RESUMO
A procedure for dissociating the rabbit aorta into single, functional smooth muscle cells is described. After removal of adventitia and intima, slices of media were incubated with purified collagenase, elastase, and soybean trypsin inhibitor in a Krebs-Ringer buffer modified with Hepes, amino acids, and a [Ca2+] of 0.2 mM. After enzymatic digestion and mechanical shear, the yield of dispersed cells was approximately 25% based on DNA recovered. Greater than 95% of the cells excluded trypan blue and approximately 80-90% adhered to tissue culture dishes. By phase contrast microscopy, most of the cells were elongate and approximately 10 micron X 30 micron in size. The remainder were either spherical or highly crenated and contracted. Electron microscopy of the cells showed that immediately after dissociation greater than 95% could be identified as smooth muscle, though most had undergone some degree of structural change compared to cells in situ. Depending on the preparation, from 5 to 50% of these cells contracted in response to agonists. Cells shortened by 10-15% and developed numerous evaginations when stimulated by angiotensin II norepinephrine, or carbamylcholine. Cells relaxed after washout of agonists and could subsequently be restimulated. Specific inhibitors of each of the agonists blocked the contractile response. Dispersed cells cultured for 1-5 days contracted in even higher numbers than the freshly prepared cells, suggesting restoration of hormone binding and/or contractile function in culture. This preparation provides a system in which the physiology of individual vascular smooth muscle cells may be studied.
Assuntos
Aorta/citologia , Músculo Liso/citologia , Animais , Aorta/ultraestrutura , Cálcio/farmacologia , Separação Celular/métodos , Técnicas de Cultura , Hialuronoglucosaminidase/metabolismo , Masculino , Colagenase Microbiana/metabolismo , Contração Muscular , Músculo Liso/fisiologia , Músculo Liso/ultraestrutura , Elastase Pancreática/metabolismo , CoelhosRESUMO
The effect of cyclic mechanical strain on growth of neonatal rat vascular smooth muscle (VSM) cells were examined. Cells were grown on silicone elastomer plates subjected to cyclic strain (60 cycle/min) by application of a vacuum under the plates. A 48 h exposure to mechanical strain increased the basal rate of thymidine incorporation by threefold and increased cell number by 40% compared with cells grown on stationary rubber plates. Strain also increased the rate of thymidine incorporation in response to alpha-thrombin (from 15- to 33-fold), but not to PDGF. As determined by thymidine autoradiography, strain alone induced a fourfold increase in labeled nuclei at the periphery of dishes, where strain is maximal, and a 2-3-fold increase at the center of dishes. Strain appeared to induce the production of an autocrine growth factor(s), since conditioned medium from cells subjected to strain induced a fourfold increase in DNA synthesis in control cells. Western blots of medium conditioned on the cells subjected to strain indicate that the cells secrete both AA and BB forms of PDGF in response to strain. Northern blots of total cell RNA from cells exposed to strain for 24 h show increased steady-state level of mRNA for PDGF-A. Lastly, polyclonal antibodies to the AA form of PDGF reduced by 75% the mitogenic effect of strain and polyclonal antibodies to AB-PDGF reduced mitogenicity by 50%. Antibodies to bFGF did not significantly reduce the strain-induced thymidine incorporation. Thus, the mechanism of strain-induced growth appears to involve the intermediary action of secreted PDGF.
Assuntos
Músculo Liso Vascular/citologia , Fator de Crescimento Derivado de Plaquetas/biossíntese , Fator de Crescimento Derivado de Plaquetas/farmacologia , Trombina/farmacologia , Animais , Animais Recém-Nascidos , Becaplermina , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Meios de Cultivo Condicionados , DNA/biossíntese , Humanos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-sis , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/farmacologia , Estresse Mecânico , Timidina/metabolismoRESUMO
The past five years have witnessed an explosion of information on the many and varied roles of H+ transport in cell function. H+ transport is involved in three broad areas of cell function: (a) maintenance and alteration of intracellular pH for initiation of specific cellular events, (b) generation of pH gradients in localized regions of the cell, including gradients involved in energy transduction, and (c) transepithelial ion transport. These processes each involve one or more of several H+ translocating mechanisms. The first section of this review will discuss these H+ translocating mechanisms and the second part will deal with the cellular functions controlled by H+ transport.
