RESUMEN
The urine concentrating mechanism in the mammalian renal inner medulla (IM) is not understood, although it is generally considered to involve countercurrent flows in tubules and blood vessels. A possible role for the three-dimensional relationships of these tubules and vessels in the concentrating process is suggested by recent reconstructions from serial sections labelled with antibodies to tubular and vascular proteins and mathematical models based on these studies. The reconstructions revealed that the lower 60% of each descending thin limb (DTL) of Henle's loops lacks water channels (aquaporin-1) and osmotic water permeability and ascending thin limbs (ATLs) begin with a prebend segment of constant length. In the outer zone of the IM (i) clusters of coalescing collecting ducts (CDs) form organizing motif for loops of Henle and vasa recta; (ii) DTLs and descending vasa recta (DVR) are arrayed outside CD clusters, whereas ATLs and ascending vasa recta (AVR) are uniformly distributed inside and outside clusters; (iii) within CD clusters, interstitial nodal spaces are formed by a CD on one side, AVR on two sides, and an ATL on the fourth side. These spaces may function as mixing chambers for urea from CDs and NaCl from ATLs. In the inner zone of the IM, cluster organization disappears and half of Henle's loops have broad lateral bends wrapped around terminal CDs. Mathematical models based on these findings and involving solute mixing in the interstitial spaces can produce urine slightly more concentrated than that of a moderately antidiuretic rat but no higher.
Asunto(s)
Capacidad de Concentración Renal/fisiología , Médula Renal/anatomía & histología , Médula Renal/metabolismo , Mamíferos/anatomía & histología , Mamíferos/metabolismo , Orina/química , Animales , Cloruros/metabolismo , Simulación por Computador , Imagenología Tridimensional , Asa de la Nefrona/anatomía & histología , Asa de la Nefrona/metabolismo , Modelos Biológicos , Modelos Teóricos , Sodio/metabolismoRESUMEN
We have previously demonstrated that cardiac myocytes isolated from the hearts of adult dogs develop rapid repetitive cytosolic Ca2+ transients, membrane depolarization, and cell contraction by mobilization of sarcoplasmic reticulum Ca2+ stores when exposed to a soluble factor from the trypomastigotes of Trypanosoma cruzi. These findings led us to investigate the regulatory mechanisms of cytosolic Ca2+ in cardiac tissues from dogs chronically infected with T. cruzi. Expression of the plasma membrane calcium pump (PMCA) RNA and protein was determined by Northern and Western blotting, respectively, followed by densitometric analyses. A 642-bp PMCA 1b complementary DNA probe derived from canine epicardial tissue hybridized to 2 major transcripts (7.3 and 5.3 kb) in canine epicardium. Expression of the dominant transcript (7.3 kb) was 77% greater in cardiac tissues obtained from dogs with chronic T. cruzi infection (140 days after inoculation) in comparison with constitutive expression levels in normal dogs. Monoclonal antibody 5F10, known to recognize all isoforms of the PMCA, was used to detect expression of the PMCA protein in epicardial tissue. Expression of a 142-kDa protein was increased by 58% in the cardiac tissues of infected dogs when compared with those from uninfected dogs. To establish a link between the upregulation of PMCA in dogs chronically infected with Chagas disease and the ventricular-based arrhythmias and myocardial failure that occur during this stage of disease both in dogs and humans, further study will be required.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Enfermedad de Chagas/metabolismo , Miocitos Cardíacos/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Northern Blotting , Western Blotting , ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/genética , ATPasas Transportadoras de Calcio/inmunología , Proteínas de Transporte de Catión , Membrana Celular/metabolismo , ADN Complementario/química , Modelos Animales de Enfermedad , Perros , Femenino , Masculino , Datos de Secuencia Molecular , Miocitos Cardíacos/ultraestructura , ATPasas Transportadoras de Calcio de la Membrana Plasmática , ARN Mensajero/metabolismo , ARN Protozoario/genética , ARN Ribosómico 18S/genética , Homología de Secuencia de Ácido Nucleico , Trypanosoma cruzi/genética , Trypanosoma cruzi/inmunología , Regulación hacia ArribaRESUMEN
