Intraspecific variation and plasticity in mitochondrial oxygen binding affinity as a response to environmental temperature.

Mitochondrial function has been suggested to underlie constraints on whole-organism aerobic performance and associated hypoxia and thermal tolerance limits, but most studies have focused on measures of maximum mitochondrial capacity. Here we investigated whether variation in mitochondrial oxygen kinetics could contribute to local adaptation and plasticity in response to temperature using two subspecies of the Atlantic killifish (Fundulus heteroclitus) acclimated to a range of temperatures (5, 15, and 33 °C). The southern subspecies of F. heteroclitus, which has superior thermal and hypoxia tolerances compared to the northern subspecies, exhibited lower mitochondrial O2 P50 (higher O2 affinity). Acclimation to thermal extremes (5 or 33 °C) altered mitochondrial O2 P50 in both subspecies consistent with the effects of thermal acclimation on whole-organism thermal tolerance limits. We also examined differences between subspecies and thermal acclimation effects on whole-blood Hb O2-P50 to assess whether variation in oxygen delivery is involved in these responses. In contrast to the clear differences between subspecies in mitochondrial O2-P50 there were no differences in whole-blood Hb-O2 P50 between subspecies. Taken together these findings support a general role for mitochondrial oxygen kinetics in differentiating whole-organism aerobic performance and thus in influencing species responses to environmental change.

Effects of a neutral endoprotease enzyme inhibitor, thiorphan, on hemodynamics and renal excretory function in four models of experimental hypertension.

Thiorphan, a neutral endoprotease (NEP) enzyme inhibitor, has been shown to enhance the effects of atrial natriuretic peptide (ANP) in vivo. In this study, we examined the effects of an intravenous (iv) infusion of thiorphan on cardiovascular hemodynamics and excretion of urine volume (UV), sodium (U(Na)V) and potassium (UKV) in four different models of experimental hypertension, namely: 1) SHR, 2) two-kidney, one clip (2K1C),3) one-kidney, 1 clip (1K1C) and. 4) 70% reduced renal mass-salt (RRM-S) hypertensive rats. SHR has normal plasma renin activity, 2K1C is renin dependent, and 1K1C and RRM-S are low renin volume dependent models of hypertension. Rats were divided into experimental and control groups. Under inactin (120 mg/kg, body weight) anesthesia, rats were instrumented to record blood pressure and dP/dt (Millar catheter) and urine was collected through a suprapubic urinary bladder catheter. Experimental animals received an iv infusion of thiorphan, 0.5 mg/kg/min for 120 minutes. Control animals received vehicle only. In some animals, vascular smooth muscle cell membrane potentials (Em) was measured in vivo. In another series of experiments, using the identical protocol, cardiac output was recorded. The thiorphan infusion produced a similar progressive decrease in blood pressure in all models of hypertension. Cardiac output did not change relative to vehicle infused control animals. Thus pressure decreased because of a decrease in total peripheral resistance. The contractility index (dP/dt/P, where P = left ventricular pressure) did not change but vascular smooth muscle cells in tail arteries hyperpolarized in all four models. In spite of a significant decrease in blood pressure, thiorphan infusion either increased or produced no change in urinary volume (UV) and sodium (U(Na)V) excretion. These data show that thiorphan, an NEP inhibitor, decreases the blood pressure of hypertensive rats due to a decrease in total peripheral resistance, perhaps by hyperpolarizing vascular smooth muscle cells. These effects are independent of the mechanism of the hypertension. Increased UV and U(Na)V in the face of decreased pressure suggests a direct renal effect.

Influence of extracellular matrix proteins on membrane potentials and excitability in NG108-15 cells.