Assuntos
Fenômenos Fisiológicos Celulares , Prótons , Ânions , Antiporters , Bactérias/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Cátions Monovalentes , Divisão Celular , Permeabilidade da Membrana Celular , Cloretos/metabolismo , Cloroplastos/metabolismo , Dicicloexilcarbodi-Imida/farmacologia , Transporte de Elétrons , Endocitose , Epitélio/metabolismo , Concentração de Íons de Hidrogênio , Insulina/farmacologia , Mitocôndrias/metabolismo , Fotoquímica , Potássio/metabolismo , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Cyclic mechanical strain (1 Hz) causes a mitogenic response in neonatal rat vascular smooth muscle cells due to production and secretion of PDGF. In this study, the mechanism for sensing mechanical strain was investigated. Silicone elastomer strain plates were coated at varying densities with elastin, laminin, type I collagen, fibronectin, or vitronectin. Strain was applied by cyclic application of a vacuum under the dishes. Cells adhered, spread, and proliferated on each matrix protein, but the mitogenic response to strain was matrix dependent. Strain increased DNA synthesis in cells on collagen, fibronectin, or vitronectin, but not in cells on elastin or laminin. When strain was applied on matrices containing both laminin and vitronectin, the mitogenic response to strain depended upon the vitronectin content of the matrix. Fibronectin, in soluble form (0-50 micrograms/ml), and the integrin binding peptide GRGDTP (100 micrograms/ml) both blocked the mitogenic response to mechanical strain in cells grown on immobilized collagen. Neither soluble laminin nor the inactive peptide GRGESP blocked the response to strain. GRGDTP did not alter the mitogenic response to exogenous PDGF or alpha-thrombin but did prevent the secretion of PDGF in response to strain. Furthermore, GRGDTP, but not GRGESP, prevented strain-induced expression of a PDGF-A chain promoter 890 bp-chloramphenicol acetyltransferase construct that was transiently transfected into vascular smooth muscle cells. Finally, the response to strain was abrogated by antibodies to both beta 3 and alpha v beta 5 integrins but not by an antibody to beta 1 integrins. Thus interaction between integrins and specific matrix proteins is responsible for sensing mechanical strain in vascular smooth muscle cells.
Assuntos
Proteínas da Matriz Extracelular/fisiologia , Integrinas/fisiologia , Músculo Liso Vascular/fisiologia , Fator de Crescimento Derivado de Plaquetas/biossíntese , Animais , Adesão Celular , Divisão Celular , Células Cultivadas , Replicação do DNA , Expressão Gênica , Fator de Crescimento Derivado de Plaquetas/genética , Ratos , Estresse MecânicoRESUMO
The effects of extracellular matrix proteins and mechanical strain on the mitogenic activity of angiotensins I and II (AI and AII) were examined in cultured rat vascular smooth muscle (VSM) cells. VSM cells on various extracellular matrices were exposed to AII (1 microM) for 48 h. On plastic, AII induced only a 1.6-fold increase in [3H]thymidine incorporation, but on fibronectin- or type I collagen-coated plastic, the response to AII was enhanced from two- to fourfold. On a type I collagen-coated silicone elastomer, to which mechanical strain was applied, [3H]thymidine incorporation dramatically increased to a maximum of 53-fold. Dup 753 (10(-5) M) blocked the AII-induced increase in DNA synthesis. AI also increased DNA synthesis in VSM cells, and this response was also enhanced by mechanical strain. Mitogenic activity of AI was blocked by ramiprilat (10(-5) M), indicating that its mitogenic activity was via conversion to AII. The synergy between AII and strain was completely eliminated by neutralizing antibodies to PDGF AB (3 micrograms/ml). Furthermore, the mitogenic effect of AII in unstrained cells was also synergistic with submaximal concentrations of PDGF AB (1 ng/ml). Thus, the synergy between AII and mechanical strain probably results from synergism between AII and PDGF secreted in response to strain.