Amino acids are apparently recycled between loops of Henle and vasa recta in rat papilla in vivo. To examine this process in the absence of metabolism, we performed continuous microinfusions of rat renal papillary ascending thin limbs (ATLs) and vasa recta in vivo, and microperflusions of isolated rat renal papillary descending thin limbs (DTLs) and ATLs in vitro using the nonmetabolizable, synthetic, neutral amino acid cycloleucine. Like naturally occurring amino acids, approximately = 25% of radiolabeled cycloleucine microinfused into ATLs in vivo was reabsorbed by a process that was not saturable or inhibitable. Also, like naturally occurring amino acids, approximately = 47% (relative to inulin) of radiolabeled cycloleucine microinfused into ascending vasa recta in vivo was transferred directly into ipsilateral tubular structures (probably DTLs) by a saturable and inhibitable process. In DTLs perfused in vitro, unidirectional bath-to-lumen fluxes (Jbl) tended to exceed unidirectional lumen-to-bath fluxes (Jlb), whereas in ATLs perfused in vitro Jlb tended to exceed Jbl, but the differences were not statistically significant. Moreover, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from ATLs in vivo but incompatible with apparent movement from vasa recta to DTLs in vivo. These in vitro observations are like those made previously for the naturally occurring neutral amino acid L-alanine. The lack of saturation and inhibition, like the previous data on L-alanine, suggest that transepithelial movement of amino acids in thin limbs of Henle's loop may occur via a paracellular route and that regulation of amino acid movement in vivo may involve vasa recta, not DTLs. They also suggest that cycloleucine is a good nonmetabolizable surrogate for the study of neutral amino acid transport in the kidney.
Asunto(s)
Cicloleucina/metabolismo , Asa de la Nefrona/metabolismo , Circulación Renal/fisiología , Alanina/farmacocinética , Animales , Transporte Biológico/fisiología , Técnicas In Vitro , Masculino , Microcirculación/fisiología , Perfusión , Ratas , Ratas Wistar , Agua/metabolismoRESUMEN
The effects of bafilomycin A(1), a blocker of V-type H(+)-ATPases, were investigated in Malpighian tubules of Aedes aegypti. Bafilomycin A(1) reduced rates of transepithelial fluid secretion and the virtual short-circuit current (vI(sc)) with an IC(50) of approximately 5 micromol l(-)(1). As vI(sc) decreased, the electrical resistance increased across the whole epithelium and across the apical membrane, indicating effects on electroconductive pathways. Bafilomycin A(1) had no effect when applied from the tubule lumen, pointing to the relative impermeability of the apical membrane to bafilomycin A(1). Thus, bafilomycin A(1) must take a cytoplasmic route to its blocking site in the proton channel of the H(+)-ATPase located in the apical membrane of principal cells. The inhibitory effects of bafilomycin A(1) were qualitatively similar to those of dinitrophenol in that voltages across the epithelium (V(t)), the basolateral membrane (V(bl)) and the apical membrane (V(a)) depolarized towards zero in parallel. Moreover, V(bl )always tracked V(a), indicating electrical coupling between the two membranes through the shunt. Electrical coupling allows the H(+)-ATPase to energize not only the apical membrane, but also the basolateral membrane. Furthermore, electrical coupling offers a balance between electroconductive entry of cations across the basolateral membrane and extrusion across the apical membrane to support steady-state conditions during transepithelial transport.