Previous studies have demonstrated that components of the extracellular matrix can induce neurite extension and cell adhesion in the neuroblastoma x glioma hybrid cell line, NG108-15. Using standard intracellular recording techniques, we examined the resting membrane potential (RMP) and membrane excitability of NG108-15 cells differentiated under serum-free media with representative extracellular matrix (ECM) protein components as the substrate. Surfaces coated with collagen IV and a laminin-1 synthetic peptide induced a significantly (P < 0.05) more hyperpolarized RMP than control polystyrene surfaces. For example, after > or =8 days in culture NG108-15 cells plated on polystyrene exhibited a RMP of -33.2+/-0.8 mV (mean+/-SEM, n=158 cells) whereas cells cultured on the laminin-1 peptide C16 and collagen IV showed a RMP of -37.6+/-0.7 mV (n=157) and -37.5+/-1.5 mV (n=68), respectively. Furthermore, the proportions of cells on ECM substrates showing membrane excitability, i.e. evoked action potentials (APs) and the capability for regular firing, were significantly greater compared to those cells cultured on polystyrene. Among excitable cells cultured on the different substrates, characteristics of the action potentials, such as AP duration, amplitude, and the maximum rate of rise, dV/dtMAX, were examined in detail. While little or no differences were observed between polystyrene and the laminin-1 peptide groups, significant differences in the AP parameters were apparent for collagen IV. For example, dV/dtMAX for polystyrene and the laminin-1 peptide C16 were only 71.7+/-24.5 V/s (n=11) and 59.0+/-8.9 V/s (n=9), respectively, whereas cells cultured on collagen IV surfaces exhibited a dV/dtMAX reaching 156.1+/-22.0 V/s (n=7). These data support a role for ECM components in the maintenance of the RMP and membrane excitability in NG108-15 cells.

Effects of head down tilt on hemodynamics, fluid volumes, and plasma Na-K pump inhibitor in rats.

BACKGROUND: Hindquarter suspension in rats has been used as a model of simulated weightlessness (SW) for ground based study of the effects of microgravity on the cardiovascular system (CVS). METHODS: Using this rat model of SW we tested the hypothesis that CVS deconditioning following spaceflight results, in part, from a decrease in the circulating concentration of sodium-potassium pump inhibitor (SPI). Control rats similarly prepared were not suspended. RESULTS: During the first hour of suspension, central venous pressure (CVP), blood pressure (BP), heart rate (HR), cardiac output (CO), plasma volume (PV), extracellular fluid volume (ECFV), urine output (UV), atrial natriuretic peptide (ANP), and the plasma level of SPI increased. Plasma renin activity (PRA) and myocardial Na+, K(+)-ATPase activity (NKA) decreased. By the end of 4 h of SW, the changes in CVP, BP, HR, ECFV, and UV persisted, but PV, plasma ANP and SPI, and myocardial NKA activity returned to control levels. By the end of 1 d of SW, ECFV and plasma SPI levels had decreased but the myocardial NKA had not increased. At day 4, CVP and BP were the same as in control sham treated rats. Plasma SPI levels were decreased at day 4 but the myocardial NKA was not different, whereas renal NKA was increased. At day 7, myocardial NKA and renal NKA were increased and vascular smooth muscle cell (VSMC) membrane potentials were hyperpolarized. CONCLUSIONS: These data indicate that prolonged SW causes a decrease in plasma SPI level which, by hyperpolarizing VSMC, may play a role in the CVS deconditioning seen in astronauts following spaceflight.

Spontaneous firing of NG108-15 cells induced by transient exposure to ammonium chloride.

1. We report that NG108-15 (neuroblastoma x glioma) cells differentiated in defined serum-free media are capable of exhibiting stable automaticity (the spontaneous occurrence of regenerative action potentials) following exposure to extracellular perfusates containing NH4Cl. 2. Membrane depolarization (4-5 mV) concomitant with an increased pHi during NH4Cl exposure are followed by hyperpolarization (5-7 mV), sub-threshold oscillations, and spontaneous firing after the removal of NH4Cl. 3. Cells cultured in 10% serum did not exhibit automaticity. Cells cultured in serum-free media are twice as likely to show automaticity as those cultured in reduced (1.5%) serum media. 4. We have examined factors that contribute to the events following NH4Cl exposure, namely, membrane depolarization and hyperpolarization, subthreshold oscillations, and automaticity. The inward currents activated at more negative potentials and the ionic currents associated with pronounced afterhyperpolarization in NG108-15 cells cultured in serum-free media provide a basis for the repetitive activity in general and automaticity in particular.

