RESUMO
The 'Triple-Whamm'-combination (TW) of renin-angiotensin-aldosteron-system-inhibitors (RAASI), diuretics and non-steroidal anti-inflammatory drugs (NSAID) can cause acute kidney injury (AKI), especially with additional risk factors like chronic kidney disease (CKD) or surgery. Thus, patients on 'Double-Whammy'-combination (DW) of RAASI and diuretics should receive postoperative NSAID only following risk-benefit-evaluation. Currently, there are no data how often surgical patients take DW/TW at admission and postoperatively. The objective of this study was to firstly assess the prevalence of DW/TW-patients, secondly, to evaluate postoperative NSAID use in DW-patients and possible effects on renal function (RF). In a seven-month retrospective study, the pre-hospital medication of patients admitted to surgical wards of a tertiary teaching hospital was screened for intake of TW-drugs and renal impairment (RI; eGFR <60 ml/min/1.73 m 2 ), respectively. For patients admitted with a DW-combination of RAASI and diuretic and undergoing surgery, postoperative NSAID use was recorded and checked against internal guidelines for postoperative pain management recommending as first line NSAID therapy ibuprofen in bone surgery and novaminsulfone in visceral surgery. If NSAID were taken, RF was followed for five days. Of 2007 patients, 343 (17.1%) presented with DW in pre-hospital medication and 28 (1.4%) with TW, which 19/28 (67.9%) took only on demand. Upon admission, RI was present in 113 (32.9%) DW-patients and 9 (33.3%) TW-patients. 227/343 (66.2%) DW-patients underwent surgery and 34/227 (15.0%) were prescribed postoperative NSAID. 24/227 (10.6%) actually received NSAID and 4/24 (16.7%) had a decrease of RF with one showing AKI. In our hospitalized surgical patients, TW-combination in pre-hospital medication was rare. The intake of DW-combination was common but only a small number actually received NSAID after surgery. When a TW-combination was given postoperatively, renal function decreased in every sixth patient. Thus, the absolute number of AKI following a TW-combination was small, however, the individual risk for TW-caused AKI should be considered when choosing postoperative pain management. Guidelines for postoperative NSAID use should consider the patient individual risk factors for AKI, thereby increasing drug safety.
Assuntos
Injúria Renal Aguda , Inibidores da Enzima Conversora de Angiotensina , Injúria Renal Aguda/induzido quimicamente , Inibidores da Enzima Conversora de Angiotensina/efeitos adversos , Anti-Inflamatórios não Esteroides/efeitos adversos , Anti-Hipertensivos , Diuréticos/efeitos adversos , Hospitais de Ensino , Humanos , Proteínas de Membrana , Proteínas Associadas aos Microtúbulos , Dor Pós-Operatória/induzido quimicamente , Dor Pós-Operatória/complicações , Dor Pós-Operatória/tratamento farmacológico , Estudos RetrospectivosRESUMO
Structured risk screening for postoperative delirium (POD) considering prehospital medication is not established. We aimed to develop a POD-risk prediction score based on known risk factors and delirium-risk increasing drugs to be used by pharmacists during medication reconciliation at hospital admission, and to test for feasibility in a retrospective cohort of surgical patients. Therefore, established POD-risk factors and drugs were extracted from the literature and a score was generated. Following this, the score was tested for feasibility in a retrospective 3-month-cohort of surgical patients. For patients with higher scores suggesting higher probability of POD, patient charts were screened for documentation of POD. For development of the score, the following POD-risk factors were defined and points assigned for score calculation: age (≥65 years=1 point/≥75 years=2), male sex (1), renal insufficiency (RI; 1), hepatic impairment (HI; Model-of-endstage-liver-disease (MELD) 10-14=1/≥15=2), delirium-risk increasing drugs (1 point per drug class), anticholinergic drug burden (ACB; ≥3=1). In the retrospective test cohort of 1174 surgical patients these factors concerned: age ≥65 years 567 patients (48%)/≥75 years 303 (26%), male 652 (55%), RI 238 (20%), MELD 10-14 106 (9%)/≥15 65 (5%), ≥ 1 delirium-risk increasing drug 418 (36%), ACB ≥3 106 (9%). The median POD-risk prediction score was 2 (range 0-9). Of 146 patients (12%) with a score ≥ 5, POD was documented for 43 (30%), no evidence for POD for 91 (62%) and data inconclusive for 12 (8%). For scores of ≥ 7, POD was documented for 50% of the patients with sufficient POD documentation. Overall, POD documentation was poor. To summarize, we developed and successfully tested the feasibility of a POD-prediction-score assessable by pharmacists at medication reconciliation at hospital admission.