Asunto(s)
Macrólidos , Túbulos de Malpighi/enzimología , Proteínas de la Membrana/fisiología , ATPasas de Translocación de Protón/fisiología , 2,4-Dinitrofenol/farmacología , Aedes , Animales , Antibacterianos/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Membrana Celular/fisiología , Polaridad Celular/efectos de los fármacos , Polaridad Celular/fisiología , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Epitelio/efectos de los fármacos , Epitelio/enzimología , Epitelio/metabolismo , Túbulos de Malpighi/citología , Túbulos de Malpighi/efectos de los fármacos , Túbulos de Malpighi/metabolismoRESUMEN
Previous studies have generally indicated that the entire descending (DTL) and ascending thin limbs (ATL) of Henle's loops in the mammalian inner medulla exhibit structurally and functionally distinct properties. In the present study, we found that about 50% of Munich-Wistar rat inner medullary thin limbs, lying at positions distinctly above the bend, had segments exhibiting structural characteristics of DTL located immediately adjacent to segments exhibiting structural characteristics of ATL. Multiple DTL-type and ATL-type segments of variable length existed along a single straight portion of these mixed tubules. Inner medullary thin limbs with repeating, sequential expression of DTL-type and ATL-type regions were also numerous in Sprague-Dawley rats, mice, and rabbits with no evidence of sexual dimorphism. RT-PCR of microdissected segments showed that the water channel aquaporin-1 (AQP1) and the urea transporter UT-A2 were expressed in pure DTL, but not in pure ATL, and in DTL-type, but not in ATL-type, regions of mixed-type thin limbs. Immunocytochemistry revealed expression of AQP1 in cells of pure DTL, but not pure ATL, and in cells of DTL-type, but not ATL-type, regions of mixed-type thin limbs. In contrast, the chloride channel ClC-K1 was expressed in pure ATL, but not pure DTL, and in ATL-type, but not DTL-type, regions of mixed-type thin limbs. Discontinuous axial expression of AQP1, UT-A2, and ClC-K1 along the straight portion of single thin limbs indicates that these nephrons possess a more heterogeneous structure than previously recognized.
Asunto(s)
Acuaporinas/metabolismo , Proteínas Portadoras/metabolismo , Canales de Cloruro/metabolismo , Médula Renal/fisiología , Asa de la Nefrona/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Animales , Acuaporina 1 , Femenino , Médula Renal/anatomía & histología , Asa de la Nefrona/anatomía & histología , Masculino , Ratones , Conejos , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Transportadores de UreaRESUMEN
The transepithelial voltage (V(t)) of isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti spontaneously oscillates in more than half the tubules. Typically, V(t) decreases and then rises at a frequency of 2 oscillations/min with a duration of 16 s. In 6 isolated perfused tubules studied in detail, V(t) oscillates between 50.5 mV and 15.7 mV in parallel with (1) oscillations of the transepithelial resistance (R(t)) between 7.61 kOmegacm and 3.63 kOmegacm, (2) oscillations of the basolateral membrane voltage of principal cells between -56.7 mV and -72.2 mV, and (3) oscillations of the apical membrane voltage between 107.2 mV and 87.8 mV. The oscillations are dependent on the Cl concentration in the extracellular solutions. As R(t) decreases during the oscillations V(t) goes to the transepithelial equilibrium potential of Cl (E(cl)) indicating transient changes in transepithelial Cl conductance as the mechanism of voltage and resistance oscillations. Since the largest voltage oscillations take place across the whole epithelium and not across cell membranes, oscillating Cl conductances are localized to a single transepithelial Cl diffusion barrier such as the paracellular pathway. This conclusion is supported by the analysis of electrically equivalent circuits that identify the shunt pathway as the site of oscillating Cl conductances.
