures rapidly formed in 8-12 h. With time, the network broke down, resulting in dense cellular cords radiating from multiple cellular clusters in 16-24 h. Finally, multicellular aggregates of cells were formed as the network underwent further retraction. Network regression was prevented when either dithiothreitol (DTT) or anti-TGF-beta 1 antibodies were added during the assay. The addition of exogenous TGF-beta 1 promoted the regression of endothelial cells into the clusters. This response to TGF-beta 1 was blocked by potent serine threonine protein kinase inhibitors, H-7 and HA100. TGF-beta 1 was released from polymerized Matrigel by incubation with Dulbecco's modified eagle's medium (DMEM) in the absence of cells. The Matrigel-conditioned DMEM inhibited endothelial DNA synthesis even in the presence of anti-TGF-beta 1 antibodies. These results suggest that TGF-beta 1 and possibly other soluble factors from Matrigel may be important for differentiation and remodeling of endothelial cells in a capillary network with possible implications for wound healing and development.


Subject(s)
Collagen/pharmacology , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiology , Laminin/pharmacology , Proteoglycans/pharmacology , Transforming Growth Factor beta/physiology , Animals , Capillaries/anatomy & histology , Cattle , Cells, Cultured , DNA/antagonists & inhibitors , DNA/biosynthesis , Drug Combinations , Endothelium, Vascular/cytology , Protein Kinase Inhibitors
7.
Pathol Res Pract ; 190(9-10): 854-63, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7534913

ABSTRACT

Expansion of the tumor-cell mass is dependent on both the degree of tumor vascularization and the rate of angiogenesis. Blood vessel growth is controlled, in part, by the matrix surrounding it, in particular, the basement membrane underlying the endothelium. Here we illustrate that laminin, a major component of basement membrane, has several biologically active sites that can bind to endothelial and tumor cells, and have the ability to regulate angiogenesis and tumor growth. We show that synthetic peptides at two sites in the laminin B1 chain (the RGD and YIGSR sequences) inhibit angiogenesis, whereas a third site in the A chain, designated SIK-VAV, stimulates vessel and tumor cell growth. By developing strategies that promote or inhibit the activities of these sites in laminin, we may obtain methods to inhibit angiogenesis and subsequent tumor growth.


Subject(s)
Basement Membrane/chemistry , Laminin/physiology , Neoplasm Invasiveness/physiopathology , Neovascularization, Pathologic/metabolism , Amino Acid Sequence , Animals , Basement Membrane/physiology , Humans , Laminin/analysis , Molecular Sequence Data
8.
Proc Natl Acad Sci U S A ; 90(5): 1937-41, 1993 Mar 01.
Article in English | MEDLINE | ID: mdl-7680481

ABSTRACT

Scatter factor (also known as hepatocyte growth factor) is a glycoprotein secreted by stromal cells that stimulates cell motility and proliferation. In vitro, scatter factor stimulates vascular endothelial cell migration, proliferation, and organization into capillary-like tubes. Using two different in vivo assays, we showed that physiologic quantities of purified native mouse scatter factor and recombinant human hepatocyte growth factor induce angiogenesis (the formation of new blood vessels). The angiogenic activity was blocked by specific anti-scatter factor antibodies. Scatter factor induced cultured microvascular endothelial cells to accumulate and secrete significantly increased quantities of urokinase, an enzyme associated with development of an invasive endothelial phenotype during angiogenesis. We further showed that immunoreactive scatter factor is present surrounding sites of blood vessel formation in psoriatic skin. These findings suggest that scatter factor may act as a paracrine mediator in pathologic angiogenesis associated with human inflammatory disease.