Assuntos
Delírio , Complicações Pós-Operatórias , Humanos , Masculino , Idoso , Estudos Retrospectivos , Estudos de Viabilidade , Delírio/induzido quimicamente , Delírio/diagnóstico , Delírio/epidemiologia , Fatores de Risco , Antagonistas ColinérgicosRESUMO
In patients with cystic fibrosis, cystic fibrosis transmembrane conductance regulator (CFTR) biomarkers, such as sweat chloride concentration and/or nasal potential difference, are used as end-points of efficacy in phase-III clinical trials with the disease modifying drugs ivacaftor (VX-770), VX809 and ataluren. The aim of this project was to review the literature on reliability, validity and responsiveness of nasal potential difference, sweat chloride and intestinal current measurement in patients with cystic fibrosis. Data on clinimetric properties were collected for each biomarker and reviewed by an international team of experts. Data on reliability, validity and responsiveness were tabulated. In addition, narrative answers to four key questions were discussed and agreed by the team of experts. The data collected demonstrated the reliability, validity and responsiveness of nasal potential difference. Fewer data were found on reliability of sweat chloride concentration; however, validity and responsiveness were demonstrated. Validity was demonstrated for intestinal current measurement, but further information is required on reliability and responsiveness. For all three end-points, normal values were collected and further research requirements were proposed. This body of work adds useful information to support the promotion of CFTR biomarkers to surrogate end-points and to guide further research in the area.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/análise , Fibrose Cística/diagnóstico , Biomarcadores/análise , Fibrose Cística/tratamento farmacológico , Humanos , Reprodutibilidade dos TestesRESUMO
Store-operated Ca(2+) entry, stimulated by depletion of intracellular Ca(2+) pools, has not been fully elucidated in vascular smooth muscle cells of pig coronary arteries. Therefore, [Ca(2+)](i) was measured in cultured cells derived from extramural pig coronary arteries using the Fura-2/AM fluorometry. Divalent cation entry was visualized with the Fura-2 Mn(2+)-quenching technique. Ca(2+) stores were depleted either by repetitive stimulation of P2Y purinoceptors with ATP (10 micromol/L), or by the sarcoendoplasmic Ca(2+)-ATPase inhibitor 2,5-Di-(tert-butyl)-1,4-benzohydroquinone (BHQ; 1 micromol/L) in Ca(2+)-free medium (EGTA 1 mmol/L). Addition of Ca(2+)(1 mmol/L) induced refilling of ATP-sensitive Ca(2+) stores and an increase in [Ca(2+)](i) in the presence of BHQ. Both could be significantly diminished by Ni(2+)(5 and 1mmol/L), La(3+)(10 micromol/L), Gd(3+)(10 micromol/L), and Mg(2+)(5.1 mmol/L). In contrast to the BHQ-mediated rise in [Ca(2+)](i), refilling of ATP-depleted stores was affected by neither flufenamate (0.1 mmol/L), nor by nitrendipine, nifedipine, and nisoldipine (each 1 micromol/L). The data suggest that after store depletion in pig coronary smooth muscle cells ATP and BHQ may converge on a common, Ni(2+)-, La(3+)-, Gd(3+)-, and Mg(2+)- sensitive Ca(2+) entry pathway, i.e. on a store-operated Ca(2+) entry. An additional contribution of the Na(+)/Ca(2+) exchanger cannot be excluded. Flufenamate-sensitive non-selective cation channels and dihydropyridine-sensitive L-type Ca(2+) channels are not involved in refilling of Ca(2+) stores after previous depletion by repetitive P2Y purinoceptor stimulation. The store-operated Ca(2+) entry in-between repetitive purinoceptor stimulation, i.e. in the absence of the agonist, may be responsible for the maintenance of agonist-induced rhythmic Ca(2+) responses.
Assuntos
Cálcio/metabolismo , Músculo Liso Vascular/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Cátions Bivalentes , Células Cultivadas , Vasos Coronários/citologia , Di-Hidropiridinas/farmacologia , Fura-2 , Hidroquinonas/farmacologia , Líquido Intracelular/metabolismo , Magnésio , Manganês , Músculo Liso Vascular/citologia , Sarcolema/metabolismo , SuínosRESUMO
We have previously demonstrated that the airway serous cell line Calu-3 employs a number of pH regulatory mechanisms required for bicarbonate secretion by these cells. The aim of the present study was to investigate the pH regulatory mechanisms of serous cells of freshly isolated submucosal glands (SMG). Porcine SMG were dissected out of pig tracheas obtained from a local slaughterhouse. Single glands were transferred into the chamber of an inverted microscope, immobilized by two holding pipettes and the serous cells loaded with the fluorescent pH probe 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Fluorescence was monitored from small areas consisting of up to 20 cells. The fluorescence ratio of the emission after excitation at 488 nm and 436 nm respectively was used to estimate cytosolic pH (pH(i)). Resting pH(i) of SMG cells in the absence of HCO(3)(-)/CO(2) was 7.1 +/- 0.16 (n=24). Addition of a solution buffered with HCO(3)(-)/CO(2) to the bath transiently acidified the cells by 0.18 +/- 0.03 (n=18). pH(i) rapidly recovered to a slightly more alkaline value than baseline pH(i). Removal of the HCO(3)(-)/CO(2) buffer strongly alkalinized SMG cells by 0.2 +/- 0.03 (n=18). To challenge pH regulatory mechanisms we exposed the cells to 20 mmol/L NH4(+) in the absence and presence of HCO(3)(-)/CO(2). In both cases we observed a rapid increase in pH(i) followed by a slight recovery. Washout of NH4(+) strongly acidified the cells. Realkalinization of pH(i) could only be observed in the presence of Na(+). This effect was inhibited by the addition of the specific Na(+)/H(+) exchanger isoform 1 (NHE1) blocker 3-methylsulfonyl-4-piperidinobenzoyl guanidine hydrochloride (HOE 694, 10-100 micromol/L) with an half maximal inhibitory concentration (IC(50)) of approximately 20 micromol/L. Full recovery of pH(i) in the presence of HOE 694 was observed when the cells were bathed in HCO(3)(-)/CO(2) solution. Addition of forskolin (5 micromol/L) in the presence of HCO(3)(-)/CO(2) did not significantly alter pH(i) or change pH(i) recovery after acid loading. We conclude that SMG cells possess both HCO(3)(-) dependent and HCO(3)(-) independent pH(i); regulatory mechanisms that require the presence of extracellular Na(+). Further studies are required to understand whether bicarbonate is only transported to regulate pH(i) or whether this transport determines the overall secretory capacity of SMG serous cells.
