Electrophysiological abnormalities produced by ammonium in isolated perfused brook trout, Salvelinus fontinalis, hearts

Electrocardiographic (ECG) abnormalities and arrhythmias have been observed in hyperammonemic patients and in animals injected with ammonium salts. Ammonium is excreted directly into the environment by fish, and it is a potent fish toxin, but the effects of ammonium on the cardiovascular system of fish are unknown. This study investigated the cardiotoxic effects of ammonium on isolated perfused fish hearts. These were compared with ammonium-induced cardiac electrophysiological abnormalities observed in humans and other experimental animals. Isolated perfused fish hearts exhibit ammonium-induced cardiac abnormalities similar to the ammonium-induced abnormalities seen in the hearts of higher vertebrates. Therefore, we conclude that the myocardium of ammonotelic species does not possess a specialized ammonium detoxification mechanism, and that ammonium is cardiotoxic to salmonid fish.     

Oxygen delivery and myocardial function in rabbit hearts perfused with cell-free hemoglobin.

Isolated rabbit hearts were perfused with Krebs-Henseleit buffer that contained 1.5 g/dl hemoglobin Ao [HbAo; PO2 at which half-saturation of hemoglobin occurs = 12 Torr], human hemoglobin cross-linked between alpha-chains with bis(3,5-dibromosalicyl)fumarate (alpha alpha-Hb; PO2 at which half-saturation of hemoglobin occurs = 30 Torr), or fatty acid-free bovine serum albumin (BSA). Myocardial performance and oxygen uptake were determined at different aortic PO2's [arterial PO2 (PaO2)] by use of an isovolumic Langendorff preparation. Function and oxygen uptake were comparable among the three different groups of hearts at an average mean PaO2 of 557 Torr. As PaO2 decreased, myocardial function was preserved better in hearts perfused with hemoglobin than in hearts perfused with Krebs-Henseleit buffer alone or with BSA. Hearts perfused with either HbAo or alpha alpha-Hb exhibited similar 10% decreases in left ventricular developed pressure and rate of change in left ventricular developed pressure at PaO2 of 141 Torr compared with a 58% decrease with BSA. However, corresponding venous PO2's were lower with HbAo (20 Torr) than with alpha alpha-Hb (35 Torr), and oxygen uptake decreased by 36% with HbAo but remained constant with alpha alpha-Hb. These data suggest that although myocardial function can be sustained over a fairly broad range of hemoglobin oxygen affinities, tissue oxygen gradients and myocardial oxygen uptake are maintained better by cell-free hemoglobin with an oxygen affinity in the normal physiological range.     

Cardiac effects of prostaglandins during global ischemia in isolated perfused cat hearts

Prostaglandin E₁,PGD₂, and 16,16 dimethyl prostaglandin E₂ were studied in an isolated perfused cat heart preparation during normal conditions and in myocardial ischemia. Under ischemic perfusion, prostaglandin E₁ showed some protective action on the release of creatine kinase into perfusate during ischemia, but none of the prostaglandins studied prevented increases in perfusate creatine kinase after reperfusion. Prostaglandin E₁ also showed significant membrane stabilizing activity reducing the rate of lysosomal hydrolase release from cat liver lysosomes. Prostaglandin E₁ may be beneficial in myocardial ischemia due to its membrane stabilizing action and perhaps to other effects but it did not exert significant protective effects on reperfusion injury of the ischemic myocardium under conditions of these experiments.     

Pressure-calcium relationships in perfused mouse hearts

We explored the relationship between left ventricular (LV) pressure and intracellular free calcium concentration ([Ca](i)) in the isolated perfused mouse heart. [Ca](i) (rhod-2) and LV pressure were recorded simultaneously. In response to increases in LV volume (Frank-Starling, FS, protocol), there were increases in developed pressure (up to 250%), with no changes in pressure morphology (rise or relaxation time) or [Ca](i) (magnitude and morphology) for up to 10 min. During transient increases in the stimulus interval at a fixed LV volume (mechanical restitution, MR, protocol), developed pressure increased significantly (31.3 +/- 1.2%), with relatively small changes in peak systolic [Ca](i) (7.4 +/- 1.4%). The relaxation of [Ca](i), however, was prolonged (30.0 +/- 5.5%), resulting in prolonged pressure relaxation (21.2 +/- 1.9%) and increased area under the calcium transient that paralleled the increase in developed pressure (1:1 ratio). A model-based analysis showed that changes in LV pressure during the MR protocol could be completely explained by altered [Ca](i); it was not necessary to invoke any changes in model parameters (i.e., dynamic processes that link calcium to pressure). For the FS data, the model predicted only a change in the gain parameter; however, this change alone cannot reproduce well-established length-dependent changes in the steady-state force-pCa relationship. In summary, the mouse myocardium appears to be unique in that significant changes in peak developed pressure can occur with little or no change in the peak [Ca](i). Additionally, unlike other mammalian species, load-dependent prolongation of pressure relaxation is absent in the mouse heart, and pressure relaxation is primarily governed by intracellular free calcium relaxation.     