Assuntos
Angiotensina II/farmacologia , Divisão Celular/fisiologia , Colágeno/farmacologia , Músculo Liso Vascular/citologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Animais Recém-Nascidos , Compostos de Bifenilo/farmacologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , DNA/biossíntese , Sinergismo Farmacológico , Fibronectinas/farmacologia , Gelatina/farmacologia , Imidazóis/farmacologia , Losartan , Músculo Liso Vascular/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ramipril/análogos & derivados , Ramipril/farmacologia , Ratos , Proteínas Recombinantes/farmacologia , Estresse Mecânico , Tetrazóis/farmacologia , Timidina/metabolismoRESUMO
Endothelin is a potent mammalian vasoconstrictive peptide with structural homology to cation channel-binding insect toxins. We tested the proposal that this peptide directly activates dihydropyridine-sensitive Ca2+ channels in cultured vascular smooth muscle (VSM) cells. First, we found that cell Ca2+ can be altered in VSM by activation of voltage-operated Ca2+ channels. KCl-induced depolarization and the dihydropyridine Ca2+ channel agonist (-) Bay K 8644 (10 microM) both raised cell Ca2+ more than twofold; the effect of KCl was blocked by the inhibitory enantiomer, (+) Bay K 8644 (40 microM). Similar responses were observed in Chinese hamster ovary (CHO) cells. Synthetic endothelin (4 x 10(-8) M) raised Ca2+ in VSM but not CHO cells from 100 +/- 17 to 561 +/- 34 nM within 12 s. Ca2+ subsequently fell to basal levels after 30 min. Half maximal Ca2+ response was at 4 x 10(-9) M endothelin. Unlike endothelin, thrombin raised Ca2+ in both VSM and CHO cells. The Ca2+ responses to endothelin and thrombin were not affected by nicardipine (1 microM), (+) Bay K 8644, or Ca2+-free solutions. Lastly, both hormones caused release of inositol phosphates in VSM cells. However, the response to thrombin was more than 10-fold larger and was more rapid than the response to endothelin; the thrombin response was sensitive to pertussis toxin, while the response to endothelin was not. Thus endothelin, like thrombin, raises cell Ca2+ in VSM by mobilization of intracellular stores and not by activation of dihydropyridine-sensitive Ca2+ channels. However, their receptors are distinct and they exhibit important differences in signal transduction.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Cálcio/análise , Endotélio Vascular/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Cálcio/metabolismo , Canais de Cálcio/fisiologia , Células Cultivadas , Endotelinas , Inositol 1,4,5-Trifosfato , Fosfatos de Inositol/metabolismo , Músculo Liso Vascular/análise , Ratos , Transdução de SinaisRESUMO
We examined the effects of 1,25-dihydroxyvitamin D3(1,25-(OH)2D3) on the proliferation of vascular smooth muscle (VSM) cells. Receptors for 1,25-(OH)2D3 were demonstrated in fresh rabbit aortic tissue and in cultured rat VSM using binding of [3H]-1,25-(OH)2D3 in sucrose density gradients of the tissue or cell homogenates. The receptor sedimented at 3.6 S, the sedimentation velocity of 1,25-(OH)2D3 receptors from other sources. 1,25-(OH)2D3 dramatically altered the growth of VSM, but this effect depended importantly on the basal conditions in which the cells were grown. In quiescent VSM deprived of serum for 72 h, 1,25-(OH)2D3 (0.1-10 nM), but not 25-(OH)D3 (up to 100 nM) increased thymidine incorporation up to 12-fold and cell number up to 2.6-fold compared with controls. The maximal effect of 1,25-(OH)2D3 on thymidine incorporation was similar to the maximal effect of the growth factors alpha-thrombin or PDGF. Furthermore, the effects of 1,25-(OH)2D3 and thrombin on thymidine incorporation in quiescent cells were markedly synergistic, yielding a 78-fold increase in thymidine incorporation when both agents were added simultaneously. In "nonquiescent cells" which were exposed to serum-free medium for only 24 h, 1,25-(OH)2D3 (10 nM) also increased DNA synthesis 10-fold compared with controls. However, in striking contrast to what was observed in quiescent cells, 1,25-(OH)2D3 diminished the mitogenic response to thrombin by as much as 50% in nonquiescent cells. 1,25-(OH)2D3 also modulated the transcription of c-myc in response to thrombin. In quiescent cells, transcription was enhanced by 1,25-(OH)2D3, whereas in nonquiescent cells, thrombin-induced c-myc transcription was blunted. Thus, 1,25-(OH)2D3 is a potent modulator of the growth of cultured VSM. The direction of this modulation depends strongly on the conditions under which the cells are cultured.
