RESUMO
Cellular mechanism(s) regulating atriopeptin secretion and processing by the atrial myocyte are currently unknown. Osmotic stretch of isolated atrial myocytes as well as potassium chloride depolarization were potent stimuli of atriopeptin secretion. Release was potentiated by buffering either extracellular calcium with EGTA or intracellular calcium with the intracellular chelator, BAPTA AM. Atrial release of atriopeptin was inhibited after administration of ionomycin which elevates intracellular calcium. Fetal or early neonatal ventricular myocytes actively synthesize atriopeptin. Atriopeptin secretion by ventricular myocytes was also markedly potentiated by osmotic stretch as well as KCl depolarization. Only the 126 amino acid prohormone was secreted by the stretch-stimulated atrial and ventricular myocyte. These data suggest that stretch of the myocyte plasma membrane is a major stimulus for atriopeptin secretion and that atriopeptin secretion is not stimulated by raising intracellular calcium and appears to be negatively modulated by this cation. Like the atrial myocyte, the ventricular myocyte possesses the cellular mechanism(s) necessary to secrete atriopeptin by a regulated mechanism.
Assuntos
Fator Natriurético Atrial/metabolismo , Cálcio/fisiologia , Membrana Celular/fisiologia , Coração/fisiologia , Animais , Animais Recém-Nascidos , Fenômenos Biomecânicos , Cálcio/farmacologia , Ácido Egtázico/farmacologia , Éteres/farmacologia , Coração/efeitos dos fármacos , Ionomicina , Potenciais da Membrana , Concentração Osmolar , Pressão Osmótica , Cloreto de Potássio/farmacologia , RatosRESUMO
Depolarization-activated outward K+ currents in isolated adult rat ventricular myocytes were characterized using the whole-cell variation of the patch-clamp recording technique. During brief depolarizations to potentials positive to -40 mV, Ca(2+)-independent outward K+ currents in these cells rise to a transient peak, followed by a slower decay to an apparent plateau. The analyses completed here reveal that the observed outward current waveforms result from the activation of two kinetically distinct voltage-dependent K+ currents: one that activates and inactivates rapidly, and one that activates and inactivates slowly, on membrane depolarization. These currents are referred to here as Ito (transient outward) and IK (delayed rectifier), respectively, because their properties are similar (although not identical) to these K+ current types in other cells. Although the voltage dependences of Ito and IK activation are similar, Ito activates approximately 10-fold and inactivates approximately 30-fold more rapidly than IK at all test potentials. In the composite current waveforms measured during brief depolarizations, therefore, the peak current predominantly reflects Ito, whereas IK is the primary determinant of the plateau. There are also marked differences in the voltage dependences of steady-state inactivation of these two K+ currents: IK undergoes steady-state inactivation at all potentials positive to -120 mV, and is 50% inactivated at -69 mV; Ito, in contrast, is insensitive to steady-state inactivation at membrane potentials negative to -50 mV. In addition, Ito recovers from steady-state inactivation faster than IK: at -90 mV, for example, approximately 70% recovery from the inactivation produced at -20 mV is observed within 20 ms for Ito; IK recovers approximately 25-fold more slowly. The pharmacological properties of Ito and IK are also distinct: 4-aminopyridine preferentially attenuates Ito, and tetraethylammonium suppresses predominantly IK. The voltage- and time-dependent properties of these currents are interpreted here in terms of a model in which Ito underlies the initial, rapid repolarization phase of the action potential (AP), and IK is responsible for the slower phase of AP repolarization back to the resting membrane potential, in adult rat ventricular myocytes.