Sites of lipase activation and prostaglandin synthesis in isolated, perfused rabbit hearts and hydronephrotic kidneys

The tissue lipids of isolated, perfused rabbit hearts and hydronephrotic kidneys were labelled with [¹⁴C]-arachidonic acid by two different techniques: direct infusion of [¹⁴C]-arachidonic acid in a protein free media into the perfused organ (method A), and recirculation of [¹⁴C]-arachidonic acid in a solution containing albumin (method B). Autoradiography of the labelled organs demonstrated that method A resulted in selective labelling of arteries and arterioles in both perfused organs as well as glomeruli in the kidney. Labelling with method B resulted in a non-specific radioisotope incorporation in both the vasculature and myocardial cells in the heart; and of the vasculature and renal tubules in the perfused kidneys. Analysis of the tissue lipids shows similar patterns of incorporation of radioactivity between methods A and B.Peptide hormone stimulation (bradykinin) and non-specific noxious stimulation (with transient ischemia) were employed to elicit lipase activation (i.e., release of [¹⁴C]-arachidonate) and prostaglandin (PG) synthesis. It was found that in both hearts and hydronephrotic kidneys, the radioactive PG release in response to bradykinin and ischemia was much higher with method A (vascular labelling) than with method B (diffuse labelling) despite the appearance of comparable amounts of bioassayable PG release, thus indicating the sites of PG synthesis in these organs is predominantly localized in the vascular tissue. Furthermore, the radioactive arachidonic acid release in response to bradykinin stimulation in the hydronephrotic kidneys was 3 times higher with method A than with method B, suggesting the predominant sites of hormone specific lipase activation in the renal cortex is also in the vasculature. However, the radioactive arachidonic acid release in response to ischemia was much higher with method B than with method A in both hearts and hydronephrotic kidneys, indicating the sites of non-specific lipase activation in these organs are more diffusely distributed, and present also in the myocardial cells and renal tubules.     

Reduction of myocardial infarction by calpain inhibitors A-705239 and A-705253 in isolated perfused rabbit hearts

Two novel calpain inhibitors (A-705239 and A-705253) were studied in isolated perfused rabbit hearts subjected to 60-min occlusion of the ramus interventricularis of the left coronary artery (below the origin of the first diagonal branch), followed by 120 min of reperfusion. The inhibitors were added to the perfusion fluid in various final concentrations from the beginning of the experiments before the coronary artery was blocked. Hemodynamic monitoring and biochemical analysis of perfusion fluid from the coronary outflow were carried out. Myocardial infarct size and the area at risk (transiently non-perfused myocardium) were determined from left ventricular slices after a special staining procedure with Evans blue and 2,3,5-triphenyltetrazolium chloride. The infarcted area (dead myocardium) was 77.9+/-2.3% of the area at risk in untreated controls ( n =12). The infarct size was significantly reduced in the presence of both calpain inhibitors. The best effect was achieved with 10 -8 M A-705253 ( n =8), which reduced ( p <0.001) the infarcted area to 49.3+/-3.9% of the area at risk, corresponding to an infarct reduction of 61.8%. No statistical difference was observed between the experimental groups in coronary perfusion, left ventricular pressure, and in the release of lactate dehydrogenase and creatine kinase from heart muscle.     

Alterations in intracellular calcium activity and contractility of isolated perfused rabbit hearts by ionophores and adrenergic agents

The fluorescent calcium indicator quin2 has been used for the first continuous measurement of the effects of pharmacological agents on intracellular calcium activity in isolated, perfused rabbit hearts. The average intracellular calcium activity was elevated after the infusion of norepinephrine, concurrent with increases in left ventricular pressure and heart rate. These changes were abolished by pretreatment of the heart with phentolamine and nadolol, alpha and beta adrenergic receptor antagonists, respectively. Pretreatment with phentolamine and nadolol did not eliminate the increases in left ventricular pressure and intracellular calcium activity caused by the infusion of the monovalent carboxylic ionophores monensin and salinomycin. It is concluded that the ionophores cause these effects by elevating intracellular sodium activity, which then raises the intracellular calcium activity of the myocardium through intracellular displacement and/or transcellular exchange. It is suggested that the use of fluorescent calcium indicators in intact organs could be useful in evaluating the role of calcium in a variety of pathological states.     