Assuntos
Calcitriol/farmacologia , Músculo Liso Vascular/citologia , Receptores de Esteroides/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Calcitriol/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Sinergismo Farmacológico , Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Músculo Liso Vascular/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-myc/genética , Proto-Oncogenes , Ratos , Receptores de Calcitriol , Trombina/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
We examined the role of intracellular and extracellular calcium on the ability of human polymorphonuclear leukocytes to migrate chemotactically and reexpress (or recycle) formyl peptide receptors when challenged with the synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular calcium was not required for either optimal chemotactic responses or receptor reexpression. Depletion and chelation of intracellular calcium resulted in significant diminution in the ability of polymorphonuclear leukocytes to release the specific granule constituents lactoferrin and vitamin B12-binding protein during the process of chemotaxis, but had no effect on the capability of these cells to respond chemotactically. Similarly, chelation of intracellular calcium did not affect the ability of these cells to reexpress a population of formyl peptide receptors. Inhibition of receptor reexpression, by a nonagglutinating derivative of wheat-germ agglutinin, was associated with inhibition of chemotactic responses to FMLP. Thus, it appears that large changes in cytosolic free calcium are not necessary for formyl peptide-induced polymorphonuclear leukocyte chemotaxis. In contrast, continuous reexpression (or recycling) of formyl peptide receptors is required for polymorphonuclear leukocyte chemotactic responses to FMLP, a process that appears to be independent from specific granule fusion with plasma membrane.
Assuntos
Cálcio/fisiologia , Quimiotaxia de Leucócito , Grânulos Citoplasmáticos/metabolismo , Citosol/fisiologia , Fura-2/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina , Neutrófilos/fisiologia , Receptores Imunológicos/metabolismo , Adulto , Benzofuranos , Quimiotaxia de Leucócito/efeitos dos fármacos , Humanos , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Neutrófilos/metabolismo , Receptores de Formil Peptídeo , Receptores Imunológicos/efeitos dos fármacos , Receptores Imunológicos/fisiologia , Espalhamento de Radiação , Temperatura , Aglutininas do Germe de Trigo/farmacologiaRESUMO
Amiloride and the more potent amiloride analog, 5-(N-t-butyl) amiloride (t-butylamiloride), were used to examine the role of the Na+/H+ antiporter in bicarbonate absorption in the in vivo microperfused rat proximal convoluted tubule. Bicarbonate absorption was inhibited 29, 46, and 47% by 0.9 mM or 4.3 mM amiloride, or 1 mM t-butylamiloride, respectively. Sensitivity of the Na+/H+ antiporter to these compounds in vivo was examined using fluorescent measurements of intracellular pH with (2', 7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein (BCECF). Amiloride and t-butylamiloride were shown to be as potent against the antiporter in vivo as in brush border membrane vesicles. A model of proximal tubule bicarbonate absorption was used to correct for changes in the luminal profiles for pH and inhibitor concentration, and for changes in luminal flow rate in the various series. We conclude that the majority of apical membrane proton secretion involved in transepithelial bicarbonate absorption is mediated by the Na+-dependent, amiloride-sensitive Na+H+ antiporter. However, a second mechanism of proton secretion contributes significantly to bicarbonate absorption. This mechanism is Na+-independent and amiloride-insensitive.