Assuntos
Miocárdio/metabolismo , Canais de Potássio/efeitos dos fármacos , 4-Aminopiridina/farmacologia , Animais , Eletrofisiologia , Ventrículos do Coração , Técnicas In Vitro , Cinética , Potenciais da Membrana/efeitos dos fármacos , Miocárdio/citologia , Ratos , Soluções , Compostos de Tetraetilamônio/farmacologiaRESUMO
In the preceding paper (Bevensee, M.O., R.A. Weed, and W.F. Boron. 1997. 110: 453-465.), we showed that a Na-driven influx of HCO causes the increase in intracellular pH (pH) observed when astrocytes cultured from rat hippocampus are exposed to 5% CO/17 mM HCO. In the present study, we used the pH-sensitive fluorescent indicator 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and the perforated patch-clamp technique to determine whether this transporter is a Na-driven Cl-HCO exchanger, an electrogenic Na/HCO cotransporter, or an electroneutral Na/HCO cotransporter. To determine if the transporter is a Na-driven Cl-HCO exchanger, we depleted the cells of intracellular Cl by incubating them in a Cl-free solution for an average of approximately 11 min. We verified the depletion with the Cl-sensitive dye -(6-methoxyquinolyl)acetoethyl ester (MQAE). In Cl-depleted cells, the pH still increases after one or more exposures to CO/HCO. Furthermore, the pH decrease elicited by external Na removal does not require external Cl. Therefore, the transporter cannot be a Na-driven Cl-HCO exchanger. To determine if the transporter is an electrogenic Na/ HCO cotransporter, we measured pH and plasma membrane voltage (V) while removing external Na, in the presence/absence of CO/HCO and in the presence/absence of 400 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). The CO/HCO solutions contained 20% CO and 68 mM HCO, pH 7.3, to maximize the HCO flux. In pH experiments, removing external Na in the presence of CO/HCO elicited an equivalent HCO efflux of 281 microM s. The HCO influx elicited by returning external Na was inhibited 63% by DIDS, so that the predicted DIDS-sensitive V change was 3.3 mV. Indeed, we found that removing external Na elicited a DIDS-sensitive depolarization that was 2.6 mV larger in the presence than in the absence of CO/ HCO. Thus, the Na/HCO cotransporter is electrogenic. Because a cotransporter with a Na:HCO stoichiometry of 1:3 or higher would predict a net HCO efflux, rather than the required influx, we conclude that rat hippocampal astrocytes have an electrogenic Na/HCO cotransporter with a stoichiometry of 1:2.
Assuntos
Astrócitos/metabolismo , Bicarbonatos/metabolismo , Proteínas de Transporte/metabolismo , Hipocampo/metabolismo , Animais , Soluções Tampão , Separação Celular , Células Cultivadas , Cloretos/metabolismo , Estimulação Elétrica , Eletrofisiologia , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Hipocampo/citologia , Concentração de Íons de Hidrogênio , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Ratos , Simportadores de Sódio-BicarbonatoRESUMO
BACKGROUND AND PURPOSE: Cerebral vascular smooth muscle cells (VSMCs) contract on extracellular pH (pH(o)) increases and relax on pH(o) decreases. These changes in tone are believed to result from changes in [Ca(2+)](i), although the responsible mechanisms are not fully understood. VSMCs also contract in response to 5-hydroxytryptamine (5-HT), which increases [Ca(2+)](i) via both Ca(2+) release and influx. We hypothesized that examining effects of pH(o) decreases on 5-HT-induced [Ca(2+)](i) changes would allow us to identify mechanisms whereby pH(o) influences tone. Accordingly, we compared [Ca(2+)](i) increases in cerebral VSMCs, evoked by 5-HT, with increases evoked by increased pH(o) and examined 5-HT-dependent [Ca(2+)](i) increases at normal and decreased pH(o). METHODS: We monitored [Ca(2+)](i,), using the Ca(2+)-sensitive dye fura 2, in cultured rat cerebral VSMCs obtained by enzymatic digestion of middle cerebral arteries and their branches (passages 1 to 3) grown on glass coverslips and superfused with physiological saline. RESULTS: Increasing pH(o) from 7.3 to 7.8 increased [Ca(2+)](i), and these increases were prevented in Ca(2+)-free solutions. Decreasing pH(o) from 7.3 to 6.9 did not alter [Ca(2+)](i) unless [Ca(2+)](i) was first raised by treatment with 5-HT (10 micromol/L). 5-HT resulted in biphasic [Ca(2+)](i) increases characterized by transient peaks blocked by the Ca(2+)-ATPase inhibitor thapsigargin (10 nmol/L) and prolonged plateaus blocked by the Ca(2+) channel blocker Ni(2+) (1 mmol/L). Acidification did not alter the transient peaks but significantly reduced 5-HT-induced Ca(2+) influx. CONCLUSIONS: We conclude that increasing pH(o) induces Ca(2+) influx in rat cerebral VSMCs and decreasing pH(o) inhibits 5-HT-stimulated Ca(2+) entry but not intracellular Ca(2+) release.