Assuntos
Bicarbonatos/metabolismo , Mucosa Respiratória/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Animais , Soluções Tampão , Cultura em Câmaras de Difusão , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Concentração de Íons de Hidrogênio , Mucosa Respiratória/citologia , Suínos , Traqueia/citologia , Traqueia/metabolismoRESUMO
Calu-3 cells secrete HCO(3)(-) in response to cAMP agonists but can be stimulated to secrete Cl(-) with K(+) channel activating agonists. Microelectrode and impedance analysis experiments were performed to obtain a better understanding of the conductances and driving forces involved in these different modes of anion secretion in Calu-3 cells. Microelectrode studies revealed apical and basolateral membrane depolarizations upon the addition of forskolin (V(ap) -52 mV vs. -21 mV; V(bl) -60 mV vs. -44 mV) that paralleled the hyperpolarization of the mucosal negative transepithelial voltage (V(T) -8 mV vs. -23 mV). These changes were accompanied by a decrease in the apical membrane fractional resistance (F(Rap)) from approximately 0.50 to 0.08, consistent with the activation of an apical membrane conductance. The subsequent addition of 1-ethyl-2-benzimidazolinone (1-EBIO), a K(+) channel activator, hyperpolarized V(ap) to -27 mV, V(bl) to -60 mV and V(T) to -33 mV. Impedance analysis revealed the apical membrane resistance (R(ap)) of the forskolin-stimulated cells was less than 20 ohm cm(2), indeed in most monolayers R(ap) fell to less than 5 ohm cm(2). The impedance derived estimate of the basolateral membrane resistance (R(bl)) was approximately 170 ohm cm(2) in forskolin treated cells and fell to 50 ohm cm(2) with the addition of 1-EBIO. Using these values for the R(bl) and the F(Rap) value of 0.08 yields a R(ap) of approximately 14 ohm cm(2) in the presence of forskolin and 4 ohm cm(2) in the presence of forskolin plus 1-EBIO. Thus, by two independent methods, forskolin-stimulated Calu-3 cells are seen to have a very high apical membrane conductance of 50 to 200 mS/cm(2). Therefore, we would assert that even at one-tenth the anion selectivity for Cl(-), this high conductance could support the conductive exit of HCO(3)(-) across the apical membrane. We further propose that this high apical membrane conductance serves to clamp the apical membrane potential near the equilibrium potential for Cl(-) and thereby provides the driving force for HCO(3)(-) secretion in forskolin-stimulated Calu-3 cells. The hyperpolarization of V(ap) and V(bl) caused by 1-EBIO provides a driving force for Cl(-) exit across the apical membrane, inhibits the influx of HCO(3)(-) on the Na(+):HCO(3)(-) cotransporter across the basolateral membrane, activates the basolateral membrane Na(+):K:2Cl(-) cotransporter and thereby provides the switch from HCO(3)(-) secretion to Cl(-) secretion.
Assuntos
Ânions/metabolismo , Pulmão/metabolismo , Pulmão/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Impedância Elétrica , Humanos , Pulmão/citologia , Microeletrodos , Mucosa/citologia , Mucosa/metabolismo , Mucosa/fisiologiaRESUMO
Pancreatic acini secret Na+, Cl- and H2O in response to secretagogues such as acetylcholine. Cl- channels in the luminal membrane are a prerequisite for this secretion. The properties of the corresponding conductance have previously been examined using whole-cell recordings. The present study attempts to examine the properties of the single channels in cell-attached and cell-free excised patches from the luminal membrane. To this end the pipettes were filled with an N-methyl-D-glucamine (NMDG+) chloride-gluconate solution. The voltage-clamp range was chosen to be pipette positive (cell negative, -60 to -130 mV) in order to increase the driving force for outward Cl- currents. Under resting conditions cell attached luminal patches had very few single-channel currents (12 out of 45 experiments). Their incidence was sharply increased by carbachol (CCH, 1 micromol/l) in 41 out of 45 experiments. The single-channel conductance of these channels was 1.97 +/- 0.05 pS. The properties of these channels in excised patches were examined further: their single-channel conductance was 2.2 +/- 0.07 pS (n = 59) and their conductance selectivity was I- > Br- > Cl- >> gluconate. None of the typical Cl- channel blockers (DIDS, NPPB, glibenclamide 100 micromol/l) blocked these channels. It is concluded that the luminal membrane of the rat pancreatic acinus possesses Cl- channels with very low conductance which are activated by carbachol.