Assuntos
Bicarbonatos/metabolismo , Proteínas de Transporte/fisiologia , Túbulos Renais Proximais/metabolismo , Absorção , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Masculino , Matemática , Microvilosidades/enzimologia , Perfusão , ATPases Translocadoras de Prótons/metabolismo , Ratos , Ratos Endogâmicos , Trocadores de Sódio-HidrogênioRESUMO
The coupling mechanism for Cl- and H+/OH- transport in renal brush-border vesicles was examined from intravesicular pH changes following imposed H+ and Cl- gradients. Vesicles were loaded with 6-carboxyfluorescein and exposed to H+ gradients and Cl-, gluconate, or sulfate gradients, each with and without a K+/valinomycin voltage clamp. Parallel experiments were performed with vesicles equilibrated with 10 mM HCO3- or 5 mM formate. Rate of H+/OH- transport was determined from the initial rate of change in 6-carboxyfluorescein fluorescence, vesicle buffer capacity and the relationship between fluorescence and vesicle pH. In contrast to gluconate or sulfate, Cl- caused enhanced H+/OH- transport under all conditions. This difference was eliminated with voltage clamping in the presence of gluconate, SO4(2-), or HCO3-, but not in the presence of formate. These findings were not affected by the method of preparation of the vesicles. Electrically coupled Cl-/OH- transport was not inhibited by 100 microM DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate) or 100 microM DBDS (4,4'-dibenzamidostilbene-2,2'-disulfonate). SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate) was found to be a protonophore at concentrations greater than 500 microM. As a control for the method, we demonstrated amiloride inhibitable, electroneutral Na+-H+ exchange (H+ flux = 107 +/- 9 nmol/s per mg, 100 mM Na+) and electroneutral, DBDS inhibitable Cl(-)-HCO3- exchange in sealed human red blood cell ghosts. Therefore, electroneutral Cl(-)-OH- or HCO3- exchange does not measurably contribute to Cl- transport in the proximal tubule brush border. Cl(-)-formate exchange with formic acid recycling appears to be the only electroneutral coupling mechanism between Cl- and OH- transport demonstrable in renal brush-border membrane vesicles.
Assuntos
Membrana Celular/metabolismo , Cloretos/metabolismo , Hidróxidos/metabolismo , Rim/ultraestrutura , Microvilosidades/metabolismo , Animais , Feminino , Formiatos/farmacologia , Concentração de Íons de Hidrogênio , Troca Iônica , Coelhos , Estilbenos/farmacologiaRESUMO
There is a large body of evidence supporting a role for GTP-binding proteins in signal transduction by growth factors. In certain cells, ligands which activate or inhibit the production of cAMP via heterotrimeric G proteins promote replication of the target cell. These mechanisms play an important role in a limited number of tumours. Ligands which activate PI hydrolysis through heterotrimeric G proteins may also promote growth in certain systems, but the precise role for PI hydrolysis remains to be determined. Receptors with intrinsic tyrosine kinases may also interact with the heterotrimeric G proteins, but it is not known if these interactions represent side reactions, or whether they are central in the responses of certain cell types. Lastly, p21ras and other small molecular weight G proteins appear to be profoundly important in growth control. The tyrosine kinase growth factor receptors may interact indirectly with these GTP binding proteins via GAP proteins. The molecular detail of this process is emerging rapidly and is likely to be worked out in the near future.
Assuntos
Divisão Celular/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Substâncias de Crescimento/metabolismo , Transdução de Sinais , Animais , Fosfatidilinositóis/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
Serotonin-induced DNA synthesis in bovine aortic smooth muscle cells was totally abolished by pretreatment of cultures with 5 ng/ml pertussis toxin. The half maximally effective concentration of toxin was approximately 10 pg/ml. Pertussis toxin did not affect platelet-derived growth factor (PDGF)-stimulated DNA synthesis and actually enhanced the mitogenic effect of the phorbol ester, phorbol 12-myristate 13-acetate. Pertussis toxin did not inhibit serotonin-stimulated inositol phosphate accumulation or increases in intracellular calcium or cAMP concentrations under conditions sufficient to completely inhibit serotonin-induced (3H)thymidine incorporation. These results demonstrate that a novel, pertussis-sensitive pathway is required for serotonin-, but not platelet-derived growth factor-induced DNA synthesis in vascular smooth muscle cells. The pertussis-sensitive step does not involve cAMP, phosphoinositide hydrolysis, mobilization of intracellular calcium, or phorbol ester-sensitive protein kinase C activity.