Assuntos
Cálcio/metabolismo , Espaço Extracelular/metabolismo , Músculo Liso Vascular/metabolismo , Serotonina/metabolismo , Ácidos/farmacologia , Álcalis/farmacologia , Animais , Células Cultivadas , Circulação Cerebrovascular/efeitos dos fármacos , Corantes Fluorescentes , Fura-2 , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Serotonina/farmacologiaRESUMO
OBJECTIVE: To test the hypotheses that: (1) integrating information processing tasks using an electronic clinical information system (ECIS) decreases time to complete these tasks by hand; and (2) structured data entry encourages generation of more detailed records and capture of specific data elements even when entry is voluntary. DESIGN: Prospective observational time analysis during medical documentation tasks. Retrospective analysis of clinical documentation completed by hand or electronically. SETTING: Eleven bed pediatric intensive care unit within an academic medical center. PARTICIPANTS: Five pediatric intensive care medicine attending physicians. MEASUREMENTS: Compared handwritten and electronic documentation to determine: (1) time spent entering data or composing notes; (2) number of descriptors documenting patients' physical exams; (3) users' preferences for structured or unstructured data entry; (4) frequency of documenting specific data elements related to nutritional support. RESULTS: Documentation time varied by user but not charting method: it took 13 % less time to document using the ECIS but this was not significant. Electronic documents were more detailed than handwritten containing 50 % more descriptors (17.8 +/- 1.4 vs 11.6 +/- 1.4) overall and some data elements that were not handwritten: information related to nutritional supplementation was recorded in 13 % of electronic documents but in none of 89 handwritten documents. CONCLUSIONS: Electronic and handwritten documentation consumed equal amounts of time. Structured entry, compared to handwriting, may encourage recording of specific or otherwise unincorporated data elements resulting in a more detailed record. This suggests that user interfaces and decision support components may influence both the types and complexity of clinical data recorded by caregivers.