Effects of soman (pinacolyl methylphosphonofluoridate) on coronary blood flow and cardiac function in swine.

The effects of soman (pinacolyl methylphosphonofluoridate) on coronary blood flow, the electrocardiogram, and cardiac function were measured in alpha-chloralose-anesthetized swine. Coronary blood flow (CBF), mean arterial blood pressure (MAP), peak systolic left ventricular pressure (IVP), maximum rate of left ventricular pressure development (dP/dtmax), cardiac output, and the ECG were monitored continuously. A dose of 2X LD50 of soman (1 LD50 = 4.6 micrograms/kg) was given at 1 LD50/min in the femoral vein, which produced an increase in coronary sinus plasma acetylcholine (ACh) from a control of 0.7 +/- 0.01 nmol/ml to a maximum 314% of control at 15 min and a decrease in CBF from a control of 99 +/- 13 ml/min/100 g to a minimum 55% of control at 15 min. The increase in ACh in the coronary sinus was significantly correlated with a decrease in CBF (r = -0.87, p < 0.001). The fall in CBF was accompanied by concomitant decreases in IVP, MAP, and dP/dtmax, with S-T segment elevation and ventricular fibrillation. The increase in coronary sinus acetylcholine concentration was significantly correlated with a 10-fold fall in coronary sinus acetylcholinesterase levels from a control of 2.47 +/- 0.97 mol acetylcholine hydrolyzed/ml blood/min and was consistent with the time course for the reduced hemodynamic measurements. These studies support the hypothesis that acetylcholine increases following soman toxicity may decrease coronary blood flow, thereby initiating ischemic electrocardiographic changes and reducing cardiac function.

Regional volume changes in canine lungs suspended in air.

NASA: The purpose of this study was to determine the effect of the absence of a pleural pressure gradient (simulating the presumed condition found in microgravity) upon regional expansion of the lung. We attempted to produce a uniform pressure over the surface of the lung by suspending excised lungs in air. Such studies should help determine whether or not absence of a pleural pressure gradient leads to uniform ventilation. A preparation in which there is no pleural pressure gradient should also be useful in studying non-gravitational effects on ventilation distribution.^ieng

Differential response of dog and pig tracheal smooth muscle to the acetylcholinesterase inhibitor soman.

The effect of the acetylcholinesterase inhibitor soman on tracheal smooth muscle (TSM) from the dog and pig was studied. In response to soman, tracheal ring preparations contract more and the resting tension for TSM preparations is higher for the dog compared with the pig. Tension induced by electrical field stimulation (EFS) and the half-time of EFS-train induced contractions have a similar dependence on soman exposure in both dog and pig TSM. These results suggest that the basal acetylcholine secretion or leakage within the TSM nerve terminal is probably higher for the dog compared with the pig.

Comparative electrophysiological properties of NG108-15 cells in serum-containing and serum-free media.

The electrophysiological properties of NG108-15 neuroblastoma x glioma hybrids were compared after culture in serum-containing medium (SCM) versus serum-free media (SFM) containing N2 or B27 supplements. The excitability of cells was media dependent (B27 > N2 > SCM). Action potential profiles of SFM cells were characterized by slower activation and prolonged after hyperpolarization which predisposed SFM cells to fire repetitively. The presence of three types of inward calcium currents was also revealed in SFM cells. These differential effects were primarily attributable to the media used with a secondary enhancement by the chemical differentiating agents used (dB-cAMP and forskolin).

Measurement of acetylcholine receptor function in microcircuit-coupled myoblasts.

An electrophysiological measurement principle for long-term, noninvasive monitoring of the nicotinic acetylcholine receptor (nAChR) function is described. The measurement is based on the ability to record agonist-induced depolarizations of clonal myoblasts that have formed high impedance seals with extracellular microcircuit electrodes. The technique appears promising for several types of assays and environmental monitoring applications.

Reversal of one-kidney, one-clip hypertension in rats. Effects on myocardial Na+, K(+)-ATPase, arterial Na(+)-K+ pump, arterial membrane potential, and plasma Na(+)-K+ pump inhibitory activity.

