RESUMEN
ATP-loaded liposomes (ATP-L) infused into Langendorff-instrumented isolated rat hearts protect the mechanical functions of the myocardium during ischemia/reperfusion. The left ventricular developed pressure (LVDP) at the end of the reperfusion in the ATP-L group recovered to 72% of the baseline (preservation of the systolic function) compared to 26%, 40%, and 51% in the groups treated with Krebs-Henseleit (KH) buffer, empty liposomes (EL), and free ATP (F-ATP), respectively. The ATP-L-treated group also showed a significantly lower left ventricular end diastolic pressure (LVEDP; better preservation of the diastolic function) after ischemia/reperfusion than controls. After incubating the F-ATP and ATP-L with ATPase, the protective effect of the F-ATP was completely eliminated because of ATP degradation, while the protective effect of the ATP-L remained unchanged. Fluorescence microscopy confirmed the accumulation of liposomes in ischemic areas, and the net ATP in the ischemic heart increased with ATP-L. Our results suggest that ATP-L can effectively protect myocardium from ischemic/reperfusion damage.
Asunto(s)
Adenosina Trifosfato/farmacología , Corazón/efectos de los fármacos , Liposomas , Contracción Miocárdica/efectos de los fármacos , Isquemia Miocárdica/patología , Isquemia Miocárdica/prevención & control , Miocardio/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/administración & dosificación , Adenosina Trifosfato/metabolismo , Animales , Portadores de Fármacos , Electroquímica , Colorantes Fluorescentes , Técnicas In Vitro , Microscopía Fluorescente , Tamaño de la Partícula , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: To test stability of insulin lispro in two insulin infusion systems over 48 h. RESEARCH DESIGN AND METHODS: We used reverse-phase and size-exclusion high-performance liquid chromatography (HPLC) to determine the purity, potency, and degree of polymerization of U100 insulin lispro (Humalog) after 24- and 48-h pump cycles conducted at 37 degrees C in five Disetronic H-TRON V100 and five MiniMed 504 pumps. Pumps were set to deliver a basal rate of 0.5 U/h and 6-U boluses at t = 0, 4, 8, 24, 24.5, 28.5, 32.5, and 48 h during each cycle. The effluent was collected into 1-ml vials, pooled at 24 or 48 h, and stored at 4 degrees C until assay. After each 48-h run period of insulin delivery, assays for potency, polymer, and purity were performed on the pooled samples from each individual cycle. m-cresol content and the pooled reservoir content were assayed in the 48-h pooled samples. RESULTS: Insulin lispro retained full HPLC potency (delta < or = 4%) at 48 h, with no degradation of insulin lispro to des-amidoinsulin forms (24 or 48 h). No increase in pumped insulin polymer concentration was observed following 24 h of pump flow. Nonsignificant increases of < or =0.09% (Disetronic) and < or =0.15% (MiniMed) from initial concentrations of 0.18% (polymer divided by total insulin) were detected in three of five pump cycles at 48 h when compared with 37 degrees C paired controls. Nonsignificant decreases (<5 and 10%, Disetronic and MiniMed, respectively) of m-cresol content occurred in both systems following 48 h storage in each device, but sterility was not compromised by this decrease (initial m-cresol concentration, 3.15 mg/ml). Pump performance was without mechanical or electrical fault throughout the study Basal and bolus insulin delivery was evaluated three times daily and remained as expected. Occlusion of catheters by insulin precipitation did not occur, and no change in pH was observed following delivery. CONCLUSIONS: We conclude that insulin lispro is suitable for prolonged infusion in these two medical devices when syringes and catheters are replaced at 48-h intervals.