Assuntos
Documentação , Eficiência Organizacional , Sistemas de Informação Hospitalar , Unidades de Terapia Intensiva Pediátrica/organização & administração , Médicos , Análise de Variância , Atitude Frente aos Computadores , Tomada de Decisões Assistida por Computador , Humanos , Análise Multivariada , Processamento de Linguagem Natural , Estudos Prospectivos , Estudos Retrospectivos , Análise e Desempenho de Tarefas , Fatores de Tempo , Estados Unidos , Interface Usuário-ComputadorRESUMO
OBJECTIVES: A set of standard processes was developed for delivering continuous drug infusions in order to improve (1) patient safety; (2) efficiency in staff workflow; (3) hemodynamic stability during infusion changes, and (4) efficient use of resources. Failure modes effects analysis (FMEA) was used to examine the impact of process changes on the reliability of delivering drug infusions. SETTING: An 11 bed multidisciplinary pediatric ICU in the children's hospital of an academic medical center staffed by board certified pediatric intensivists. The hospital uses computerized physician order entry for all medication orders. METHODS: A multidisciplinary team characterized key elements of the drug infusion process. The process was enhanced to increase overall reliability and the original and revised processes were compared using FMEA. Resource consumption was estimated by reviewing purchasing and pharmacy records for the calendar year after full implementation of the revised process. Staff satisfaction was evaluated using an anonymous questionnaire administered to staff nurses in the ICU and pediatric residents who had rotated through the ICU. RESULTS: The original process was characterized by six elements: selecting the drug; selecting a dose; selecting an infusion rate; calculating and ordering the infusion; preparing the infusion; programming the infusion pump and delivering the infusion. The following practice changes were introduced: standardizing formulations for all infusions; developing database driven calculators; extending infusion hang times from 24 to 72 hours; changing from bedside preparation by nurses to pharmacy prepared or premanufactured solutions. FMEA showed that the last three elements of the original process had high risk priority numbers (RPNs) of >225 whereas the revised process had no elements with RPNs >100. The combined effect of prolonging infusion hang times, preparation in the pharmacy, and purchasing premanufactured solutions resulted in 1500 fewer infusions prepared by nurses per year. Nursing staff expressed a significant preference and pediatric residents unanimously expressed a strong preference for the revised process. CONCLUSIONS: Standardization of infusion delivery reduced the frequency for completing the most unreliable elements of the process and reduced the riskiness of the individual elements. Both contribute to a safer system.
Assuntos
Infusões Intravenosas/normas , Unidades de Terapia Intensiva Pediátrica/normas , Erros de Medicação/prevenção & controle , Sistemas de Medicação no Hospital/normas , Gestão da Segurança , Centros Médicos Acadêmicos , Adolescente , Criança , Pré-Escolar , Sistemas de Informação em Farmácia Clínica , Connecticut , Quimioterapia Assistida por Computador , Registros Hospitalares , Hospitais Pediátricos/normas , Humanos , Equipe de Assistência ao Paciente , Avaliação de Processos em Cuidados de Saúde , Análise de Sistemas , Gestão da Qualidade TotalRESUMO
Hydrops fetalis occurs when the rate of interstitial fluid production by capillary ultrafiltration exceeds the rate of interstitial fluid return to the circulation via lymphatic vessels. Developmental differences in the microcirculation and lymphatic system of the fetus, as compared with mature subjects, renders the fetus susceptible to interstitial fluid accumulation. These differences include greater capillary permeability, more compliant interstitial compartment, and greater influence of venous pressures on lymphatic return. The balance between interstitial fluid production and removal is most commonly disrupted as a consequence of homeostatic mechanisms serving to preserve adequate systemic delivery of metabolic substrate when cardiocirculatory function is impaired. The pathophysiology of two conditions of impaired cardiocirculatory function, atrial tachycardia and severe anemia, serve as examples of the mechanisms by which these homeostatic mechanisms perturb the balance of interstitial fluid movement.