Assuntos
Membrana Celular/fisiologia , Canais de Cloreto/fisiologia , Pâncreas/fisiologia , Animais , Carbacol/farmacologia , Canais de Cloreto/efeitos dos fármacos , Técnicas de Patch-Clamp , RatosRESUMO
The aim of this study was to examine the question of whether activation of wt-CFTR (wild-type cystic fibrosis transmembrane conductance regulator) by cAMP and the opening of a Cl- conductance is paralleled by exocytosis and corresponding increases in membrane capacitance. To this end three types of Chinese hamster ovary (CHO) cells were examined: a control group of CHO cells; a group of CHO cells stably expressing wt-CFTR at high levels (also called BQ2-CHO); and a group of CHO cells stably expressing the frequent mutation DeltaF508-CFTR. Whole-cell patch-clamp studies were performed to measure the membrane voltage (Vm), the membrane conductance (Gm) and the membrane capacitance (Cm). Cm was assessed by a two-frequency lock-in amplifier method. Forskolin (Fsk, 0.1 micromol/l) and isobutylmethylxanthine (IBMX, 0.1 mmol/l) were used to increase cytosolic cAMP. It is shown that Fsk and IBMX had no effect on Vm and Gm in control CHO and DeltaF508-CFTR-CHO cells. Fsk and IBMX depolarized wt-CFTR-expressing CHO cells significantly (from -40 +/- 1.5 to -32 +/- 1.6 mV, n = 41) and enhanced Gm strongly from 5.0 +/- 0.9 to 36 +/- 3.9 nS (n = 65). The conductance increase was mostly for Cl-, because under stimulated conditions a reduction in bath Cl- concentration depolarized these cells further and significantly from -26 +/- 1.8 to -10 +/- 1.2 mV (n = 16). This conductance had the characteristic wt-CFTR selectivity of Br- > Cl- > I- (n = 16). Despite this large increase in the Fsk- and IBMX-induced conductance Cm was not altered significantly (15.5 versus 15.7 pF, n = 50). These data indicate that stable overexpression of wt-CFTR but not of DeltaF508-CFTR in CHO cells induces a cAMP-activated Cl- conductance. The activation of this large conductance obviously proceeds with little if any exocytosis.
Assuntos
Canais de Cloreto/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Exocitose/fisiologia , Ovário/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Células CHO/efeitos dos fármacos , Células CHO/metabolismo , Colforsina/farmacologia , Cricetinae , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Condutividade Elétrica , Feminino , Mutação , Ovário/citologia , Ovário/efeitos dos fármacosRESUMO
A number of agonists increase intracellular Ca2+ activity, [Ca2+]i, in pancreatic ducts, but the influx/efflux pathways and intracellular Ca2+ stores in this epithelium are unknown. The aim of the present study was to characterise the Ca2+ influx pathways, especially their pH sensitivity, in native pancreatic ducts stimulated by ATP and carbachol, CCH. Under control conditions both agonists led to similar changes in [Ca2+]i. However, these Ca2+ transients, consisting of peak and plateau phases, showed different sensitivities to various experimental manoeuvres. In extracellular Ca2+-free solutions, the ATP-induced [Ca2+]i peak decreased by 25%, but the CCH-induced peak was unaffected; both plateaus were inhibited by 90%. Flufenamate inhibited the ATP-induced peak by 35%, but not the CCH-evoked peak; the plateaus were inhibited by 75-80%. La3+ inhibited the ATP-induced plateau fully, but that induced by CCH by 55%. In resting ducts, an increase in extracellular pH, pHe, by means of HEPES and HCO3-/CO2 buffers, increased [Ca2+]i; a decrease in pHe had the opposite effect. In stimulated ducts the pH-evoked effects on Ca2+ influx were more pronounced and depended on the agonist used. At pHe 6.5 both ATP- and CCH-evoked plateaus were inhibited by about 50%. At pH 8.0 the ATP-stimulated plateau was inhibited by 27%, but that stimulated by CCH was increased by 72%. Taken together, we show that CCH stimulates Ca2+ release followed by Ca2+ influx that is moderately sensitive to flufenamate, La3+, depolarisation, it is inhibited by low pH, but stimulated by high pH. ATP stimulates Ca2+ release and probably an early Ca2+ influx, which is more markedly sensitive to flufenamate and La3+, and is both inhibited by low and high pH. Thus our study indicates that there are at least two separate Ca2+ influx pathways in pancreatic ducts cells.
Assuntos
Cálcio/metabolismo , Ductos Pancreáticos/metabolismo , Animais , Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Carbacol/farmacologia , Eletrofisiologia , Feminino , Ácido Flufenâmico/farmacologia , Concentração de Íons de Hidrogênio , Membranas Intracelulares/fisiologia , Nifedipino/farmacologia , Ductos Pancreáticos/efeitos dos fármacos , Ratos , Ratos Wistar , Verapamil/farmacologiaRESUMO
Extracellular adenosine 5'-triphosphate (ATP) has been described to act as a regulator in many cells and tissues, including epithelia, and in the gastrointestinal tract ATP is one of the substances involved in non-cholinergic non-adrenergic control. However, very little is known about the effect of ATP on pancreatic ducts, which normally secrete bicarbonate-rich fluid in response to secretin. Hence, the aim of our present study was to test the effect of ATP and other nucleotides on intracellular Ca2+ activity ([Ca2+]i) of pancreatic ducts, and thereby get information about purinergic receptors that might play a role in the regulation of pancreatic bicarbonate transport. Native intralobular ducts were obtained from rat pancreas and [Ca2+]i in 10-20 cells was measured using the fura-2 method. ATP (10(-4) mol/l) evoked a characteristic biphasic Ca2+ transient in duct cells. Nucleotides, used to classify the P2 receptors, acted with the following potency on the peak Ca2+ in many ducts: uridine 5'-triphosphate (UTP) >/= ATP >inosine 5'-triphosphate >/= 2-methylthio-ATP > beta,gamma-methyl-ATP > adenosine. However, although the peak [Ca2+]i responses to ATP and UTP were similar, the plateau [Ca2+]i was nearly doubled with UTP. Moreover, in about one-third of the ducts studied, UTP had no effect on cell Ca2+, while the response to ATP was normal. In further experiments we found that removal of extracellular Mg2+ increased the peak [Ca2+]i evoked in response to ATP. 2'&3'-O-(4-benzoylbenzoyl) ATP (BzATP) evoked a monophasic and slower increase in [Ca2+]i, which was inhibited by removal of extracellular Ca2+, or by addition of 4,4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (DIDS). Taken together, our data indicate that there are two types of purinergic receptors on pancreatic ducts through which ATP can act. These are pharmacologically known as P2U and P2Z receptors and may correspond to P2Y2 and P2X7 receptors.