Subject(s)
Hepatocyte Growth Factor/physiology , Neovascularization, Pathologic , Animals , Cattle , Collagen , Cornea/blood supply , Drug Combinations , Endothelium, Vascular/cytology , Humans , Laminin , Mice , Plasminogen Activators/metabolism , Proteoglycans , Psoriasis/metabolism , Rats
9.
J Biol Chem ; 268(5): 3184-93, 1993 Feb 15.
Article in English | MEDLINE | ID: mdl-8381420

ABSTRACT

The pre-steady state time course of amiloride-sensitive Na+o uptake by the Na(+)-H+ exchanger in renal brush border membrane vesicles (BBMV) exhibits a burst phase at 0 degrees C which corresponds to the initial turnover of the exchanger (Otsu, K., Kinsella, J. L., Sacktor, B. S., and Froehlich, J. P. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4818-4822). Investigation of the Na+o dependence of the Na(+)-H+ exchanger between 1 and 10 mM Na+ revealed that activation of the burst phase involves at least two Na+ transport sites interacting with positive cooperativity. In this study, characterization of the Na+ transport sites contributing to the burst phase was extended to include Na+ concentrations below 1 mM. Between 0.1 and 1 mM Na+ the amplitude of the burst phase in acid-loaded BBMV (pHi 5.7; pHo 7.7) exhibited a sigmoidal dependence on [Na+]o, consistent with the presence of a second class of high affinity Na+ transport sites with cooperative binding characteristics. In contrast, steady state Na+ uptake obeyed Michaelis-Menten kinetics, similar to the behavior observed previously at higher (1-10 mM) Na+o concentrations. Treatment of the vesicles with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, which induced the formation of an inside-negative membrane potential, increased the burst amplitude but had no effect on the steady state uptake velocity. Experiments performed with alkaline-loaded BBMV (pHi 7.7; pHo 7.7), which permit only a single turnover of the exchanger, gave a simple hyperbolic dependence of the burst amplitude on [Na+]o (0.5-5 mM). We propose that the change in multiplicity of Na+ transport sites and membrane potential sensitivity that occurs in the transition between the pre-steady state and the steady state of Na+ uptake in acid-loaded vesicles reflects the presence of an oligomer which operates according to a "flip-flop" mechanism. The minimum subunit composition inferred from the biphasic [Na+]o dependence of the burst amplitude is a dimer at low (< 1 mM) Na+o levels and a tetramer at high [Na+]o. Communication between the subunits producing the complex [Na+]o dependence is controlled by the intravesicular (cytoplasmic) H+ modifier site. Under alkaline conditions (pH 7.7), where this site is unoccupied, the subunits behave as independent units and cease operation after the first turnover. Occupation of the H+ modifier site activates a conformational interaction between the subunits that leads to cooperative Na+o binding, alternation of the transport sites, and repetitive cycling of the Na(+)-H+ exchanger.


Subject(s)
Carrier Proteins/metabolism , Kidney Cortex/metabolism , Microvilli/metabolism , Sodium/metabolism , Amiloride/pharmacology , Animals , Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Cell Fractionation , Hydrogen-Ion Concentration , Kinetics , Mathematics , Membrane Potentials , Microvilli/drug effects , Microvilli/ultrastructure , Models, Biological , Rabbits , Sodium-Hydrogen Exchangers
10.
Symp Soc Exp Biol ; 47: 227-34, 1993.
Article in English | MEDLINE | ID: mdl-8165567

ABSTRACT

Scatter factor (SF), a fibroblast-derived cytokine characterized by its ability to convert non-motile epithelial cells to a motile fibroblast-like phenotype, is identical to hepatocyte growth factor (HGF), a broad-spectrum mitogen. SF is a heterodimeric glycoprotein that is homologous to plasminogen and other blood coagulation proteases but lacks proteolytic activity. Its receptor is the c-met proto-oncogene product, a growth factor receptor-like transmembrane tyrosine kinase. This unique cytokine is also synthesized and secreted by vascular smooth muscle cells and acts on endothelial cells to stimulate migration, protease production, invasion, proliferation, and differentiation into capillary-like tubes in vitro. SF-containing implants in mouse subcutaneous tissue and rat cornea induce directed ingrowth of new blood vessels from surrounding tissue, with maximal angiogenic responses at doses of 100-200 ng of SF. Immunoreactive SF is expressed at sites of neovascularization within human psoriatic plaques. These findings suggest that SF may play a significant role in the formation and repair of blood vessels under physiologic and pathologic conditions.