RESUMEN
An unusual 120-kDa alkaline peptidase contained in a trypomastigote soluble fraction (TSF) of Trypanosoma cruzi is associated with the induction of repetitive Ca2+ transients and subsequent invasion by the parasite of a number of mammalian cell lines, including tissue culture L6E2 myoblasts (B. A. Burleigh and N. W. Andrews, J. Biol. Chem. 270:5172-5180, 1995; S. N. J. Moreno, J. Silva, A. E. Vercesi, and R. Docampo, J. Exp. Med. 180:1535-1540, 1994; A. Rodríguez, M. G. Rioult, A. Ora, and N. W. Andrews, J. Cell Biol. 129:1263-1273, 1995; I. Tardieux, M. H. Nathanson, and N. W. Andrews, J. Exp. Med. 179:1017-1022, 1994). Using single cell spectrofluorometry and whole-cell patch clamping, we show that TSF produces rapid repetitive cytosolic Ca2+ transients (each associated with cell contraction) in primary cardiac myocytes isolated from dogs. The response of myocytes to TSF was dose dependent in that increasing numbers of cells responded to increasing concentrations of TSF. The TSF-induced Ca2+ transients could be obliterated when TSF was heated or treated with trypsin or the protease inhibitor leupeptin. Aprotinin, pepstatin A, and E-64 did not affect TSF activity. The TSF-induced Ca2+ transients and trypomastigote cell invasion could not be inhibited by alpha (prazosin)- or beta (propanolol)-adrenergic blockers or L-type Ca2+ channel blockers (verapamil, nisoldipine, or cadmium) or by removal of extracellular Ca2+. However, inhibition of pertussis toxin-sensitive G proteins and Ca2+ release from the sarcoplasmic reticulum (with thapsigargin or ryanodine) prevented the TSF-induced Ca2+ transients and cell invasion by trypomastigotes. These data suggested that cardiac myocyte pertussis toxin-sensitive G proteins are associated with the regulation of TSF-induced Ca2+ transients and myocyte invasion by trypomastigotes but are independent of Ca2+ entry into the cytosol via L-type Ca2+ channels. The Ca2+ transients are dependent on release of Ca2+ from sarcoplasmic reticulum Ca2+ stores, but this release is not dependent on extracellular Ca2+ or on the classic model of Ca2+ -induced Ca2+ release in cardiac myocytes. Further, subthreshold depolarizations, together with cell contraction as demonstrated by whole-cell patch clamping, occurred with each Ca2+ transient. However, the depolarizations were of magnitude insufficient to generate an action potential, providing further evidence for a lack of dependence on L-type Ca2+ channels and other voltage-dependent channels (Na+ and K+ channels) in the generation of TSF-induced Ca2+ transients. Our findings suggest that primary canine cardiac myocytes respond to TSF and parasite invasion in ways similar to those of the in vitro cell lines studied to date. Since cardiac myocytes are primary targets for T. cruzi in the vertebrate host, our study indicates that TSF may play a role in the pathogenesis of Chagas' disease in humans.
Asunto(s)
Calcio/metabolismo , Cisteína Endopeptidasas/toxicidad , Miocardio/metabolismo , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/patogenicidad , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Células Cultivadas , Enfermedad de Chagas/etiología , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/aislamiento & purificación , Citosol/metabolismo , Perros , Proteínas de Unión al GTP/metabolismo , Humanos , Transporte Iónico/efectos de los fármacos , Peso Molecular , Transducción de Señal/efectos de los fármacos , SolubilidadRESUMEN
The vitamin D hormone, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], was shown to increase intestinal plasma membrane calcium pump (PMCA) gene expression. The present study was done to determine whether gene transcription is involved in this process. Nuclei were isolated from duodena of vitamin-D-deficient chicks given 1,25(OH)2D3 intracardially at various times before experiment. The abundance of PMCA RNA in the nuclear and total cellular fractions, measured by a ribonuclease protection assay, was significantly increased above control values at 1.5 hr. and maximized at 3 hr. post-dose. As shown, cross-contamination of nuclear PMCA RNA by cytosolic RNA cannot account for these results. These studies are the first to show that 1,25(OH)2D3 regulates expression of a plasma membrane calcium pump gene by increasing the rate of transcription.