Asunto(s)
Hipoglucemiantes/química , Sistemas de Infusión de Insulina , Insulina/análogos & derivados , Cromatografía Líquida de Alta Presión , Cresoles/análisis , Estabilidad de Medicamentos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/normas , Insulina/administración & dosificación , Insulina/química , Insulina/normas , Insulina Lispro , Polímeros/análisis , Conservadores Farmacéuticos/análisis , Valores de Referencia , Temperatura , Factores de TiempoRESUMEN
An acute coronary occlusion causes severe low-flow ischemia in the occluded region. Calcium antagonists have the potential to reduce the rate of ischemic injury by decreasing myocardial oxygen demand, as well as by other mechanisms, especially when given prior to the onset of ischemia. However, their clinical use may be limited by their negative inotropic effects. The purpose of this study was to assess the effects of felodipine as a potentially protective agent against myocardial ischemia and reperfusion injury, independent of any negative inotropic actions, when given after the onset of low-flow ischemia. Isolated isovolumic (balloon-in-LV), blood-perfused rabbit hearts, paced at a constant heart rate, were subjected to 90 minutes of low-flow ischemia at a coronary perfusion pressure of 10 mmHg, which reduced coronary blood flow to 22-24% of baseline. After 15 minutes of low-flow ischemia, hearts received 2 x 10(-6) M felodipine (n = 7) or no drug (controls, n = 8). Felodipine was given until 15 minutes of reperfusion. During low-flow ischemia both groups of hearts had identical coronary blood flow, heart rate, left ventricular (LV) developed pressure, lactate production, and O2 consumption. However, felodipine markedly protected against ischemic diastolic dysfunction. At the end of low-flow ischemia, LV end-diastolic pressure (LVEDP) had increased from 10 +/- 1 to 28 +/- 5 mmHg in the felodipine group, while in the controls LVEDP increased to 48 +/- 8 mmHg (p < 0.05). During 30 minutes of reperfusion, felodipine had a beneficial effect upon coronary blood flow (initial postischemic hyperemia 245 +/- 38% of baseline in the felodipine group vs. 124 +/- 18% in the controls; p < 0.01) Felodipine markedly improved the recovery of contractile function [LV developed pressure recovered from a baseline of 104 +/- 4 to 75 +/- 6 mmHg (72%) in the felodipine group vs. 34 +/- 10 mmHg (32%) in the control group; p < 0.01], as well as diastolic function (LVEDP = 25 +/- 4 mmHg in the felodipine group vs. 61 +/- 10 mmHg in the controls; p < 0.05), and ATP levels (8.5 +/- 1.4 mumoles/g d.w. in the felodipine group vs. 3.9 +/- 1.4 mumoles/g d.w. in the control group, p < 0.05). Felodipine, given after the onset of low-flow ischemia, protects the myocardium during both ischemia and reperfusion by mechanisms other than reducing myocardial oxygen demand.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Felodipino/farmacología , Corazón/efectos de los fármacos , Daño por Reperfusión Miocárdica/prevención & control , Vasodilatadores/farmacología , Adenosina Trifosfato/análisis , Animales , Circulación Coronaria/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Felodipino/uso terapéutico , Técnicas In Vitro , Ácido Láctico/metabolismo , Contracción Miocárdica/efectos de los fármacos , Isquemia Miocárdica/tratamiento farmacológico , Oxígeno/metabolismo , Conejos , Presión Ventricular/efectos de los fármacosRESUMEN
We investigated the role of pulmonary granulocyte sequestration in the development of early failure of the autoperfused working heart-lung preparation. A significant decline in the total circulating leukocyte count in 21 preparations at 60 minutes of perfusion (5.0 to 1.4 x 10(3)/microL; 28% of baseline; p less than 0.001) was observed. Differential cell counts in 14 of these preparations revealed a predominant decrease in granulocyte count (8.7% of baseline) and a moderate decline in lymphocyte count (46% of baseline). In study I, indium 111-labeled autologous granulocytes were injected intravenously into 10 adult New Zealand White rabbits. In group I (n = 5), an autoperfused working heart-lung preparation was harvested and perfused for 60 minutes. In group II (controls, n = 5), the heart-lung block was harvested following 60 minutes of in situ perfusion. Organ blocks were imaged before and after saline flush. There was a significant decline in granulocyte counts at 60 minutes of perfusion in group I versus no change in group II (I, 2.3 +/- 0.4 to 0.3 +/- 0.1; p less than 0.01; II, 1.7 +/- 0.2 to 2.3 +/- 0.5; not significant; x 10(3)/microL +/- standard error of the mean). Postflush lung activity was significantly increased in group I versus group II (I, 3,751 +/- 566; II, 1,867 +/- 532; p less than 0.05; counts +/- standard error of the mean). In study II, 15 autoperfused preparations were divided into two groups. Group I (n = 10) preparations were controls. Group II (n = 5) animals were depleted of leukocytes by pretreating with nitrogen mustard. Group I (controls) produced a bimodal survival distribution (Ia, 8.2 +/- 1.0; Ib, 26.4 +/- 2.0; hours +/- standard error of the mean). Group II survival was significantly longer than that of group Ia and similar to that of group Ib (II, 25.3 +/- 2.2; p less than 0.01 versus group Ia, not significant versus group Ib; hours +/- standard error of the mean). Hemodynamic profiles for group II closely paralleled those of group Ib. In conclusion, pulmonary sequestration of granulocytes occurs early in the autoperfused working heart-lung preparation (within 60 minutes of autoperfusion), and preoperative leukocyte depletion prolongs survival of the autoperfused working heart-lung preparation by eliminating the subset group Ia (short survivors) seen in untreated preparations.