Assuntos
DNA/biossíntese , Músculo Liso Vascular/metabolismo , Toxina Pertussis , Serotonina/farmacologia , Fatores de Virulência de Bordetella/farmacologia , Animais , Ácidos Araquidônicos/metabolismo , Cálcio/análise , Cálcio/metabolismo , Bovinos , Células Cultivadas/efeitos dos fármacos , AMP Cíclico/biossíntese , AMP Cíclico/metabolismo , DNA/efeitos dos fármacos , Hidrólise , Mitose/efeitos dos fármacos , Músculo Liso Vascular/citologia , Fosfatidilinositóis/metabolismoRESUMO
Calcitonin (CT), a polypeptide hormone, regulates calcium homeostasis by activating surface receptors coupled to stimulation of adenylyl cyclase in bone and kidney cells. CT has also been reported to increase cytoplasmic Ca2+ in osteoclasts and renal tubule cells. Signaling pathways activated by a recombinant porcine renal calcitonin receptor transiently expressed in HEK-293 cells were studied. In cells expressing the recombinant CT receptor, salmon CT stimulated cAMP accumulation (EC50, 0.16 nM) and synthesis of inositol phosphates (IP; EC50, 3.7 nM). Two other recombinant receptors, the m1-muscarinic acetylcholine receptor and the LH receptor, activated synthesis of either IP or cAMP, respectively, but not both. Stable expression of the CT receptor in a CT receptor-deficient cell line, M18, restored the cells' ability to increase cytoplasmic Ca2+ in response to salmon CT. These results show that a single recombinant CT receptor can independently activate effector pathways mediated by cAMP and IP/Ca2+.
Assuntos
Calcitonina/farmacologia , Cálcio/metabolismo , AMP Cíclico/metabolismo , Fosfatos de Inositol/metabolismo , Receptores de Superfície Celular/fisiologia , Transdução de Sinais , Animais , Calcitonina/metabolismo , Linhagem Celular , Humanos , Cinética , Plasmídeos , Receptores da Calcitonina , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/genética , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
OBJECTIVE: The cellular basis of the cardioprotective effects of estrogen are largely unknown. An inhibitory effect on vascular smooth muscle (VSM) growth has been proposed. We examined the effect of 17beta-estradiol (E2) on mechanical strain-induced mitogenesis in human fetal VSM cells. METHODS AND RESULTS: Cells were grown on fibronectin-coated plates with silicone-elastomer bottoms, and exposed to cyclic mechanical strain (60 cycles/min), with and without E2 (1 nmol/l), for 48 h. [3H]-Thymidine incorporation was measured during the last 6 h. Strain induced 1.5-2 fold increases in DNA synthesis that were attenuated by antibodies to platelet-derived growth factor (PDGF) AA and BB. Strain also induced increases both in mRNA and protein levels of Sp-1, a transcription factor that binds to the PDGF-A gene promoter site. E2 attenuated strain-induced mitogenesis, and also increases in mRNA and protein levels of Sp-1. The estrogen receptor (ER) antagonist ICI 182,780 (100 nmol/l) reversed the inhibitory effect of E2 on strain-induced increases in DNA synthesis and Sp-1 protein. RT-PCR analysis showed presence of both ER-alpha and -beta in these cells. CONCLUSIONS: Estrogen inhibits strain-induced mitogenesis in human VSM cells via an ER mediated process involving down-regulation of the transcription factor Sp-1.