Assuntos
Cálcio/metabolismo , Ductos Pancreáticos/metabolismo , Receptores Purinérgicos/fisiologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Marcadores de Afinidade , Animais , Feminino , Ductos Pancreáticos/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores Purinérgicos P2/fisiologia , Uridina Trifosfato/farmacologiaRESUMO
The gene KCNQ1 encodes a K(+) channel alpha-subunit important for cardiac repolarization, formerly known as K(v)LQT1. In large and small intestine a channel complex consisting of KCNQ1 and the beta-subunit KCNE3 (MiRP2) is known to mediate the cAMP-activated basolateral K(+) current, which is essential for luminal Cl(-) secretion. Northern blot experiments revealed an expression of both subunits in lung tissue. However, previous reports suggested a role of KCNE1 (minK, Isk) but not KCNE3 in airway epithelial cells. Here we give evidence that KCNE1 is not detected in murine tracheal epithelial cells and that Cl(-) secretion by these cells is not reduced by the knock-out of the KCNE1 gene. In contrast we show that a complex consisting of KCNQ1 and KCNE3 probably forms a basolateral K(+) channel in murine tracheal epithelial cells. As described for colonic epithelium, the current through KCNQ1 complexes in murine trachea is specifically inhibited by the chromanol 293B. A 293B-sensitive current was present after stimulation with forskolin and agonists that increase Ca(2+) as well as after administration of the pharmacological K(+) channel activator, 1-EBIO. A 293B-inhibitable current was already present under control conditions and reduced after administration of amiloride indicating a role of this K(+) channel not only for Cl(-) secretion but also for Na(+) reabsorption. We conclude that at least in mice a KCNQ1 channel complex seems to be the dominant basolateral K(+) conductance in tracheal epithelial cells.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/fisiologia , Traqueia/química , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Cloretos/metabolismo , AMP Cíclico/fisiologia , Indóis/farmacologia , Canais de Potássio KCNQ , Canal de Potássio KCNQ1 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canais de Potássio/química , Canais de Potássio/genética , Subunidades ProteicasRESUMO
Oxygen radicals are known to be mediators of renal injury under several pathophysiological conditions. We have examined the effect of hydrogen peroxide (H2O2) on intracellular calcium activity ([Ca2+]i) in mesangial cells in primary culture. Mesangial cells were loaded with 1 mumol/liter fura-2, and kept in a Ringer-like solution. Fura-2 fluorescence was measured in an inverted microscope at 37 degrees C. Angiotensin II (0.1 nmol/liter) and ATP (0.1 mumol/liter) induced a rapid transient increase of [Ca2+]i, which was followed by a sustained plateau (N = 37 and N = 24). In contrast, the addition of H2O2 (0.01 to 10 mmol/liter, N = 157) caused a time- and concentration-dependent slow increase of [Ca2+]i, which reached a stable [Ca2+]i plateau after 3 to 10 minutes (ED50: 100 mumol/liter). After the removal of H2O2 [Ca2+]i decreased partially and reached a stable value approximately 90% above the resting [Ca2+]i value. Addition of 100 mumol/liter H2O2 to an extracellular Ca(2+)-free solution resulted either in no rise of [Ca2+]i in some experiments (N = 7), or [Ca2+]i oscillations in others (N = 10). In the presence of H2O2 (> 25 mumol/liter), the angiotensin II or ATP mediated increases in [Ca2+]i were almost completely inhibited (N = 15 and N = 10). The cations Ni2+ and La3+ and the Ca(2+)-antagonist verapamil (10 mumol/liter) did not inhibit the H2O2 mediated increase of -Ca2+-i (N = 6 to 9). Flufenamate (100 mumol/liter), an inhibitor of non-selective cation channels inhibited the H2O2 induced increase of [Ca2+]i by 63 +/- 11% (N = 7). Preincubation of the cells with a disulphide reducing agent (dithiothreitol, 500 mumol/liter, N = 5) or an iron-chelator (deferoxamine, 100 mumol/liter, N = 5) attenuated the H2O2 mediated effect by 95 +/- 15% and 74 +/- 6%, respectively. The H2O2 mediated [Ca2+]i increase was completely inhibited when mesangial cells were preincubated with 1 mumol/liter U-83836E, an inhibitor of lipid peroxidation (N = 7), and inhibited by 84 +/- 6% when the cells were pretreated with 1 mmol/liter pyruvate (N = 5). The data indicate that H2O2: (i) increases [Ca2+]i in mesangial cells by a mechanism distinct from angiotensin II or ATP and (ii) that it inhibits the [Ca2+]i response to both agonists.