Subject(s)
Angiogenesis Inducing Agents , Endothelium, Vascular/growth & development , Hepatocyte Growth Factor/physiology , Animals , Capillaries/cytology , Cell Division/physiology , Cell Movement/physiology , Endothelium, Vascular/cytology , Humans , Mice , Proto-Oncogene Mas , Rats
11.
Circulation ; 86(6 Suppl): III68-73, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1330366

ABSTRACT

Endothelial and smooth muscle cells normally exist in a quiescent differentiated state. After injury to the vessel, these cells dedifferentiate, migrate, and proliferate as needed for repair. In culture on plastic, both endothelial and smooth muscle cells exhibit the dedifferentiated phenotype. We have found that laminin and reconstituted basement membrane proteins (Matrigel) induce a very rapid cessation of endothelial cell proliferation followed by alignment and subsequent reorganization into tubelike structures. We have also found that smooth muscle cells in culture exhibit a differentiated phenotype when exposed to Matrigel. The molecular mechanisms involved in smooth muscle differentiation resemble those of skeletal muscle, in which proliferation and differentiation appear to be mutually exclusive states controlled by both positive and negative transcriptional regulators. The dedifferentiated smooth muscle cells produce proteases and exhibit a migratory and invasive phenotype capable of destroying normal tissue architecture. These studies suggest that the modulation of endothelial and smooth muscle cells between a differentiated and dedifferentiated phenotype is regulated by extracellular matrix components as well as by cytokines. Model systems such as those described here should allow the identification of molecular events controlling the differentiation of vascular cells and facilitate the development of therapeutic agents that maintain healthy vessels.


Subject(s)
Endothelium, Vascular/cytology , Muscle, Smooth, Vascular/cytology , Basement Membrane/cytology , Cell Differentiation , Cell Division , Cytological Techniques , Endothelium, Vascular/physiology , Gene Expression , Models, Biological , Muscle Proteins/genetics , Muscle Proteins/metabolism , Muscle, Smooth, Vascular/physiology , MyoD Protein , Transcription Factors/metabolism
12.
J Cell Physiol ; 153(3): 614-25, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1280280

ABSTRACT

Endothelial cells are known to bind to laminin, and two peptides derived from the laminin A (CTFALRGDNP) and B1 (CDPGYIGSR) chains block the capillary-like tube formation on a laminin-rich basement membrane matrix, Matrigel. In the present study, we have used various in vitro and in vivo assays to investigate the angiogenic-biologic effects of a third active site in the laminin A chain, CSRARKQAASIKVAVSADR (designated PA22-2) on endothelial cells. The SIKVAV-containing peptide was as active as the YIGSR-containing peptide for endothelial cell attachment but was less active than either the RGD-containing peptide or intact laminin. Endothelial cells seeded on this peptide appeared fibroblastic with many extended processes, unlike the normal cobblestone morphology observed on tissue culture plastic. In addition, in contrast to normal tube formation on Matrigel, short irregular structures formed, some of which penetrated the matrix and sprouting was more apparent. Analysis of endothelial cell conditioned media of cells cultured in the presence of this peptide indicated degradation of the Matrigel and zymograms demonstrated active collagenase IV (gelatinase) at 68 and 62 Kd. A murine in vivo angiogenesis assay and the chick yolk sac/chorioallantoic membrane assays with the peptide demonstrated increased endothelial cell mobilization, capillary branching, and vessel formation. These data suggest that the -SIKVAV-site may play an important role in initiating branching and formation of new capillaries from the parent vessels, a behavior that is observed in vivo in response to tumor growth or in the normal vascular response to injury.