We have previously reported that myocardial microsomal Na+,K(+)-ATPase activity, arterial wall ouabain-sensitive 86Rb uptake, and arterial smooth muscle cell membrane potentials are decreased and plasma Na(+)-K+ pump inhibitory activity is increased in rats during the fifth week of one-kidney, one-clip hypertension. We here report measurements of these four parameters and blood pressure following unclipping. A new series of rats with one-kidney, one-clip hypertension was prepared. Each animal was paired with a one-kidney, sham-clipped (nonconstricting clip) control rat. After 5 weeks, the clips were removed. In the hypertensive animals arterial pressure promptly (within 3 h) returned to normal and remained at the level for 7 observation days. On the third day following unclipping, all four parameters were not significantly different from those in the paired control animals. On the seventh day following unclipping, three of the four parameters were not significantly different from those in the paired control animals and arterial ouabain sensitive 86Rb uptake was slightly increased relative to the value in the control animals. These studies invite further inquiry into the possible role of plasma Na(+)-K+ pump inhibitory activity in the genesis and maintenance of the hypertension in this model.

Effects of three sodium-potassium adenosine triphosphatase inhibitors.

Reports from several laboratories suggest the presence of an ouabainlike compound in plasma and various animal tissues, particularly during acute volume expansion and in low-renin hypertension. It has been hypothesized that this compound, through inhibition of the Na(+)-K+ pump, can constrict blood vessels, enhance vasoconstriction in response to agonists, increase cardiac contractility, raise blood pressure, and cause natriuresis/diuresis and therefore is implicated in the pathophysiology of the low-renin, volume-expanded type of hypertension. However, so far, only two steroid Na(+)-K+ pump inhibitors (namely, a bufodienolide derivative [resibufogenin], obtained from toad skin and plasma and a factor with the same carbon, oxygen, and hydrogen content as ouabain obtained from the plasma of volume-expanded humans) have been purified and structurally characterized. To determine whether such endogenous Na(+)-K+ pump inhibitors can in fact produce the above effects on the cardiovascular and renal systems, we infused commercially available bufalin (aglycone, identical to resibufogenin except for one H+), ouabain, and ouabagenin (aglycone) at equimolar doses in normotensive rats. Relative to ouabain, bufalin produced significantly greater dose-dependent increases in blood pressure, left ventricular rate of pressure change, heart rate, and excretion of urinary volume and sodium. Ouabagenin was without effect on any of these parameters. These data indicate that a Na(+)-K+ pump inhibitor can cause an increase in blood pressure despite potent diuretic and natriuretic effects and that, in rats, bufalin is much more potent in this respect than ouabain or ouabagenin.

Action potential-induced fluorescence changes resolved with an optical fiber carrying excitation light.

With the use of a single, implantable, optical fiber, to excite fluorescence and detect changes from voltage-sensitive dyes, transmembrane potential changes were measured without the need for a clear line-of-sight path between the excitation light, the tissue, and the detector. In a previous study, we were required to use signal averaging and could detect only cardiac action potentials from frog. In the present study we improved this system so that unaveraged cardiac action potentials were resolved with high fidelity, and action potentials from single nerve axons were detected. Endeavors to optimize the signal-to-noise ratio resulted in the selection of a larger core fiber with a rounded tip, styryl dyes, and filters based upon fluorescence spectra of the dyes when bound to membrane (rather than in solution). The frog gave signals nearly comparable in magnitude and signal-to-noise ratio to those seen with systems that use a fluorescence microscope. Action potential-induced signals could be detected in single lobster axons with the intracellular injection of a dye. The improvement in the signal-to-noise ratio allowed the use of a reduced-intensity excitation illumination which produced less bleaching of the dye.

Effect of increased dietary calcium on the development of reduced renal mass saline hypertension in rats.