Asunto(s)
Calcitriol/farmacología , ATPasas Transportadoras de Calcio/genética , Regulación de la Expresión Génica/efectos de los fármacos , Mucosa Intestinal/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Membrana Celular/metabolismo , Pollos , Cinética , Masculino , Hibridación de Ácido Nucleico , ARN/metabolismo , Sondas ARN , Deficiencia de Vitamina D/metabolismoRESUMEN
A peptide termed culekinin depolarizing peptide (CDP) was isolated from approximately 1.2 million mosquitos (94% Culex salinarius). The peptide was isolated on the basis of a rapid myotropic assay that utilized a hindgut preparation from Leucophaea maderae and a transepithelial voltage assay that used mosquito Malpighian tubules from Aedes aegypti. A 15% trifluoroacetic acid extraction from the mosquitos, two solid phase extraction steps, and six HPLC steps resulted in the isolation of 9.7 nmol of CDP. This value corresponds to approximately 8 fmol/mosquito. Edman degradation indicated the following sequence for CDP: Asn-Pro-Phe-His-Ser-Trp-Gly-NH2. The sequence was confirmed as the suspected C-terminal amide form of the peptide, since native and synthetic CDP had identical chemical and biological properties. CDP is a member of the leucokinin family of neuropeptides. The leucokinins have been found in three other insect species (Leucophaea maderae, Acheta domesticus and Locusta migratoria) where these peptides were isolated by their myotropic properties alone. CDP shares a C-terminal sequence homology (i.e., Phe-X-Ser-Trp-Gly-NH2) with the rest of the leucokinins. CDP corresponds to the strongest tubule depolarizing activity in the C. salinarius extract. These findings agree with previous structure-activity studies that suggest that mosquitos would contain a leucokinin-like factor that had Phe-His-Ser-Trp-Gly-NH2 as the C-terminal pentapeptide. This is the first leucokinin isolated from blood feeding or holometabolous insects.
Asunto(s)
Aedes/fisiología , Cucarachas/fisiología , Túbulos de Malpighi/metabolismo , Neuropéptidos/fisiología , Secuencia de Aminoácidos , Animales , Intestinos , Transporte Iónico/fisiología , Datos de Secuencia Molecular , Contracción Muscular/fisiología , Neuropéptidos/aislamiento & purificaciónRESUMEN
Isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti spontaneously secrete NaCl, KCl and water across an epithelium of modest transepithelial resistance (40-80 omega cm2) and high transepithelial voltage (30-70 mV, lumen positive). Transepithelial electrochemical potentials indicate that Na and K are secreted by active and Cl by passive transport mechanisms. The addition of synthetic leucokinin-VIII (LK-VIII, insect myotropic peptide) to the peritubular bath significantly increases the rates of transepithelial NaCl, KCl and water secretion. In parallel, LK-VIII depolarizes the transepithelial voltage from 59.3 to 5.7 mV, decreases the transepithelial resistance from 57.7 to 9.9 omega cm2, and renders the basolateral and apical membrane voltages nearly equipotential (approximately -90 mV). Unilateral step changes of the [Cl] in the peritubular bath or tubule lumen elicit small transepithelial Cl diffusion potentials in the absence of LK-VIII but large transepithelial Cl diffusion potentials, up to 85% of Nernst equilibrium potentials, in the presence of LK-VIII. In Malpighian tubules treated with dinitrophenol for estimates of the shunt resistance Rsh, LK-VIII reduces Rsh from 52.5 to 5.8 omega cm2. Bilateral reductions of the Cl concentration in tubule lumen and peritubular bath fully restore Rsh to 55.8 omega cm2 in the presence of LK-VIII. LK-VIII has no effects when presented from the luminal side. These results suggest that LK-VIII increases the Cl conductance of the epithelial shunt via a receptor located at the basolateral side of the epithelium.
