Howard J. Bryant

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.

Effect of 810 nm light on nerve regeneration after autograft repair of severely injured rat median nerve.

Destruction of large segments of peripheral nerves results in chronic loss of sensation and paralysis. For this type of severe injury, the defect can be bridged by nerve grafts. However, even with state‐of‐the‐art microsurgical techniques, there is minimal recovery of sensation and motor function. Light therapy (LT) has been shown to improve functional outcome after surgical intervention to repair injured nerves using different techniques. Our objective was to investigate the effect of LT on peripheral nerve regeneration and function after severe median nerve injury and microsurgical autologous nerve graft repair using fibrin glue.

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).

The development and evaluation of a low-cost microdensitometer for use with the 2-deoxy-D-glucose method of functional brain mapping.

The conversion of a standard laboratory compound microscope into a microdensitometer for use in the 2-deoxy-D-glucose autoradiographic method of functional mapping in the brain is described. A solid-state detector was attached to the camera port of a Zeiss Universal Microscope and minor modifications to the microscope optics were made to produce a microdensitometer with a field of view 0.15 mm in diameter. Details are presented showing the modifications to the microscope which do not permanently destroy its functional ability to perform as a viewing microscope. A simple electronic circuit is presented to digitally display the output of the photo detector. Calibration of the instrument in terms of optical density or 2-deoxy-D-glucose activity is also described. The primary design goal was the construction of a simple, reliable, inexpensive microdensitometer that could be assembled by laboratory personnel. This densitometer should allow laboratories on modest budgets to have access to quantitative methods for the study of brain functional activity at a cost considerably less than the price of a commercial microdensitometer.

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.

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.

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.

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.

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

Summary1. We report that NG108-15 (neuroblastoma × 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), subthreshold 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.

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 pento-barbital, 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.