A diet fortified with calcium carbonate has been reported to reduce blood pressure in low-renin and salt-sensitive hypertensive patients. We have therefore examined the effect of increased dietary calcium on the development of reduced renal mass-saline hypertension in rats, a classical, low-renin, volume, and sodium-dependent model of hypertension. Rats with 70-75% reduction in renal mass were divided into experimental and control groups. The experimental rats were fed a sodium-free diet supplemented with calcium carbonate (2.0% calcium) and drank 1% saline for 5 weeks. Control rats consumed the salt-free diet and drank 1% saline for the same period. In control rats, as previously observed, blood pressure progressive increased from a control value of 120.0 +/- 1.2 to 174.2 +/- 1.2 mm Hg by the fifth week. In contrast, in the calcium-supplemented rats the development of hypertension was significantly attenuated; the blood pressure only increased from 117.0 +/- 1.2 to 134.0 +/- 3.8 mm Hg by the fifth week. This was associated with a 30% decrease in saline intake by the fifth week, with proportionate decreases in urine volume and sodium excretion but not potassium excretion. Urinary magnesium excretion increased. No such changes were seen in control rats. At the end of the treatment period, plasma levels of sodium, potassium, calcium, creatinine, BUN, and protein were not different, but plasma chloride and magnesium were lower in experimental rats; vascular smooth muscle cell membrane potentials were also not different. These data show that dietary calcium carbonate can attenuate the development of reduced renal mass-saline hypertension in the rat, possibly in part by altering sodium and water intake.

Pulmonary mechanical responses to cholinesterase inhibitor.

Lung static and dynamic compliances, and lung and upper airway resistances were measured in pentobarbital-anesthetized dogs before and after intravenous administration of 2 LD50 of the organophosphate cholinesterase inhibitor pinacolyl methylphosphonofluoridate (GD), followed by 1 mg of atropine 8 min later. Dynamic compliances and resistances were estimated by a linear regression model and by a Fourier analysis technique, with the two methods giving comparable results. GD caused a maximum increase in lung resistance of about 20 times control values, and about an 80% decrease in lung dynamic compliance. Frequency dependence of lung compliance and resistance was increased by GD administration. Following GD administration, upper airway opening pressure increased, indicating the presence of laryngospasm. Upper airway resistance during the latter portion of the breath, when the airway was open, decreased after GD administration, concurrent with the increase in carinal pressure that occurred as the result of increased lung impedance. These results suggest that the GD-induced decrease in upper airway resistance was due to passive distension of the upper airway. Physiological deadspace decreased by a maximum of about 65% following GD administration. Administration of atropine resulted in a prompt and almost complete reversal of all of the GD-induced effects on pulmonary mechanical properties and ventilation. The results of this study suggest that the major pulmonary mechanical effects of GD in the dog are caused by constriction of smooth muscle at different levels of the respiratory tract.

Sustained antihypertensive effect of chronic oral administration of 6-iodo-amiloride, a sodium channel blocker, in spontaneously hypertensive rats.

We have previously shown that a 10-min intravenous infusion of 6-iodo-amiloride, an analogue of the sodium channel blocker amiloride, causes a sustained decrease in blood pressure in two genetic models of hypertension, spontaneously hypertensive rats (SHR) and Dahl salt-sensitive (DS) rats. In contrast, the same infusion produced only a transient decrease in blood pressure in two renal models of hypertension, viz. one-kidney, one clip, and reduced renal mass-saline rats. With these findings, we suggested that 6-iodo-amiloride has potential both as a diagnostic probe and as a therapeutic agent in genetic models of hypertension. The aim of the present study was to examine the effectiveness of 6-iodo-amiloride as a long-term antihypertensive agent and determine the mechanism of its antihypertensive action. We administered 6-iodo-amiloride to SHR for 4 weeks in the drinking fluid (tap water). The treatment with 6-iodo-amiloride caused a significant decrease in blood pressure but had no effect on urine volume or urinary excretion of sodium and potassium. These data strongly suggest that 6-iodo-amiloride is an effective long-term antihypertensive agent in genetic types of hypertension.

Effects of triiodothyronine on resting membrane potential of primary cultured rat submandibular gland cells.