Asunto(s)
Granulocitos/fisiología , Corazón , Pulmón , Preservación de Órganos/métodos , Supervivencia Tisular , Animales , Análisis de los Gases de la Sangre , Presión Sanguínea , Gasto Cardíaco , Granulocitos/diagnóstico por imagen , Recuento de Leucocitos , Rendimiento Pulmonar , Recuento de Plaquetas , Arteria Pulmonar/fisiología , Conejos , CintigrafíaRESUMEN
We studied the role of leukocyte redistribution and eicosanoid changes in the early stages of instituting 16 rabbit autoperfused working heart-lung preparations (AWHLP). Physiological changes occurring during the transition from the intact animal to the AWHLP may determine the survival and viability of the organ blocks for transplantation. White blood cell (WBC) count decreased from 5,160/microL to 1430/microL (P less than .01) at 60 min of autoperfusion. Differential WBC counts performed in ten of these AWHLP revealed a 63% decrease in lymphocyte count and an 88% decrease in the granulocyte count at 60 min. Thus, the predominant leukocyte remaining in the circulation was the lymphocyte. Blood samples were collected from the intact animal and from the AWHLP for assay of the stable metabolites of thromboxane A2 (TxA2) and prostacyclin (PGI2). Transition from the in situ heart-lung block to the in vitro AWHLP stage caused significant changes in these metabolites. The PGI2 metabolite 6-ketoprostaglandin F1a (6KPGF1a) increased from 2680 +/- 487 to 4339 +/- 478 (pg/mL), P less than .05, while the TxA2 metabolite, thromboxane B2 (TxB2) decreased from 618 +/- 105 to 289 +/- 63 (pg/mL). However, assays of 11-dehydro-TxB2 (11-DHT), a longer lived metabolite of TxA2 (n = 7) increased (668.4 +/- 84.6 to 946.4 +/- 43.7, P less than .05). The transition from the in situ heart-lung block of the intact animal to the AWHLP involves significant physiological changes. Redistribution of leukocytes occurs with a predominant decrease in the granulocyte count, while levels of bioactive lipid mediators show a distinct large rise in the PGI2 metabolites and a lesser increase in TxA2 metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Eicosanoides/biosíntesis , Trasplante de Corazón-Pulmón/fisiología , Recuento de Leucocitos , Animales , Recuento de Eritrocitos , Trasplante de Corazón-Pulmón/patología , Preservación de Órganos , Perfusión , Recuento de Plaquetas , ConejosRESUMEN
Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxypeptidase that converts angiotensin I into the potent vasoconstrictor angiotensin II. We have used cDNA and genomic sequences to assemble a composite cDNA, ACE.315, encoding the entire amino acid sequence of mouse converting enzyme. ACE.315 contains 4838 base pairs and encodes a protein of 1278 amino acids (147.4 kDa) after removal of a 34-amino acid signal peptide. Within the protein, there are two large areas of homologous sequence, each containing a potential Zn-binding region and catalytic site. These homologous regions are approximately half the size of the whole ACE protein and suggest that the modern ACE gene is the duplicated product of a precursor gene. Mouse ACE is 83% homologous to human ACE in both nucleic acid and amino acid sequence, and like human ACE, contains a hydrophobic region in the carboxyl terminus that probably anchors the enzyme to the cell membrane (Soubrier, F., Alhenc-Gelas, F., Hubert, C., Allegrini, J., John, M., Tregear, G., and Corvol, P. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9386-9390). Northern analysis of mouse kidney, lung, and testis RNA demonstrates that the testicular isozyme of ACE is encoded by a single, smaller RNA (2500 bases) than the two message sizes found in kidney or lung (4900 and 4150 bases), and that this testicular RNA hybridizes to the 3' portion of ACE.315.
Asunto(s)
Secuencia de Bases , Peptidil-Dipeptidasa A , Homología de Secuencia de Ácido Nucleico , Secuencia de Aminoácidos , Animales , Northern Blotting , ADN , Riñón/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , ARN Mensajero/análisisRESUMEN
Angiotensin-converting enzyme (ACE) is an Zn(II)-containing dipeptidyl carboxypeptidase that converts angiotensin I to the potent vasoconstrictor, angiotensin II. Using oligonucleotide probes based on the amino acid sequence of mouse kidney ACE, cDNA encoding this protein has been isolated. One cDNA, ACE.31, encodes the N-terminal 332 amino acids of mouse ACE, a portion of the protein containing a putative 34-amino acid leader sequence and the N terminus of the mature protein. Northern analyses with cloned ACE cDNA revealed that both mouse kidney and lung express two ACE mRNAs, one of 4900 and another of 4150 bases. Southern analysis suggests that cDNA ACE.31 is the product of a single gene, and thus these data add evidence to the hypothesis that the converting enzymes produced by epithelial and endothelial cells are identical.