Assuntos
Permeabilidade Capilar/fisiologia , Espaço Extracelular/fisiologia , Hidropisia Fetal/fisiopatologia , Sistema Linfático/fisiologia , Fenômenos Fisiológicos da Nutrição , Oxigênio/farmacocinética , Humanos , Hidropisia Fetal/etiologia , Microcirculação/fisiologia , UltrafiltraçãoRESUMO
The effects of alpha 1-adrenergic agonists on the waveforms of action potentials and voltage-gated ionic currents were examined in isolated adult rat ventricular myocytes by the whole-cell patch-clamp recording technique. After "puffer" applications of either of two alpha 1 agonists, phenylephrine and methoxamine, action-potential durations were increased. In voltage-clamped cells, phenylephrine (5-20 microM) or methoxamine (5-10 microM) reduced the amplitudes of Ca2+-independent voltage-activated outward K+ currents (Iout); neither the kinetics nor the voltage-dependent properties of Iout were significantly affected. The effects of phenylephrine or methoxamine on Iout were larger and longer-lasting at higher concentrations and after prolonged or repeated exposures; in all experiments, however, Iout recovered completely when puffer applications were discontinued. The suppression of Iout is attributed to the activation of alpha 1-adrenergic receptors, as neither beta- nor alpha 2-adrenergic agonists had measurable effects on Iout; in addition, the effect of phenylephrine was attenuated in the presence of the alpha antagonist phentolamine (10 microM), but not in the presence of the beta antagonist propranolol (10 microM). Voltage-gated Ca2+ currents, in contrast, were not altered measurably by phenylephrine or methoxamine and no currents were activated directly by these agents. Suppression of Iout was also observed during puffer applications of either of two protein kinase C activators, phorbol 12-myristate 13-acetate (10 nM-1 microM) and 1-oleoyl-2-acetylglycerol (60 microM). We conclude that the activation of alpha 1-adrenergic receptors in adult rat ventricular myocytes leads to action-potential prolongation as a result of the specific suppression of Iout and that this effect may be mediated by activation of protein kinase C.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Coração/fisiologia , Canais de Potássio/fisiologia , Animais , Células Cultivadas , Cinética , Potenciais da Membrana , Metoxamina/farmacologia , Fenilefrina/farmacologia , Ésteres de Forbol/farmacologia , Canais de Potássio/efeitos dos fármacos , Ratos , Função VentricularRESUMO
1. Direct observations of perfused cerebral arterioles in vivo and in vitro have demonstrated that alkalinization of blood or cerebrospinal fluid (CSF) causes arteriolar constriction. Inasmuch as such alkalinizations lead to increases in intracellular pH (pHi) as well as interstitial pH (pHo), it is possible that increases in either pHi or pHo (or both) underlie alkalinization-induced cerebral vasoconstriction. In order to test the hypothesis that changes in pHi alone underline alkalinization-induced cerebral vasoconstriction, we simultaneously measured vessel diameter and pHi (using the pH-sensitive dye, SNAFL) in isolated cerebral arterioles from adult rats during imposed alterations in pHo and pHi. 2. Penetrating cerebral arterioles from the distribution of the middle cerebral artery were hand dissected, cannulated on one end and occluded distally. Vessels were inflated hydrostatically to 60 cmH2O under no-flow conditions. Confocal microscopy verified specific pH-sensitivity dye staining of the vascular smooth muscle cells within the vessel wall. 3. Extracellular alkalinization from pH 7.3 to 7.8 caused pHi to increase by 0.06 +/- 0.01 of a pH unit, and vessel diameter to decrease by 21.8 +/- 1.8% (mean +/- S.E.M.). 4. Intracellular alkalinization at constant pHo was produced by exposure to weak bases, including NH3 and trimethylamine, or by exposure to, followed by withdrawal of, weak acids, including CO2 and acetic acid. None of these treatments evoked vasoconstriction even though each of them caused increases in pHi greater than those observed in the same vessels during exposure to the pHo 7.8 solution. 5. We conclude that, at least in cerebral arterioles, alkalinization-induced vasoconstriction is mediated by an increase in pHo, not pHi [corrected].
Assuntos
Álcalis/metabolismo , Circulação Cerebrovascular , Espaço Extracelular/metabolismo , Membranas Intracelulares/metabolismo , Vasoconstrição , Animais , Arteríolas , Concentração de Íons de Hidrogênio , Masculino , RatosRESUMO
Inhaled nitric oxide (iNO) is a pulmonary vasodilator that recruits blood flow to well-ventilated lung areas in the presence of lung disease. iNO may improve oxygenation by decreasing intrapulmonary shunt or may worsen oxygenation by reversing hypoxic pulmonary vasoconstriction, therapy increasing ventilation-perfusion mismatch. Recent studies have examined the mechanisms for gas exchange alterations with iNO. Moreover, several randomized controlled trials have explored the magnitude of the effect on oxygenation and the beneficial influence on clinical outcome in neonatal patients with hypoxemic respiratory failure. It is not known to what extent an increase in oxygenation affects clinical outcome in older patients. The potential benefit of iNO therapy must be weighed against the potential risks of inactivating surfactant and platelet function as well as influencing endogenous pulmonary vasoregulation. Well-designed studies will be important to determine whether the improvement in oxygenation outweighs these as well as unknown risks.