The effect of triiodothyronine (T3) on the resting membrane potential was measured in primary cultured rat submandibular gland cells. The resting membrane potential was 29.5 +/- 0.71 mV. The hormone T3, at concentrations of 10(-9) M or greater, hyperpolarized the cells 5.8 mV (p less than 0.05). Hyperpolarization was complete within 24 hours. Ouabain (1 mM) depolarized the cells 5.9 mV. Cells exposed to T3 and ouabain had the same membrane potential as cells treated with ouabain alone. These data suggest that the hyperpolarization observed can be, in part, attributed to triiodothyronine-induced synthesis of (Na-K)-adenosine triphosphatase.

Humoral sodium transport inhibitor in acute volume expansion and low renin hypertension.

This review summarizes our bioassay methods for determining the level of humoral sodium pump inhibiting factor after acute volume expansion in experimental animals and humans, and in low renin experimental and human essential hypertension. In brief, ouabain-sensitive 86Rb uptake and membrane potential in blood vessels from normal animals are measured after incubation in plasma supernate from experimental subjects and animals and their respective controls. The data show that humoral sodium pump inhibitor is elevated after acute volume expansion in normal animals (dogs and rats) and in normal humans. The level of inhibitor is also elevated in patients with low renin essential hypertension and in experimental animals with low renin, volume-dependent types of hypertension, namely, one-kidney, one wrapped hypertension in dogs, and one-kidney, one clip and reduced renal mass-saline hypertension in rats. Humoral sodium pump inhibiting factor inhibits the Na+-K+ pump in the cardiovascular system. Such inhibition by other means (hypokalemia, cardiac glycosides) activates the system. Therefore, we also discuss the possible role of humoral sodium pump inhibitor in low renin volume-dependent hypertension.

Effects of 6-iodo-amiloride, a sodium channel blocker, on cardiovascular parameters in spontaneously hypertensive and Wistar-Kyoto rats.

6-Iodo-amiloride, an analogue of the sodium channel blocker amiloride, was infused intravenously for 10 min in anaesthetized Okamoto spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats in doses ranging from 0.08 to 0.38 mg/100 g body weight. Systemic arterial blood pressure and urine flow were measured for 120 min. In SHR, 6-iodo-amiloride produced a prompt, sustained, dose-dependent decrease in pressure. The lower doses were associated with increased urine flow and sodium excretion, while higher doses were not. Paradoxically, in WKY all doses produced a small dose-independent sustained increase in pressure and were associated with diuresis and natriuresis. 6-Iodo-amiloride had no effect on cardiac output, dP/dt or heart rate in isolated working hearts from SHR or WKY. However, addition of 6-iodo-amiloride to physiological salt solution bathing an isolated Wistar rat tail artery produced hyperpolarization of impaled vascular smooth muscle cells. These studies show that 6-iodo-amiloride is a vasodilatory antihypertensive agent in SHR, and that this can be associated with natriuresis and diuresis.

Determinants of alveolar ventilation during high-frequency transtracheal jet ventilation in dogs.

The effectiveness of transtracheal jet ventilation is a function of gas delivery pressure (drive pressure), duty cycle (insufflation time/total cycle time), and respiratory frequency. Nine dogs, anesthetized with sodium pentobarbital, were ventilated through a cricothyrotomy cannula using a controller that allowed separate setting of drive pressure, duty cycle, and frequency. PaO2 and PaCO2 were measured after achieving steady-state gas exchange at 15 to 22 different combinations of drive pressure, duty cycle, and frequency in each dog. There were slight increases in PaCO2 and larger decreases in PaO2 as frequency was increased from 10 to 200 cycle/min. Increases in drive pressure and duty cycle resulted in reduced PaCO2 and increased PaO2. Multiple linear regression showed good correlation between PaCO2 and drive pressure, duty cycle, and frequency. The distribution of air flow between alveolar and physiologic dead space, upper airway leakage, and entrainment was determined for each set of conditions. Changes in alveolar ventilation corresponding to the blood gas changes resulted from interaction of dead-space ventilation and upper airway leakage, which varied with breath duration. Decreases in leakage during short breaths tended to compensate for the increased fractional dead-space ventilation at high frequency, thus minimizing the effects of frequency changes on gas exchange.