Assuntos
Cálcio/metabolismo , Mesângio Glomerular/citologia , Peróxido de Hidrogênio/farmacologia , Trifosfato de Adenosina/farmacologia , Angiotensina II/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Antídotos/farmacologia , Antioxidantes/farmacologia , Catalase/farmacologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Cromanos/farmacologia , Citoplasma/metabolismo , Desferroxamina/farmacologia , Ditiotreitol/farmacologia , Ácido Flufenâmico/farmacologia , Mesângio Glomerular/enzimologia , Lantânio/farmacologia , Níquel/farmacologia , Piperazinas/farmacologia , Ácido Pirúvico/farmacologia , Ratos , Reagentes de Sulfidrila/farmacologia , Vasoconstritores/farmacologia , Vasodilatadores/farmacologia , Verapamil/farmacologiaRESUMO
The aim of this study was to examine whether the stable expression of wild-type cystic fibrosis transmembrane conductance regulator (CFTR) in Chinese hamster ovary (CHO) cells alters the properties of these cells towards hypotonic cell swelling and ATP. According to many previous studies this was not expected a priori, since overexpression of CFTR should not affect the conductive pathways upregulated by the purinergic agonist or cell swelling. Three types of CHO cells were examined: a control group of normal CHO cells; a group of CFTR-CHO cells stably expressing wild-type CFTR at high levels (CHO-CFTR), and a group delta F508-CFTR-CHO cells, stably expressing the frequent mutation delta F508 CFTR (CHO-delta F508). Whole cell patch-clamp studies were performed to measure the membrane voltage (Vm), the membrane conductance (Gm), and the membrane capacitance (C(m)). Hypotonic cell swelling (Hypo, 150 mosm/l) was used, because it activates Cl- and K+ channels and enables the cell to extrude KCl in many cells, and ATP because it is known to activate Ca(2+)-regulated channels in a large variety of cells. Hypo depolarized all three types of cells. This depolarization was accompanied by an increase in Cl- conductance. The selectivity of the conductance was I- > or = Br- > or = Cl- in CHO cells, but Cl- = Br- = I- in the CFTR cells. Even more surprising: ATP (100 mumol/l) hyperpolarized CHO and delta F508 cells and predominantly enhanced K+ conductance, whilst it depolarized and increased mostly a Cl- conductance in CFTR cells. The selectivity of this anion conductance was atypical for ATP: Br- > Cl- > I-. C(m) was increased by ATP and Hypo in all three types of cells. ATP enhanced cytosolic Ca2+ ([Ca2+]i) in all three types of cells but did not enhance cAMP. These data indicate that the expression of CFTR profoundly alters the properties of CHO cells. Agonists which stimulate characteristic Ca(2+)-regulated channels now enhance a Cl- conductance resembling the properties of CFTR-Cl- conductance.
Assuntos
Trifosfato de Adenosina/farmacologia , Canais de Cálcio/fisiologia , Membrana Celular/fisiologia , Canais de Cloreto/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Canais de Potássio/fisiologia , Animais , Células CHO , Cálcio/metabolismo , Tamanho Celular , Cloretos/metabolismo , Cricetinae , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Condutividade Elétrica , Soluções Hipotônicas , Concentração Osmolar , Técnicas de Patch-Clamp , Potássio/metabolismoRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl(-) channel whose activity is controlled by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We found that CFTR immunoprecipitates from Calu-3 airway cells contain endogenous PKA, which is capable of phosphorylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by the PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFTR could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). Both the catalytic and type II regulatory subunits of PKA are identified by immunoblotting CFTR immunoprecipitates, demonstrating that the endogenous kinase associated with CFTR is PKA, type II (PKA II). Phosphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a protein kinase A anchoring protein (AKAP) is responsible for the association between PKA and CFTR. Ezrin may function as this AKAP, since it is expressed in Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is immunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of Calu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl(-) current, but Ht31P does not. Taken together, these data demonstrate that PKA II is linked physically and functionally to CFTR by an AKAP interaction, and they suggest that ezrin serves as an AKAP for PKA-mediated phosphorylation of CFTR.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fosfoproteínas/metabolismo , Sequência de Bases , Linhagem Celular , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas do Citoesqueleto , Primers do DNA , Testes de Precipitina , Ligação ProteicaRESUMO
Transport of salt and water in various tissues is under control of the mineralocorticoid hormone aldosterone. As a liphophilic hormone, aldosterone diffuses through the plasma membrane and, then, binds to cytosolic mineralocorticoid receptors in the target cells. After binding to nuclear pore complexes, the activated receptor is translocated to the nucleus where transcription processes are initiated. After a lag period of about 20 minutes hormone-specific early mRNA transcripts leave the nucleus through nuclear pores. Some of the steps in this cascade can be followed by electrophysiology in Xenopus laevis oocyte nuclei. In addition to the genomic pathway, aldosterone exerts a rapid pre-genomic response that involves an increase in intracellular calcium. In this study, we tested for the potential role of Ca(2+) in the genomic response of the hormone. We measured the electrical resistance across the nuclear envelope in response to aldosterone, in presence and absence of intracellular Ca(2+). Nuclear envelope electrical resistance reflects receptor binding to the nuclear pore complexes ("early" resistance peak, 2 minutes after aldosterone), ongoing transcription ("transient" resistance drop, 5-15 minutes after aldosterone) and mRNA export ("late" resistance peak, 20 minutes after aldosterone). Pre-injection of the Ca(2+) chelator EGTA eliminated all electrical responses evoked by aldosterone. The transient resistance drop and the late resistance peak, induced by the hormone, were prevented by the transcription inhibitor actinomycin D, coinjected with aldosterone, while the early resistance peak remained unaffected. We conclude that (i). the presence of intracellular Ca(2+) is a prerequisite for the genomic action of aldosterone. (ii). Intracellular calcium plays a role early in the signaling cascade, either in agonist-receptor interaction, or receptor transport/docking to the nuclear pore complexes.