Subject(s)
Endothelium, Vascular/metabolism , Laminin/metabolism , Neovascularization, Pathologic/chemically induced , Peptide Fragments/metabolism , Amino Acid Sequence , Animals , Cell Movement , Cells, Cultured , Chick Embryo , Collagen/classification , Collagen/metabolism , Culture Media , Drug Combinations , Endothelium, Vascular/cytology , Endothelium, Vascular/ultrastructure , Extraembryonic Membranes/blood supply , Extraembryonic Membranes/drug effects , Humans , Laminin/genetics , Microscopy, Electron, Scanning , Molecular Sequence Data , Peptide Fragments/genetics , Proteoglycans , Yolk Sac/blood supply , Yolk Sac/drug effects
13.
J Biol Chem ; 267(12): 8089-96, 1992 Apr 25.
Article in English | MEDLINE | ID: mdl-1373720

ABSTRACT

The pre-steady state time dependence of Na+ accumulation by the Na(+)-H+ exchanger in renal brush border membrane vesicles was investigated at 0 degree C by a manual mixing technique using amiloride to quench the reaction. Dilution of acid-loaded (pHi 5.7) vesicles into an alkaline medium (pHo 7.7) containing 1 mM 22Na+ produced a time course of amiloride-sensitive Na+ uptake that consisted of three distinct phases: 1) a lag, 2) a monoexponential "burst," and 3) a linear or steady state phase. Experiments testing for the presence of 22Na+ backflux, residual Na+ binding to the membrane, and hysteresis were negative, lending support to the hypothesis that the burst phase corresponds to Na+ translocation during the initial turnover of Na(+)-H+ exchanger. Lowering the internal pH increased the amount of na+ uptake in each of the phases without affecting the apparent burst rate, whereas lowering the external pH inhibited Na+ uptake while increasing the duration of the lag phase. The pattern of inhibition produced by external H+ was of the simple competitive type, indicating that Na+ and H+ share a common binding site. Steady state Na+ uptake showed a sigmoidal dependence on internal pH (Hill coefficient = 1.67), consistent with the presence of an internal allosteric H+ activation site. Alkaline loading conditions (pHi 7.7), which favor desaturation of the internal H+ binding sites, completely abolished Na+ uptake in the steady state. In contrast, Na+ accumulation during the burst phase was reduced to 25% of an acid-loaded (pHi 5.7) control. The persistence of the burst phase and the disappearance of steady state Na+ uptake under alkaline loading conditions suggest that recycling of the H(+)-loaded exchanger is a late event in the transport cycle that follows Na+ translocation (ping-pong mechanism) and controls the steady state rate of Na+ accumulation. Activation of the recycling step involves sequential binding of H+ to the allosteric and transport sites, thus accounting for the cooperative dependence of steady state Na+ uptake on the internal [H+].


Subject(s)
Carrier Proteins/metabolism , Kidney Cortex/metabolism , Microvilli/metabolism , Allosteric Site , Amiloride/pharmacology , Animals , Biological Transport , Gramicidin/pharmacology , Hydrogen-Ion Concentration , Kidney Cortex/drug effects , Microvilli/drug effects , Rabbits , Sodium/metabolism , Sodium-Hydrogen Exchangers
14.
Exp Cell Res ; 199(1): 56-62, 1992 Mar.
Article in English | MEDLINE | ID: mdl-1370939

ABSTRACT

Human umbilical vein endothelial cells differentiate within 12 h to form capillary-like networks of tube structures when the cells are plated on Matrigel, a mixture of basement membrane proteins. Nothing is known about the intracellular signaling events involved in this differentiation. As a first step to define the process, we investigated the possible role of protein kinase C activation by beta-phorbol 12-myristate 13-acetate (PMA) in regulating the formation of the tube structures. In this model, PMA increased tube formation several-fold in a dose-dependent manner with half-maximum stimulation of tube formation at approximately 5 nM PMA. In the absence of serum, essentially little or no tubes were formed on Matrigel unless PMA was added to the medium. Only active phorbol analogs increased tube formation, while the protein kinase C inhibitor, H-7, blocked tube formation. The protein kinase C activators and inhibitors were effective only when added at or just after plating of the cells and did not affect already formed tubes. This study suggests that protein kinase C is involved in the early events of in vitro endothelial cell tube formation on Matrigel.