Assuntos
Estradiol/farmacologia , Mitose/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Fator de Transcrição Sp1/metabolismo , Técnicas de Cultura de Células , Divisão Celular/fisiologia , DNA/biossíntese , Regulação para Baixo , Feminino , Humanos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/fisiologia , RNA Mensageiro/genética , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse MecânicoRESUMO
Proliferation of cultured neonatal vascular smooth muscle (VSM) cells is enhanced by exposure to cyclic mechanical strain, in part through autocrine action of secreted platelet-derived growth factor (PDGF). We examined transcription factors and DNA response elements that may participate in the induction of PDGF-A gene transcription by mechanical strain. PDGF-A mRNA increased gradually over 4 to 24 hours exposure to cyclic (1 Hz) strain. This was due, at least in part, to increased transcription since a full length (890 bp) PDGF-A promoter reporter construct was induced 3.5-fold in transfected VSM cells exposed to strain for 24 hours. A series of PDGF-A promoter truncation reporter constructs was used to identify potential regions of the promoter involved in regulation by strain. Strain-responsive regions were found between -262 bp and -92 bp and between -92 bp and -41 bp of the promoter. Since these regions are GC-rich and contain response elements for Egr-1 and Sp-1, we examined expression of these transcription factors in response to strain. mRNA for both factors increased over 0.5 to 4 hours of strain, while protein expression for both increased gradually over a 24 hours period. Gel shift assays with a probe specific for Egr-1 demonstrated at least 1 prominent new shifted band after 4 to 12 hours exposure to strain. An Sp-1 probe demonstrated constitutive shifted bands that did not change in response to strain. Thus, GC-rich regions in the proximal 92 bp of the PDGF-A promoter contain mechanical strain-responsive elements that bind Egr-1 and possibly Sp-1.
Assuntos
Proteínas Imediatamente Precoces , Músculo Liso Vascular/fisiologia , Fator de Crescimento Derivado de Plaquetas/biossíntese , Fator de Crescimento Derivado de Plaquetas/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Animais Recém-Nascidos , Divisão Celular , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Proteína 1 de Resposta de Crescimento Precoce , Regulação da Expressão Gênica , Genes Reporter , Cinética , Músculo Liso Vascular/citologia , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Fator de Transcrição Sp1/biossíntese , Estresse Mecânico , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , TransfecçãoAssuntos
Antiporters , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Eritrócitos/metabolismo , Hipertensão/etiologia , Contração Muscular , Animais , Humanos , Hipertensão/sangue , Hipertensão/genética , Hipertensão/metabolismo , Tono Muscular , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Ratos , Ratos Endogâmicos SHR , Trocadores de Sódio-HidrogênioRESUMO
The net H+/OH- permeability of rabbit renal proximal tubule brush border membrane vesicles was determined by measuring the rate of collapse of preformed pH gradients using acridine orange. The membranes were voltage clamped using valinomycin and [K+]in = [K+]out. Internal buffer capacity was determined by titration of lysed vesicles and by titration of measured Na+/H+ exchange rates with exogenously added buffers. Both methods revealed an intravesicular buffer capacity of 125-135 mM/pH unit at pH 6.0 and 20 degrees C. Using this buffer capacity, the net H+/OH- permeability was found to be 5 X 10(-3) cm/s in brush border vesicles prepared by Mg2+ aggregation. The rate of collapse of pH gradients in brush border vesicles prepared by sucrose density gradient centrifugation was virtually identical to the rate in vesicles prepared with Mg2+, indicating that the high H+/OH- permeability was not an artifact of Mg2+ preparation. Activation energy of the H+/OH- permeability pathway was 4.9 kcal/mol, whereas activation energy of the Na+/H+ antiporter was 11.4 kcal/mol. Since the rate of H+/OH- diffusion was not affected by amiloride, it is concluded that H+/OH- permeate through brush border membranes by a pathway separate from the Na+/H+ antiporter. This pathway is not inhibited by dicyclohexylcarbodiimide at concentrations up to 2 mM but is inhibited by 0.2-5 mM p-chloromercuribenzenesulfonate, suggesting the presence of a sulfhydryl group in the pathway.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Hidrogênio/metabolismo , Hidróxidos/metabolismo , Túbulos Renais Proximais/metabolismo , Microvilosidades/metabolismo , Laranja de Acridina , Animais , Soluções Tampão , Dicicloexilcarbodi-Imida/farmacologia , Feminino , Concentração de Íons de Hidrogênio , Radical Hidroxila , Permeabilidade , CoelhosRESUMO
This discussion was selected from the weekly staff conferences in the Department of Medicine, University of California, San Francisco. Taken from a transcription, it has been edited by Homer A. Boushey, MD, Professor of Medicine, and Nathan M. Bass, MD, PhD, Associate Professor of Medicine, under the direction of Lloyd H. Smith, Jr, MD, Professor of Medicine and Associate Dean in the School of Medicine.