Assuntos
Aldosterona/farmacologia , Cálcio/metabolismo , Membrana Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Membrana Nuclear/fisiologia , Oócitos/fisiologia , RNA Mensageiro/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Ácido Egtázico/farmacologia , Impedância Elétrica , Regulação da Expressão Gênica/efeitos dos fármacos , Espaço Intracelular/metabolismo , Membrana Nuclear/metabolismo , Oócitos/efeitos dos fármacos , Receptores de Mineralocorticoides/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Xenopus laevisRESUMO
N-Acetyl-L-cysteine (NAC) is a widely used mucolytic drug in patients with a variety of respiratory disorders including cystic fibrosis (CF). The beneficial effects of NAC are empirical and the exact mechanism of action in the airways remains obscure. In the present study we examined the effects on whole-cell (wc) conductance (Gm) and voltage (Vm) of NAC and the congeners S-carboxymethyl-L-cysteine (CMC) and S-carbamyl-L-cysteine (CAC) and L-cysteine in normal and CF airway epithelial cells. L-Cysteine (1 mmol/l) had no detectable effect. The increase in Gm (delta Gm) by the other compounds was concentration dependent and was (all substances at 1 mmol/l) 3.8 +/- 1.4 nS (NAC; n = 11), 4.2 +/- 1.0 nS (CMC; n = 16) and 3.8 +/- 1.6 nS (CAC; n = 18), respectively. The changes in Gm were paralleled by an increased depolarization (delta Vm) when extracellular Cl- concentration was reduced to 34 mmol/l: under control conditions = -4.1 +/- 2.1 versus 10.2 +/- 2.1 mV in the presence of NAC, CMC, CAC (n = 36). In the presence of NAC, CMC and CAC, the reduction in Cl- concentration was paralleled by a reduction of Gm by 2.1 +/- 0.4 nS (n = 35), indicating that all substances acted by increasing the Cl- conductance. Analysis of intracellular pH did not reveal any changes by any of the compounds (1 mmol/l). A Cl- conductance was also activated in HT29 colonic carcinoma and CF tracheal epithelial (CFDE) cells but not in CFPAC-1 cells, which do not express detectable levels of delta F508-CFTR, suggesting that the presence of CFTR may be a prerequisite for the induction of Cl- currents. Next we examined the ion currents in Xenopus oocytes microinjected with CFTR-cRNA. Water-injected oocytes did not respond to activation by forskolin and 3-isobutyl-1-methylxanthine (IBMX) (delta Gm = 0.08 +/- 0.04 microS; n = 10) and no current was activated when these oocytes were exposed to NAC or CMC. In contrast, in CFTR-cRNA-injected oocytes Gm was enhanced when intracellular adenosine 3',5'-cyclic monophosphate (cAMP) was increased by forskolin and IBMX (Gm = 4.5 +/- 1.3 microS; n = 8). Gm was significantly increased by 0.74 +/- 0.2 microS (n = 11) and 0.46 +/- 0.1 microS (n = 10) when oocytes were exposed to NAC and CMC, respectively (both 1 mmol/l). In conclusion, NAC and its congeners activate Cl- conductances in normal and CF airway epithelial cells and hence induce electrolyte secretion which may be beneficial in CF patients. CFTR appears to be required for this response in an as yet unknown fashion.
Assuntos
Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/fisiologia , Animais , Brônquios/citologia , Brônquios/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Neoplasias do Colo/patologia , Fibrose Cística/fisiopatologia , Condutividade Elétrica , Células Epiteliais , Humanos , Concentração de Íons de Hidrogênio , Oócitos/fisiologia , Sistema Respiratório/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , XenopusRESUMO
Acetylcholine-controlled exocrine secretion by pancreatic acini has been explained by two hypotheses. One suggests that NaCl secretion occurs by secondary active secretion as has been originally described for the rectal gland of Squalus acanthias. The other is based on a "push-pull" model whereby Cl- is extruded luminally and sequentially taken up basolaterally. In the former model Cl- uptake is coupled to Na+ and basolateral K+ conductances play a crucial role, in the latter model, Na+ uptake supposedly occurs via basolateral non-selective cation channels. The present whole-cell patch-clamp studies were designed to further explore the conductive properties of rat pancreatic acini. Pilot studies in approximately 300 cells revealed that viable cells usually had a membrane voltage (Vm) more hyperpolarized than -30 mV. In all further studies Vm had to meet this criterion. Under control conditions Vm was -49 +/- 1 mV (n = 149). The fractional K+ conductance (fK) was 0.13 +/- 0.1 (n = 49). Carbachol (CCH, 0.5 micromol/l) depolarized to -19 +/- 1.1 mV (n = 63) and increased the membrane conductance (Gm) by a factor of 2-3. In the seeming absence of Na+ [replacement by N-methyl-D-glucamine (NMDG+)] Vm hyperpolarized slowly to -59 +/- 2 mV (n = 90) and CCH still induced depolarizations to -24 +/- 2 mV (n = 34). The hyperpolarization induced by NMDG+ was accompanied by a fall in cytosolic pH by 0.4 units, and a very slow and slight increase in cytosolic Ca2+. fK increased to 0.34. The effect of NMDG+ on Vm was mimicked by the acidifying agents propionate and acetate (10 mmol/l) added to the bath. The present study suggests that fK makes a substantial contribution to Gm under control conditions. The NMDG+ experiments indicate that the non- selective cation conductance contributes little to Vm in the presence of CCH. Hence the present data in rat pancreatic acinar cells do not support the push-pull model.