Asunto(s)
Transporte Iónico/efectos de los fármacos , Tuftsina/farmacología , Aedes , Animales , Líquidos Corporales/metabolismo , Cloruros/metabolismo , Dinitrofenoles/farmacología , Conductividad Eléctrica , Electrofisiología , Epitelio/efectos de los fármacos , Epitelio/fisiología , Técnicas In Vitro , Túbulos de Malpighi/efectos de los fármacos , Túbulos de Malpighi/fisiología , Potenciales de la MembranaRESUMEN
In the course of electrophysiological studies of Malpighian tubules of the mosquito Aedes aegypti, we have found unusual effects of 2,4-dinitrophenol (DNP) that offer new insights into the electrogenic and conductive properties of the tubule. DNP (10(-4)M) depolarized the basolateral membrane voltage from -58.0 to -3.3 mV, and it depolarized the apical membrane voltage from 110.6 to 8.9 mV. In parallel the transepithelial electrical resistance increased from 11.4 to 16.8 k omega.cm, and the fractional resistance of the apical membrane increased from 0.32 to 0.57. On the assumption that measures of transepithelial resistance in the presence of DNP approach the shunt resistance, the experimental results indicate the following characteristics for the equivalent circuit of the tubule: 1) a shunt resistance that is approximately one-half the transcellular resistance, 2) low and high electromotive forces, respectively, at the basolateral and apical membranes of principal cells, 3) an electrogenic pump at the apical membrane, and 4) a basolateral membrane voltage that is due mostly to the voltage developed by current flow across the basolateral membrane resistance.
Asunto(s)
Dinitrofenoles/farmacología , Túbulos de Malpighi/efectos de los fármacos , 2,4-Dinitrofenol , Aedes , Animales , Electrofisiología , Técnicas In Vitro , Túbulos de Malpighi/fisiología , Modelos Biológicos , Desacopladores/farmacologíaRESUMEN
The effects of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and bumetanide (both 10(-4) M) on transepithelial Na+, K+, Cl-, and fluid secretion and on tubule electrophysiology were studied in isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti. Peritubular DBcAMP significantly increased Na+, Cl-, and fluid secretion but decreased K+ secretion. In DBcAMP-stimulated tubules, bumetanide caused Na+, Cl-, and fluid secretion to return to pre-cAMP control rates and K+ secretion to decrease further. Peritubular bumetanide significantly increased Na+ secretion and decreased K+ secretion so that Cl- and fluid secretion did not change. In bumetanide-treated tubules, the secretagogue effects of DBcAMP are blocked. In isolated Malpighian tubules perfused with symmetrical Ringer solution, DBcAMP significantly hyperpolarized the transepithelial voltage (VT) and depolarized the basolateral membrane voltage (Vbl) with no effect on apical membrane voltage (Va). Total transepithelial resistance (RT) and the fractional resistance of the basolateral membrane (fRbl) significantly decreased. Bumetanide also hyperpolarized VT and depolarized Vbl, however without significantly affecting RT and fRbl. Together these results suggest that, in addition to stimulating electroconductive transport, DBcAMP also activates a nonconductive bumetanide-sensitive transport system in Aedes Malpighian tubules.
Asunto(s)
Bucladesina/farmacología , Bumetanida/farmacología , Electrólitos/metabolismo , Túbulos de Malpighi/fisiología , Aedes , Animales , Cloruros/metabolismo , Epitelio/efectos de los fármacos , Epitelio/fisiología , Femenino , Cinética , Túbulos de Malpighi/efectos de los fármacos , Métodos , Modelos Biológicos , Potasio/metabolismo , Sodio/metabolismoRESUMEN
Leucokinins are octapeptides isolated from heads of the cockroach Leucophaea maderae. In the cockroach they increase motility of the isolated hindgut. Surprisingly, synthetic leucokinins have biological activity in a different insect and in a different tissue. In isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti, leucokinins depolarize the transepithelial voltage. This effect on voltage is dependent on extracellular Cl. One leucokinin, LK-8, the effects of which were studied further in isolated Malpighian tubules, was found to inhibit transepithelial fluid secretion at low concentrations (10(-11) M threshold), and to stimulate fluid secretion at high concentrations (3.5 x 10(-9) M threshold). Together, the depolarizing effects on voltage and the stimulation of fluid secretion suggest that leucokinins increase the Cl permeability of the tubule wall thereby increasing the availability of Cl for secretion with Na, K and water. Structure-function comparisons of the seven leucokinins studied suggest that the active region of the octapeptide is segregated to the C-terminal pentapeptide. In view of the known effects of leucokinins on hindgut motility in the cockroach, our finding of effects in mosquito Malpighian tubules suggests that leucokinins may be widely distributed in insects where they may have diverse functions in a variety of organs.