D. L. Kunze

Dual effects of dihydropyridines on whole cell and unitary calcium currents in single ventricular cells of guinea‐pig.

We studied the effects of dihydropyridine Ca channel ligands (DHPs), mainly nitrendipine and Bay K8644, on whole cell and single channel Ca currents on single myocytes isolated from the adult guinea‐pig ventricle. Nitrendipine had dual effects, stimulatory or inhibitory, depending upon the membrane potential. At low frequencies (less than 0.03 Hz) and negative holding potentials (‐90 mV or more), nitrendipine increased the Ca currents in a dose‐dependent manner. The dose‐response curve was best fitted by a Langmuir adsorption isotherm model which was the sum of two independent one‐to‐one drug‐receptor sites with median effective doses (ED50S) of 1.0 X 10(‐9) M and 1.4 X 10(‐6) M respectively. When the membrane potential was held at ‐30 mV or less, nitrendipine inhibited the Ca currents, also in a dose‐dependent manner. The dose‐response curve was fitted by a single binding site model having a median inhibitor concentration (IC50) of 1.5 X 10(‐9) M. At holding potentials between ‐70 and ‐40 mV, nitrendipine produced mixed effects on Ca currents; an increase occurred initially and this was followed by a decrease. When rundown was excluded, Bay K8644 showed only stimulatory effects on the Ca currents between holding potentials of ‐120 and ‐30 mV. When the test potential was zero or +10 mV the Ca currents reached peak values and the dose‐response curve was best fitted by a single binding site model having an ED50 of 3 X 10(‐8) M. When the effects were measured at negative test potentials of ‐30 to ‐10 mV, the curve was best fitted by a two‐site model with ED50S of 3 X 10(‐9) and 9 X 10(‐7) M. At the single Ca channel level the stimulatory effect of nitrendipine was due to an increased probability that a Ca channel which had opened once would reopen, a reduction in records without activity and an increase in the mean open time. There were no changes in unit conductance. Inhibitory effects were due to a large increase in nulls. At lower concentrations the main effect of Bay K8644 was an increase in the probability of opening. At doses above 10(‐6) M, a pronounced increase in the open time was observed. The effects we observed are attributed to at least two sites for DHP related to Ca channels; one with high affinity and one with a lower affinity. The low affinity site mediates a stimulatory effect due to greatly prolonged openings.(ABSTRACT TRUNCATED AT 400 WORDS)

Rate-dependent changes in extracellular potassium in the rabbit atrium.

We measured levels of potassium ion in the extracellular space of isolated superfused rabbit atria continuously with double-barreled microelectrodes of which on barrel was a K+ liquid ion-exchanger microelectrode and the other a potential-sensing micropipette. Increases in heart rate resulted in transient increases in extracellular potassium ([K+]0). When the quiescent atrium was stimulated the maximal increase was 0.4 mM at rates of 60/min, 0.7 mM at 90/min, 0.9 mM at 120/min, 1.3 mM at 200/min, and 1.8 mM at 300/min. The increase was not sustained during continued stimulation but declined toward prestimulation levels. When the stimulus was terminated the extracellular potassium activity decreased below bathing solution values by 0.2 mM after 60/min, 0.5 mM after 90/min, 0.7 mM ater 120/min, 0.9 mM after 200/min, and 1.0 mM after 300/min and subsequently returned to a value equal to that of the bathing solution. The magnitude of the decline in extracellular potassium activity during prolonged stimulation was markedly decreased when the bathing solution contained either zero potassium, ouabain, LiCl, or a decreased Po2 such that an elevation in [K+]0 persisted during stimulation. Moreover, the reduction in [K+]0 that followed the cessation of stimulation also was inhibited. These results support a role of the Na-K pump in maintaining extracellular potassium activity during changes in cardiac rate.

Rapid resetting of low pressure vagal receptors in the superior vena cava of the rat.