Assuntos
Pâncreas/fisiologia , Animais , Carbacol/farmacologia , Cloretos/metabolismo , Condutividade Elétrica , Eletrofisiologia , Meglumina/farmacologia , Pâncreas/citologia , Pâncreas/efeitos dos fármacos , Técnicas de Patch-Clamp , Projetos Piloto , Canais de Potássio/fisiologia , RatosRESUMO
Previously we have shown that stimulation of in vitro perfused rectal gland tubules (RGT) of the dog-fish Squalus acanthias by adenosine 3',5'-cyclic monophosphate (cAMP), (as a cocktail comprising 0.1 mmol/l dibutyryl-cAMP, 10 micromol/l forskolin and 0.1 mmol/l adenosine, hereafter termed STIM) leads to an increase in cytosolic Ca2+ ([Ca2+]i) and that this assists Cl- secretion by enhancing basolateral K+ conductance. In the present study we examined the mechanism of the cAMP-induced increase in [Ca2+]i. [Ca2+]i was measured using the fura-2 technique in isolated in vitro perfused RGT. As before, STIM enhanced [Ca2+]i. This elevation of [Ca2+]i was prevented completely when STIM was added in the presence of the Na+2Cl-K+ cotransport inhibitor furosemide (0.5 mmol/l). This suggests that the increase in [Ca2+]i induced by STIM is caused by a concomitant increase in cytosolic Na+ ([Na+]i) and not by the activation of second messenger cascades. Furosemide prevents this increase in [Na+]i and hence the elevation of [Ca2+]i. Moreover, the plateau phase of the [Ca2+]i transient produced by carbachol (CCH, 0.1 mmol/l) was augmented strongly when bath Na+ was reduced to 5 mmol/l. These data suggest that the level of [Ca2+]i is determined by Na(+)-dependent Ca2+ export, most likely via a Na+/Ca2+ exchanger. The increase in [Na+]i accompanying stimulation of Cl- secretion reduces the rate of Ca2+ export leading to an elevation of [Ca2+]i, as does a reduction in bath Na+ which augments the [Ca2+]i plateau produced by CCH.
Assuntos
Cação (Peixe)/metabolismo , Glândula de Sal/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Cálcio/metabolismo , Carbacol/farmacologia , AMP Cíclico/farmacologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Diuréticos/farmacologia , Corantes Fluorescentes , Fura-2 , Furosemida/farmacologia , Técnicas In Vitro , Agonistas Muscarínicos/farmacologia , Glândula de Sal/efeitos dos fármacos , Sódio/metabolismo , Estimulação QuímicaRESUMO
Effects of cAMP on Cl- secretion, intracellular Cl- activity and cell volume were studied in isolated perfused rectal gland tubules (RGT) of Squalus acanthias with electrophysiological and fluorescence methods. Recording of equivalent short-circuit current (Isc) showed that cAMP stimulates Na+Cl- secretion in a biphasic manner. The first and rapid phase corresponds to Cl- exit via the respective protein-kinase-A- (PKA-) phosphorylated Cl- conductance. The inhibitory effect of the loop diuretic furosemide (0.5 mmol/l, n=12) indicates that second phase reflects the delayed (1-2 min) activation of the Na+2Cl-K+ cotransporter. During the first phase cytosolic Cl- activity, as monitored by 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) fluorescence, fell to 78% (n=23) of the control value. Concomitantly, a transient fall in cell volume was recorded by calcein fluorescence to 92% (n=5) of the control value. Preincubation of the RGT with phalloidin (0.1 mmol/l, n=6) or cytochalasin D (0.1 mmol/l, n=4) almost completely prevented the development of the second phase of Isc activation. When cytosolic Cl- activity was increased by exposing the RGT to a high K+ concentration (25 mmol/l), in the presence of mannitol to prevent volume increases, stimulation was unaffected and biphasic. In contrast, when cell volume was clamped to an increased value (115%, n=8) by removing extracellular NaCl, the second phase was abolished completely (n=11). These data suggest that the primary and key process for triggering the Na+2Cl-K+ cotransport is transient cell shrinkage.
Assuntos
Proteínas de Transporte/metabolismo , Tamanho Celular/fisiologia , Cloretos/metabolismo , Potássio/metabolismo , Glândula de Sal/metabolismo , Sódio/metabolismo , Animais , Bário/metabolismo , Bucladesina/farmacologia , Cálcio/metabolismo , Proteínas de Transporte/efeitos dos fármacos , Colforsina/farmacologia , Citocalasina D/metabolismo , Cação (Peixe) , Masculino , Masoprocol/farmacologia , Faloidina/farmacologia , Inibidores de Proteínas Quinases , Glândula de Sal/citologia , Glândula de Sal/efeitos dos fármacos , Simportadores de Cloreto de Sódio-PotássioRESUMO
1. The secretagogue-activated K(+) conductance is indispensable for the electrogenic Cl(-) secretion in exocrine tissue. In this study, we investigated the effect of secretin and other cAMP-mediated secretagogues on the slowly activating voltage-dependent K(+) current (I(Ks)) of rat pancreatic acinar cells (RPAs) with the whole-cell patch clamp technique. 2. Upon depolarization, RPAs showed I(Ks) superimposed upon the instantaneous background outward current. Secretin (5 nM), vasoactive intestinal peptide (5 nM), forskolin (5 microM), isoprenaline (10 microM) or 3-isobutyl-1-methylxanthine (IBMX, 0.1 mM) increased the amplitude of I(Ks) two- to fourfold. 3. The physiological concentration of secretin (50 pM) had a relatively weak effect on I(Ks) (160 % increase), which was significantly enhanced by transient co-stimulation with carbachol (CCh) (10 microM). However, the secretin-induced production of cAMP, which was measured by enzyme-linked immunosorbent assay, was not augmented by co-stimulation with CCh. 4. This study is the first to demonstrate the regulation of K(+) channels in RPAs by cAMP-mediated agonists. The I(Ks) channel is a common target for both Ca(2+) and cAMP agonists. The vagal stimulation under the physiological concentration of secretin facilitates I(Ks), which provides an additional driving force for Cl(-) secretion.