Subject(s)
Collagen/physiology , Endothelium, Vascular/cytology , Laminin/physiology , Neovascularization, Pathologic , Protein Kinase C/metabolism , Proteoglycans/physiology , Tetradecanoylphorbol Acetate/pharmacology , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine , Blood , Cell Differentiation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Drug Combinations , Humans , Isoquinolines/pharmacology , Kinetics , Piperazines/pharmacology , Protein Kinase C/antagonists & inhibitors
15.
Semin Nephrol ; 10(4): 330-8, 1990 Jul.
Article in English | MEDLINE | ID: mdl-2166325

ABSTRACT

A number of transport systems in the proximal tubule now have been shown to change activity in response to glucocorticoids. Two of them, the Na(+)-H+ exchanger and the Na(+)-dependent phosphate transporter, are apparently directly regulated by specific receptors in the proximal tubule. Glucocorticoid regulation of both transport systems is now known to be dependent upon the synthesis of new mRNA and protein. When one surveys the variety of transporter mechanism and metabolic pathways under glucocorticoid control, it seems clear that this group of hormones usually thought of as controlling glucose metabolism may in fact be playing a central role in maintaining acid-base homeostasis. Future research will clarify this role for glucocorticoids.


Subject(s)
Glucocorticoids/physiology , Kidney Tubules, Proximal/physiology , Water-Electrolyte Balance/physiology , Acid-Base Equilibrium/physiology , Animals , Biological Transport/physiology , Carrier Proteins/physiology , Glucocorticoids/pharmacology , Humans , Phosphate-Binding Proteins , Phosphates/metabolism , Sodium-Hydrogen Exchangers
16.
Am J Physiol ; 258(2 Pt 2): F333-8, 1990 Feb.
Article in English | MEDLINE | ID: mdl-2155539

ABSTRACT

Cultured opossum kidney (OK) cells were used to determine whether thyroid hormone has a direct stimulatory effect on Na(+)-H+ exchange activity in an intact cellular preparation. Na+ uptake or intracellular pH recovery was measured in confluent monolayer cells following acid loading with NH4Cl. Triiodo-L-thyronine (T3) had no effect on cell number or protein and DNA contents but stimulated amiloride-sensitive Na+ uptake in a dose- and time-dependent manner. Maximal stimulation of Na+ uptake was observed at 10(-7) M T3 and 10(-6) M L-thyroxine (T4) with half-maximal effects at 10(-9) M T3 and 3 x 10(-8) M T4. The T3 specific binding capacity of OK cells was 96 +/- 15 fmol/mg DNA with a KD of 1.1 +/- 0.2 x 10(-9) M T3. Neither T3 nor T4 had any effect on amiloride-insensitive Na+ uptake. In kinetic studies of Na+ uptake, T3 increased the Vmax from 123 +/- 22 to 157 +/- 24 nmol.mg-1.min-1 without changing the Michaelis-Menten kinetics (Km) for Na+ (21 +/- 1 in control and 22 +/- 4 mM in T3-treated cells). Studies of intracellular pH (pHi) showed that the resting pHi and the buffering capacity were unaffected by T3. However, after an acid load, OK cells treated with T3 exhibited a greater rate of Na(+)-dependent pH recovery than untreated control cells. These results indicate that thyroid hormone can stimulate Na(+)-H+ exchange activity directly in renal cell line without apparent changes in pHi, cellular hypertrophy, or hyperplasia.