The discharge characteristics of mechanoreceptors located in the left superior vena cava were determined in an in vitro preparation developed from the rat. Two classes of receptors with afferent fibers in the vagus were identified on the basis of their response to steps of pressure. Slowly adapting receptors (n = 18) discharged as long as pressure was above threshold and exhibited a biphasic decline in discharge frequency in response to a pressure step. There was a rapid, initial decline in discharge frequency, complete within 15 seconds, followed by a slow, gradual decline complete within 5 minutes. Rapidly adapting receptors (n = 15) discharged irregularly or ceased firing after 5–10 seconds of a step increase in pressure. Following a 15-minute increase in perfusion pressure from 0 to 5 mm Hg, the threshold pressure of the slowly adapting receptors was increased and maximal discharge frequency and slope were decreased, whereas, in the rapidly adapting receptors, the threshold pressure was increased but maximal discharge frequency and slope were unchanged. Additional perfusion of the vessel at 5 mm Hg for up to 60 minutes produced no further increase in the degree of resetting. The resetting was reversible with discharge returning to control levels after a 15- to 25-minute return to 0 mm Hg. To determine whether the resetting of discharge following the acute pressure increase reflected a change within the receptor or an alteration of the vessel wall due to sustained distension, we examined the passive mechanical properties of the superior vena cava. Pressure-radius relationships were determined with an ocular micrometer. A 15- minute increase in pressure had no significant effect upon the mechanical properties of the vessel. Rapid resetting in these low pressure mechanoreceptors occurs with no discernible alteration in the vessel wall.

Sodium sensitivity of baroreceptors. Reflex effects on blood pressure and fluid volume in the cat.

Thesodium sensitivity of the carotid sinus baroreceptor reflex was demonstrated in anesthetized cats. Decreases in carotid perfusate sodium concentration |Na+]o of 5% and 12.5% attenuated the depressor response to increased carotid sinus pressure. At constant sinus pressure, increases in systemic pressure and heart rate were produced when the carotid perfusate |Na+]o was switched from 145 mM (control) to 138 or 127 miu, and this was accompanied by an increase in urine volume. The changes in |Na+]0 had no effect on the static pressure-volume relationship of the carotid sinus, indicating an action on the baroreceptors that already has been confirmed directly by studies of baroreceptor discharge. Chemoreceptor involvement, examined by recording chemoreceptor discharge, was negligible. The increase in urine volume was not dependent on intact renal sympathetic nerves, and renal denervation alone produced an increase in urine volume during control perfusion. At 87.5% carotid sinus |Na + |(, the urine volume increased from 28 ± 2.0 (SEM) to 31 ± 3.0 μl/min in innervated kidneys and decreased from 32 ± 3.1 to 27 ± 4.6 in denervated kidneys. Urine sodium excretion was higher in denervated kidneys (1.61 ± 0.21 /u.Eq/min compared to 1.40 ± 0.14 μEq/min for innervated kidneys) and was unchanged in innervated kidneys (1.36 ± 0.18 αEq/min) as carotid perfusate sodium was decreased. However, sodium excretion from denervated kidneys was increased (1.86 ± 0.3 μEq/min) as carotid perfusate sodium was lowered. Renal sympathetic discharge also was increased as carotid sinus sodium was reduced. Thus, reducing extracellular [Na+]0 by as little as 5% produces significant baroreceptor reflexes, including a rise in blood pressure, and diuresis with no change in total sodium excretion. These studies indicate a role of the sodium sensitivity of baroreceptors in the regulation of blood pressure, body fluid volume, and body sodium.

Calcium and magnesium sensitivity of the carotid baroreceptor reflex in cats.

The systemic blood pressure response to changes in carotid sinus pressure has been examined during variations in the calcium and magnesium concentration of the perfusate of the isolated sinus. The threshold for the blood pressure response was increased by 16 ± 0.8% when calcium was raised from 1.1 mM (control) to 2.2 mM and increased by 27 ± 1.8% when calcium was raised to 3.3 mM. The threshold was reduced by 21 ± 0.7% in zero calcium and 13 ± 0.8% in 0.55 mM Ca 2+. The sensitivity (slope of the linear portion of the pressure response curve) of the reflex was not significantly changed except at 3.3 mM Ca2+ where it was reduced by 16 ± 2.9%. Magnesium had directionally similar effects of a lesser magnitude. Earlier work described the reduced sensitivity and threshold of the baroreceptor reflex when sodium concentration in the carotid perfusate was reduced. The present study shows that reducing calcium attenuates the effect of lowered sodium and increasing calcium augments it. These results are discussed in terms of possible ionic influences on the receptor potential and spike generation, Circ Res 45: 815-821, 1979

Changes in extracellular potassium activity in response to decreased pH in rabbit atrial muscle.