Gonadotropin-releasing hormone: effects on the in vitro perfused rabbit ovary

Gonadotropin-releasing hormone (GnRH) has been shown to inhibit ovulation in gonadotropin-primed hypophysectomized rats and steroid production in cultured rat granulosa cells. To determine if similar effects of GnRH can be observed in another species, the extracorporeal perfused rabbit ovary was utilized. Two groups of rabbit ovaries were exposed to GnRH in a pulsatile fashion at two dose levels (Group I, 2.56 X 10(-8) M; Group II, 2.56 X 10(-7) M). Contralateral ovaries were not perfused with GnRH. Human chorionic gonadotropin (hCG) was added to the perfusate of all ovaries 30 min after the onset of perfusion. Ovulation occurred in all ovaries exposed to hCG in the presence or absence of GnRH. Ovulatory efficiency was similar in both the experimental and control groups. No statistical difference could be determined in the time of ovulation, stage of maturity of oocytes, or percent of degeneration of ovulated or follicular oocytes. Progesterone production was not inhibited in the GnRH-treated ovaries. In contrast to observations in the rat, GnRH does not exhibit a direct inhibitory effect on ovulation or steroid production in the rabbit.     

辣椒素对家兔房室结细胞自发活动的电生理效应

本工作旨在研究辣椒素对家兔房室结细胞自发活动的电生理效应及其作用机制.应用经典玻璃微电极记录方法,观察到辣椒素(1～30 μmol/L)剂量依赖性地抑制房室结起搏细胞的动作电位幅度,零相最大上升速度(Vmax),舒张期除极速度和起搏放电频率,而且延长复极化90%时间(APD90).应用L型钙通道开放剂Bay K8644(0.5 μmol/L),以及提高灌流液中钙离子浓度(5 mmol/L),均可抑制辣椒素对起搏细胞的电生理效应.辣椒素受体阻断剂钌红(10μmol/L)对辣椒素(10μmol/L)的上述电生理效应并无影响.上述结果表明,辣椒素能抑制家兔房室结的自发活动,此效应可能与其抑制钙离子内流有关,但并非由辣椒素受体介导.     

Effects of tissue absorbance on NAD(P)H and Indo-1 fluorescence from perfused rabbit hearts

The effects of tissue optical absorbance on intracellular NAD(P)H and Indo-1 fluorescence emission have been evaluated in the perfused rabbit heart. These results demonstrate that the tissue optical absorbance significantly modifies the emission characteristics of these fluorophores. This tissue 'inner filter' effect, observed with both probes, changed as a function of tissue oxygenation and redox state in a wavelength-dependent manner. Pathlength calculations from these results indicate that this inner filter effect could occur with a mean pathlength of 310 microns due to the extremely high extinction coefficient of heart tissue. It is concluded that tissue optical absorbance significantly affects the fluorescent emission characteristics of both intrinsic and extrinsic probes in the intact heart, under a variety of conditions. Several potential methods of correcting for these tissue inner filter effects are discussed.     

Endothelial adenosine transporter characterization in perfused guinea pig hearts

Adenosine (Ado), a smooth muscle vasodilator and modulator of cardiac function, is taken up by many cell types via a saturable transporter, blockable by dipyridamole. To quantitate the influences of endothelial cells in governing the blood-tissue exchange of Ado and its concentration in the interstitial fluid, one must define the permeability-surface area products (PS) for Ado via passive transport through interendothelial gaps [PS(g)(Ado)] and across the endothelial cell luminal membrane (PS(ecl)) in their normal in vivo setting. With the use of the multiple-indicator dilution (MID) technique in Krebs-Ringer perfused, isolated guinea pig hearts (preserving endothelial myocyte geometry) and by separating Ado metabolites by HPLC, we found permeability-surface area products for an extracellular solute, sucrose, via passive transport through interendothelial gaps [PS(g)(Suc)] to be 1.9 +/- 0.6 ml. g(-1). min(-1) (n = 16 MID curves in 4 hearts) and took PS(g)(Ado) to be 1. 2 times PS(g)(Suc). MID curves were obtained with background nontracer Ado concentrations up to 800 micrometer, partially saturating the transporter and reducing its effective PS(ecl) for Ado. The estimated maximum value for PS(ecl) in the absence of background adenosine was 1.1 +/- 0.1 ml. g(-1). min(-1) [maximum rate of transporter conformational change to move the substrate from one side of the membrane to the other (maximal velocity; V(max)) times surface area of 125 +/- 11 nmol. g(-1). min(-1)], and the Michaelis-Menten constant (K(m)) was 114 +/- 12 microM, where +/- indicates 95% confidence limits. Physiologically, only high Ado release with hypoxia or ischemia will partially saturate the transporter.     