Subject(s)
Carrier Proteins/metabolism , Kidney/metabolism , Thyroxine/pharmacology , Triiodothyronine/pharmacology , Animals , Cell Line , Cell Nucleus/metabolism , Dose-Response Relationship, Drug , Kidney/cytology , Kidney/ultrastructure , Kinetics , Opossums , Osmolar Concentration , Receptors, Thyroid Hormone/metabolism , Sodium-Hydrogen Exchangers , Time Factors
17.
Am J Physiol ; 255(6 Pt 2): F1183-90, 1988 Dec.
Article in English | MEDLINE | ID: mdl-3202182

ABSTRACT

In this study, we compared results obtained in senescent rats with young rats given an equivalent acid load. We examined the renal changes by giving equivalent acid loads for 48 h to both 6- and 24-mo-old rats. The basal excretion of ammonium was the same in both groups, whereas titratable acids, phosphate, and Ca2+ excretions were increased in the senescent animal. After administration of the acid load, ammonium, phosphate, Ca2+, and titratable acid excretions increased in both age groups, but there were greater absolute increases in ammonium and titratable acid excretions in the young rats. The total acid excreted by the 24-mo rats was reduced 50 (day 1) and 25% (day 2) compared with the young rats, which was reflected by the more severe acidosis in those animals. The portion of total acid excreted as titratable acids in senescent animals was also increased during acidosis when compared with the young animals. In isolated proximal tubule brush-border membrane vesicles, acidosis increased Na+-H+ exchange and decreased Na+-dependent phosphate transport in both age groups. We also found that the basal activity of the Na+-H+ exchanger was not changed with age but the Na+-dependent phosphate transporter was less in the 24-mo rat. The results suggest that physiological regulation of these renal processes remains intact in the aged rat but the responses may be reduced or delayed in the senescent animal.


Subject(s)
Acidosis/physiopathology , Kidney/physiopathology , Acclimatization , Aging , Animals , Body Weight , Drinking Behavior , Feeding Behavior , Glomerular Filtration Rate , Kidney/growth & development , Male , Organ Size , Rats , Rats, Inbred Strains , Reference Values
18.
J Biol Chem ; 262(15): 7092-7, 1987 May 25.
Article in English | MEDLINE | ID: mdl-3034881

ABSTRACT

The inactivation of rat renal brush border membrane Na+-H+ exchange by the covalent carboxylate reagent N,N'-dicyclohexylcarbodiimide (DCCD) was studied by measuring 1 mM Na+ influx in the presence of a pH gradient (pHi = 5.5; pHo = 7.5) and H+ influx in the presence of a Na+ or Li+ gradient ([Na+]i = 150 mM; [Na+]o = 1.5 mM). In the presence of DCCD, the rate of Na+ uptake decreased exponentially with time and transport inhibition was irreversible. At all DCCD concentrations the loss of activity was described by a single exponential, consistent with one critical DCCD-reactive residue within the Na+-H+ exchanger. Among several carbodiimides the most hydrophobic carbodiimide, DCCD, was also the most effective inhibitor of Na+-H+ exchange. With 40 nmol of DCCD/mg of protein, at 20 degrees C for 30 min, 75% of the amiloride-sensitive 1 mM Na+ uptake was inhibited. Neither the equilibrium Na+ content nor the amiloride-insensitive Na+ uptake was significantly altered by the treatment. The Na+-dependent H+ flux, measured by the change in acridine orange absorbance, was also decreased 80% by the same DCCD treatment. If 150 mM NaCl, 150 mM LiCl, or 1 mM amiloride was present during incubation of the brush border membranes with 40 nmol of DCCD/mg of protein, then Li+-dependent H+ flux was protected 50, 100, or 100%, respectively, compared to membranes treated with DCCD in the absence of Na+-H+ exchanger substrates. The combination of DCCD and an exogenous nucleophile, e.g. ethylenediamine and glycine methyl ester, increased Na+-dependent H+ flux in the presence of 80 nmol of DCCD/mg of protein, compared to the transport after DCCD treatment alone. These findings suggest that the Na+-H+ exchanger contains a single carboxylate residue in a hydrophobic region of the protein, and the carboxylate and/or a nearby endogenous nucleophilic group is critical for exchange activity.