Assuntos
Cuidados Críticos/métodos , Óxido Nítrico/uso terapêutico , Insuficiência Respiratória/terapia , Vasodilatadores/uso terapêutico , Administração por Inalação , Asfixia Neonatal/terapia , Criança , Humanos , Recém-Nascido , Óxido Nítrico/efeitos adversos , Óxido Nítrico/farmacologia , Troca Gasosa Pulmonar/efeitos dos fármacos , Síndrome do Desconforto Respiratório/terapia , Vasodilatadores/farmacologiaRESUMO
Alkalizing perivascular fluid constricts, whereas acidification dilates, cerebral arterioles. It is not known whether vascular smooth muscle cells (VSMCs), endothelium, or neuronal elements sense pH changes. We hypothesized that VSMCs themselves transduce extracellular pH (pHo) changes. We examined the motor responses of cultured adult rat middle cerebral arterial VSMCs during pHo and intracellular pH (pHi) changes. Motor responses were inferred from the deformation pattern of a silicone substratum, dimethylpolysiloxane, which wrinkles as cells contract. pHi was measured with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Cultured VSMCs retained motor responses to vasoconstrictors (5-hydroxytryptamine and K+), and to sodium nitroprusside, which were typical of intact arterioles. VSMCs contracted with increasing and relaxed with decreasing pHo. Hypocapnia contracted VSMCs when the pHo increased, and hypercapnia relaxed VSMCs when the pHo decreased. However, at a constant pHo, changes in PCO2 caused opposite responses despite equivalent changes in pHi. Thus VSMCs contract with increased pHo and relax with decreased pHo just as intact arterioles do. These responses do not reflect changes in pHi or PCO2. pHi changes paradoxically alter VSMC tone in the direction opposite that caused by pHo changes.
Assuntos
Arteríolas/fisiologia , Artérias Cerebrais/fisiologia , Concentração de Íons de Hidrogênio , Músculo Liso Vascular/fisiologia , Vasoconstritores/farmacologia , Vasodilatadores/farmacologia , Animais , Arteríolas/efeitos dos fármacos , Dióxido de Carbono/farmacologia , Técnicas de Cultura de Células/métodos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Artérias Cerebrais/efeitos dos fármacos , Circulação Cerebrovascular/fisiologia , Fluoresceínas , Corantes Fluorescentes , Microscopia de Vídeo , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Nitroprussiato/farmacologia , Potássio/farmacologia , Ratos , Serotonina/farmacologia , Fatores de TempoRESUMO
To understand why the adult human heart expresses three isoforms of the sodium pump, we generated transgenic mice (TGM) with 2.3- to 5. 5-fold overexpression of the human alpha(3)-isoform of Na-K-ATPase in the heart. Hearts from the TGM had increased maximal Na-K-ATPase activity and ouabain affinity compared with control hearts, even though the density of Na-K-ATPase pump sites (of all isoforms) was similar to that of control mice. In perfused hearts, contractility both at baseline and in the presence of ouabain tended to be greater in TGM than in controls. Surface electrocardiograms in anesthetized TGM had a steeper dependence of Q-T on sinus cycle length, and Q-T intervals measured during atrial pacing were significantly longer in TGM. Q-T dispersion during sinus rhythm also tended to be longer in TGM. Thus TGM overexpressing human alpha(3)-isoform have several of the phenotypical features of human long Q-T syndrome, despite the absence of previously described mutations in Na(+) or K(+) channels.