Direct detection of paramagnetic species in adriamycin perfused rat hearts

Direct detection of paramagnetic species in control and adriamycin-perfused rat hearts has been carried out. Depending on the flow rate of the perfusion solution (8,4,2 and 1 ml/min) different paramagnetic species were observed: Fe(III)(g = 2.12) at 4 ml/min; three types of oxygen centered radicals of which two in control hearts (g = 2.05 g = 2.038 g = g = 2.008) and the third one (g = 2.03 g = 2.005) in adriamycin perfused hearts, at 2 ml/min. The latter radical was the only one observed at perfusion rate of 1 ml/min both in control and adriamycin treated systems. A relationship between the intracellular enzymatic reductive activation of the anthracycline and the occurrence of ischemic conditions (4,2 and 1 ml/min) in myocardial tissues is proposed basing on the relative amounts of the paramagnetic species above mentioned.     

一氧化氮对家兔房室结细胞自发活动的电生理效应

应用经典玻璃微电极技术,观察一氧化氮(NO)对家兔房室结细胞自发活动的电生理效应及其作用机制。结果显示：(1)NO供体硝普(SNP,1-1000μmol／L)及SIN-1(100,1000μmol／L)剂量依赖性地抑制房室结细胞的动作电位幅值(APA)、零相最大人士升速度(Vmax)、4期自动除极速度(VDD)及自发放电频率(RSF);(2)应用L型钙通道开放剂Bay K8644(0．25μmol／L),可拮抗SNP对房室结细胞的电生理效应;(3)提高灌流液中钙离子浓度(5mmol／L)也可逆转SNP对起搏细胞的抑制效应;(4)用无钙K-H液灌流房室结,可完全阻断SNP对房事结细胞的抑制效应。(5)应用鸟苷酸环化酶阻断剂甲基美蓝(50μmol／L)对SNP的上述电生理效应无影响。以上结果提示,NO可能是通过cGMP非依赖性途径减弱钙离子内流,进而抑制了家兔房室结细胞的自发电活动。     

植物性雌激素三羟异黄酮对家兔房室结细胞自发活动的电生理效应

本研究旨在应用经典玻璃微电极方法观察植物性雌激素三羟异黄酮（genistein,GST）对家兔房室结细胞自发活动的电生理效应及其作用机制。GST（10－100μmol/L）不仅以剂量依赖性方式抑制房室结起搏细胞的动作电位0相最大上升速度（Vmax）、4期去极化速率（VDD）、自发起搏频率（RPF）和动作电位幅值（APA）,而且延长复极化90％的时间（APD90）。如提高灌流液中钙离子浓度以及应用L型钙通道开放剂Bay K8644(0.25μmol/L）,则可拮抗GST对起搏细胞的上述电理效应,但NO合酶阻断剂L－NAME（0.5nmol/L）对GST的效应并无影响,以上结果提示,GST可抑制家兔房室结的自发活动,这些效应可能与其抑制钙离子内流有关,但此作用机制中并无NO参与。     

降钙素基因相关肽对家兔离体窦房结电生理活动的影响

用常规微电极方法研究了降钙素基因相关肽（ＣＧＲＰ）对家兔窦房结起搏细胞的电生理作用,并进一步探讨这种作用与钙电流的关系。结果：⑴低浓度ＣＧＲＰ（１ｎｍｏｌ／Ｌ）对窦房结动作电位各参数无显著影响;中等浓度ＣＧＲＰ（１０ｎｍｏｌ／Ｌ）可增加最大舒张期电位、动作电位幅度、０期最大除极化速率和４期自动除极速率,缩短窦性周期、动作电位复极化５０％和９０％时间,这些作用经２０ｍｉｎ达到高峰;高浓度ＣＧＲＰ（２     

Synthesis of stress-induced protein in isolated and perfused rat hearts

Isolated and perfused rat hearts were examined by two-dimensional gel electrophoresis and liquid scintillation counting for alterations in protein synthesis following incubation with L-[3H]leucine at 0.5-2.5, 2.5-4.5, or 4.5-6.5 h of perfusion. When 35-mL volumes of three different buffers were recycled for a 2-h period from 0.5 to 2.5 h, by fluorography little effect was seen on the normal patterns of protein synthesis and there was a moderate synthesis of a stress-induced protein (heat-shock protein) with a molecular mass of 71 X 10(3) daltons (SP71). However, hearts perfused with Krebs-improved Ringer 1 bicarbonate had the highest incorporation of L-[3H]leucine. When buffers were recycled for 30-min periods from 0.5 to 2.5 h, SP71 was synthesized in hearts perfused with Krebs-Henseleit original Ringer bicarbonate. Hearts perfused in a similar fashion with Krebs-improved Ringer 1 bicarbonate had the lowest incorporation of label into SP71 and in fact SP71 was undetectable on fluorograms. Overall protein synthesis was decreased and the ratio of SP71 to the total synthesis was increased at 4.5-6.5 h of perfusion when 35-mL volumes of Krebs-improved Ringer 1 bicarbonate was recycled for 2-h periods. A similar result was observed at 2.5-4.5 h of perfusion when this buffer was recycled for either the duration of the experiment or 30-min periods.