Subject(s)
Carbodiimides/pharmacology , Carrier Proteins/antagonists & inhibitors , Dicyclohexylcarbodiimide/pharmacology , Kidney/metabolism , Protons , Sodium/metabolism , Amiloride/pharmacology , Animals , Biological Transport/drug effects , Hydrogen-Ion Concentration , Kidney/drug effects , Kinetics , Lithium/pharmacology , Microvilli/drug effects , Microvilli/metabolism , Rats , Sodium/pharmacology , Sodium-Hydrogen Exchangers
19.
Am J Physiol ; 252(4 Pt 2): R681-6, 1987 Apr.
Article in English | MEDLINE | ID: mdl-3032004

ABSTRACT

Amiloride-sensitive Na+-H+ exchange activity in brush-border membrane vesicles isolated from male rat proximal tubules was decreased in the senescent rat (24 mo) compared with the young adult (6 mo). There was no significant loss in Na+-H+ exchange activity in the kidneys of animals between 6 and 18 mo of age. Amiloride-insensitive Na+ uptake and the rate of pH gradient dissipation were not altered during aging. The decrease in sodium-dependent phosphate transport preceded the decline in Na+-H+ exchange activity by at least 6 mo. Sodium-dependent glucose transport was not significantly altered during aging. Thus various renal plasma membrane transport functions were affected differently in the aging rat. The decrease in Na+-H+ exchange activity during aging contrasted with the increase in exchange activity reported previously in acute ablation models of chronic renal failure.


Subject(s)
Aging , Carrier Proteins/metabolism , Kidney Cortex/metabolism , Amiloride/pharmacology , Animals , Biological Transport , Glucose/metabolism , Hydrogen-Ion Concentration , Microvilli/metabolism , Phosphates/metabolism , Rats , Sodium-Hydrogen Exchangers
20.
J Membr Biol ; 91(2): 183-91, 1986.
Article in English | MEDLINE | ID: mdl-3018258

ABSTRACT

Na+-H+ exchange activity in renal brush border membrane vesicles isolated from hyperthyroid rats was increased. When examined as a function of [Na+], treatment altered the initial rate of Na+ uptake by increasing Vm (hyperthyroid, 18.9 +/- 1.1 nmol Na+ X mg-1 X 2 sec-1; normal, 8.9 +/- 0.3 nmol Na+ X mg-1 X 2 sec-1), and not the apparent affinity KNa+ (hyperthyroid, 7.3 +/- 1.7 mM; normal, 6.5 +/- 0.9 mM). When examined as a function of [H+] and at a subsaturating [Na+] (1 mM), hyperthyroidism resulted in the proportional increase in Na+ uptake at every intravesicular pH measured. A positive cooperative effect on Na+ uptake was found with increased intravesicular acidity in vesicles from both normal and hyperthyroid rats. When the data were analyzed by the Hill equation, it was found that hyperthyroidism did not change the n (hyperthyroid, 1.2 +/- 0.06; normal, 1.2 +/- 0.07) or the [H+]0.5 (hyperthyroid, 0.39 +/- 0.08 microM; normal, 0.44 +/- 0.07 microM) but increased the apparent Vm (hyperthyroid, 1.68 +/- 0.14 nmol Na+ X mg-1 X 2 sec-1; normal 0.96 +/- 0.10 nmol Na+ X mg-1 X 2 sec-1). The uptake of Na+ in exchange for H+ in membrane vesicles from normal and hyperthyroid animals was not influenced by membrane potential. H+ translocation or debinding was rate limiting for Na+-H+ exchange since Na+-Na+ exchange activity was greater than Na+-H+ exchange activity. Hyperthyroidism caused a proportional increase and hypothyroidism caused a proportional decrease in Na+-Na+ and Na+-H+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Carrier Proteins/metabolism , Kidney/metabolism , Thyroid Hormones/pharmacology , Animals , Biological Transport, Active/drug effects , Hyperthyroidism/metabolism , In Vitro Techniques , Kidney/drug effects , Kinetics , Male , Microvilli/metabolism , Rats , Rats, Inbred Strains , Sodium/metabolism , Sodium-Hydrogen Exchangers
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