Assuntos
Eletrólitos/metabolismo , Hipertensão/etiologia , Animais , Cálcio/metabolismo , Humanos , Hipertensão/fisiopatologia , Hipertensão/terapia , Lítio/metabolismo , Contração Muscular , Músculo Liso Vascular , Potássio/metabolismo , Sódio/metabolismoRESUMO
The role for intracellular Ca2+ in modulating activity of the Na+/H+ exchanger was studied in cultured vascular smooth muscle cells. Na+/H+ exchange was activated by four distinct stimuli: 1) phorbol 12-myristate 13-acetate, 2) thrombin, 3) cell shrinkage, and 4) intracellular acid loading. [Ca2+]i was independently varied between 40 and 200 nM by varying the bathing Ca2+ from 10 nM to 5.0 mM. Thrombin-induced intracellular Ca2+ transients were blocked with bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM). In the absence of stimulators of Na+/H+ exchange, varying [Ca2+]i above or below the basal level of 140 nM did not activate Na+/H+ exchange spontaneously. However, varying [Ca2+]i did affect stimulus-induced activation of Na+/H+ exchange. Activation of the exchanger by phorbol 12-myristate 13-acetate was blunted by reduced intracellular Ca2+ (half-maximal activity at 50-90 nM [Ca2+]i), consistent with a Ca2+ requirement for protein kinase C (Ca2+/phospholipid-dependent enzyme). Activation of the exchanger by thrombin in protein kinase C-depleted cells was also sensitive to reduced intracellular Ca2+ (half-maximal activity at 90-140 nM [Ca2+]i) and was increased 40% by raising [Ca2+]i to 200 nM. Activation of the exchanger by cell shrinkage or intracellular acid loads was not significantly affected over the range of [Ca2+]i tested. Thus, altered [Ca2+]i does not itself affect Na+/H+ exchange activity in vascular smooth muscle but instead modulates activation of the transporter by particular stimuli.
Assuntos
Cálcio/fisiologia , Proteínas de Transporte/metabolismo , Músculo Liso Vascular/metabolismo , Animais , Animais Recém-Nascidos , Cálcio/farmacologia , Células Cultivadas , Concentração de Íons de Hidrogênio , Cinética , Proteína Quinase C/metabolismo , Ratos , Trocadores de Sódio-Hidrogênio , Trombina/farmacologiaRESUMO
We compared the mechanisms by which thrombin and platelet-derived growth factor (PDGF) activate phospholipase C in cultured vascular smooth muscle cells. Thrombin caused a transient (less than 5 min) increase in inositol trisphosphate (IP3) while PDGF caused a sustained (greater than 10 min) increase. Both pertussis toxin and phorbol 12-myristate 13-acetate (PMA) inhibited the thrombin-induced increase in IP3 but neither agent affected the PDGF-induced increase in IP3. To examine the role of GTP binding (G) proteins in the activation of phospholipase C by these two hormones, GTP analogues were introduced into saponin-permeabilized cells. In the absence of hormones, guanosine 5'-O-(3-thiotrisphosphate) (GTP gamma S) caused a progressive increase in IP3 release which was inhibited 55% by PMA (200 ng/ml). In the presence of thrombin, GTP gamma S caused synergistic increase in IP3 release. The synergism between GTP gamma S and thrombin was virtually eliminated by 10 min prior exposure to PMA (200 ng/ml). When PDGF was the hormonal agonist, GTP gamma S also caused synergistic increase in IP3 release and guanosine 5'-O-(2-thiodiphosphate) blunted PDGF-induced IP3 release. However, in contrast to thrombin, the synergism between GTP gamma S and PDGF was unaffected by PMA. Thus, thrombin and PDGF activate phospholipase C by signal transduction systems which differ in kinetic properties and in sensitivity to PMA and pertussis toxin. Despite these differences, both systems appear to involve GTP binding proteins at some step.