The extracellular and intracellular potassium (K+) actitivies of isolated superfused rabbit atrial muscle wtre measured using K+-sensitive liquid ion exchanger microelectrodes. When the pH of the bathing medium was decreased from 7.5 to 6.8. intracellular K+ activity fell and extracellular K + activity rose from a mean control level of 3.6 mm to a new steady state level of 3.9 mM after I hour. When the pH was further decreased to 6.1, extracellular Inactivity increased to a mean of 4.9 mM. Following the change in pH, the increase in extracellular K+ activity occurred over a period of 30-40 minutes at which time a stable talue was reached and maintained for the next hour. On return to normal pH the extracellular K+ activity returned to control with a time constant of 20 minutes or less. Measurements of infracellular K+ activity over 1 hour showed a mean loss of 3 mM at pH 6.8, and a mean loss of 8 mM at pH 6.1. The loss was reversible within 20 minutes of return to control pH. The increase in extracellular K+ activity was accompanied by a decrease in resting membrane potential as well as decreases in maximum dv/dt and overshoot of the action potential. The action potential contour underwent complex changes consisting of decrease in the plateau and a prolongation of the time to full repolarization.

Single Calcium Channels and Their Inactivation

Inactivation of single Ca channels in snail neurons was examined to test the idea that entering Ca ions react directly with the channel to produce this effect. Simulations of specific models were used for comparison with the experimental data. The Ca-dependent model predicts time-dependent changes in the single-channel events which were not found experimentally. It is possible that Ca-dependent inactivation is mediated by a Ca-binding protein that is associated with but not part of the channel itself.

Ionic Activities in Identifiable Aplysia Neurons

This paper reports the values of K+, Na+ and Cl- activities measured with ion-selective microelectrodes in certain identifiable neurons in the abdominal ganglion of Aplysia californica (nomenclature according to Frazier, et al., 1967). These measurements allow calculation of the equilibria potentials of the three ions thought to be most important in regulating intracellular voltage. The ion-selective microelectrode technique makes such measurements possible in small cells; moreover these values can be compared amongst neurons which are anatomically and functionally distinct.

Dynamic discharge characteristics of low pressure receptors in the rat.

An in vitro preparation was used to examine the discharge of low pressure receptors in response to a pulsatile pressure stimulus. Both slowly adapting and rapidly adapting receptors were examined. After an increase in the mean level and/or dP/dt of a pulsatile pressure input, the discharge of slowly adapting receptors reached a steady state level within 2 minutes. An increase in mean pressure, with constant pulsatile amplitude and dP/dt, produced a sustained increase in the number of spikes/cycle, in the average number of spikes/second, and in the average frequency during the burst within the cycle. In slowly adapting receptors, an increase in dP/dt at a constant mean pressure and pulsatile amplitude produced a decrease in the number of spikes/cycle and an increase in both the average number of spikes/second and the frequency during the burst. Rapidly adapting receptors showed very different characteristics, responding to a pulsatile pressure input above threshold with an irregular discharge which averages approximately 1 spike/cycle at all mean pressure levels. Although rapidly adapting receptors transiently increased their discharge in response to an increase in mean pressure, within 60 seconds discharge returned to the pre-increase level. Rapidly adapting receptors continued to discharge with an average of 1 spike/cycle as dP/dt was increased. Since dP/dt was raised by increasing the frequency of the pulsatile input, the number of spikes/second increased. In the steady state, slowly adapting receptors transmit information reflecting mean pressure levels. They are also sensitive to the rate of change of pressure. Rapidly adapting receptors, on the other hand, are insensitive to mean pressure but do respond to the frequency of the stimulus.

pH Related Potassium Movements in Rabbit Atrium

A relationship between the distribution of hydrogen and potassium ions across cell membranes has been documented by numerous studies using both in vivo and in vitro preparations (Adler and Fray, 1977). Potassium appears to leave the cells of both skeletal and cardiac muscle when extracellular acidosis is produced and to enter the cells under conditions of extracellular alkalosis. There has as yet been no explanation for the mechanism of the exchange. We were particularly interested in this problem because a period of myocardial ischemia results in both an extracellular acidosis and a movement of potassium from the myocardial cells (Case et al., 1969). The specific question we asked was whether the decrease in extracellular pH could be the initiating factor in potassium loss. To investigate this, we chose an isolated rabbit atrial preparation which could be superfused with solutions of various composition while recording both intracellular and extracellular potassium activity.