Properties of sodium and potassium channels of the squid giant axon far below 0 degrees C

Squid giant axon could be excited in concentrated glycerol solutions containing normal concentrations of electrolytes, when osmolalities of solutions inside and outside the axon were matched. These glycerol solutions did not freeze at the temperature as low as -19 degrees C. The nerve excitation in these solutions were observed at this low temperature. The excitation process at this low temperature was slowed down and time constants of the excitation kinetics were several hundredfold larger than those in normal seawater at 10 degrees C, under which temperature the squid habituated. The temperature coefficients for the electrophysiological membrane parameters under this condition were larger than those in normal seawater above 0 degrees C. The Q10 value for the conduction velocity was 2.0 and that of the duration of the action potential was around 8.5. The time course of the membrane currents was also slowed with the Q10 value of around 5 and the magnitude decreased with the Q10 value of around 2 as the temperature was lowered. The Q10 values for the kinetics of the on process of the Na-channel were around 4.5 and were almost the same as those of the off process of the Na-channel in the wide range of the temperature below 0 degrees C. The Q10 value of the on process of K-channel was around 6.5 and was larger than those for Na-channel. The Q10 values increased gradually as the temperature was lowered.     

Properties of sodium and potassium channels of the squid giant axon far below 0°C

Summary Squid giant axon could be excited in concentrated glycerol solutions containing normal concentrations of electrolytes, when osmolalities of solutions inside and outside the axon were matched. These glycerol solutions did not freeze at the temperature as low as –19°C. The nerve excitation in these solutions were observed at this low temperature. The excitation process at this low temperature was slowed down and time constants of the excitation kinetics were several hundredfold larger than those in normal seawater at 10°C, under which temperature the squid habituated. The temperature coefficients for the electrophysiological membrane parameters under this condition were larger than those in normal seawater above 0°C. The Q₁₀ value for the conduction velocity was 2.0 and that of the duration of the action potential was around 8.5. The time course of the membrane currents was also slowed with the Q₁₀ value of around 5 and the magnitude decreased with the Q₁₀ value of around 2 as the temperature was lowered. The Q₁₀ values for the kinetics of the on process of the Na-channel were around 4.5 and were almost the same as those of the off process of the Na-channel in the wide range of the temperature below 0°C. The Q₁₀ value of the on process of K-channel was around 6.5 and was larger than those for Na-channel. The Q₁₀ values increased gradually as the temperature was lowered.     

Temperature effects on the Na and Ca currents in rat and hedgehog ventricular muscle

Cardiac transmembrane potentials and Na and Ca currents were recorded at different temperatures in rat and hedgehog ventricular muscle. At 35 degrees C in both species resting potential was about -80 mV and upstroke velocity (Vmax) of the action potential above 100 V/s. The shape of the action potential in hedgehog ventricular cells at 35 degrees C was similar to that in the rat showing a fast repolarization phase. When temperature was decreased, the membrane resting potential depolarized and action potential amplitude and Vmax declined. In rat ventricular cells at 10 degrees C, the resting potential was about -40 to -50 mV and Vmax was reduced to about 5 V/s. In hedgehog ventricular cells, however, the transmembrane potentials and Vmax were better maintained at low temperature. Phase 3 of the action potential was markedly prolonged below 20 degrees C in hedgehog but not in rat ventricular cells. When temperature was decreased to 10 degrees C the availability curve of the Na current shifted toward more negative potentials and ICa.peak declined in rat ventricular cells. In hedgehog cardiac preparations, the Na current was less influenced by the cooling and ICa.peak did not change very much at low temperatures. A transient inward current usually considered to induce cardiac arrhythmias could be recorded in rat ventricular cells below 20 degrees C but not in hedgehog preparations. These features of hedgehog cardiac membranes may contribute to the cold tolerance and the resistance to ventricular fibrillation during the hypothermia in mammalian hibernators.     

Permeability of human blood platelets to glycerol

The permeability of human platelets to glycerol was determined at 37 degrees C, 25 degrees C, and 0 degrees C from the rate of change of cell volume after abrupt addition of 0.5 mol/liter glycerol in phosphate-buffered saline. Intracellular water volume was measured employing both tritiated water and a photometric method. Intracellular glycerol was measured employing tritiated glycerol. The glycerol permeability coefficient derived from the tracer cell volume data was 4.0 +/- 0.7 X 10(-7) cm/s at 37 degrees C, and 1.1 +/- 0.4 X 10(-7) cm/s at 25 degrees C, and the photometric data gave a permeability coefficient of 5.4 +/- 0.4 X 10(-7) cm/s at 37 degrees C. The activation energy between 23 degrees C and 37 degrees C for glycerol permeation was 19.8 kcal/mol. The cells were virtually impermeable to glycerol at 0 degrees C. The minimum intracellular water volume attained after the addition of 0.5 mol/liter glycerol at 37 degrees C determined by the photometric method was 47.8% of normal water volume, whereas the minimum water volume calculated assuming that glycerol exerted its full osmotic effect (i.e., sigma = 1) was 45.6%. The reflexion coefficient was therefore assumed to be unity. Neither method of cell volume determination could be used with 1 or 2 mol/liter glycerol: adequate separation of the cells from the labeled medium could not be achieved in the tracer method; in the photometric method, it was apparent that transmittance (660 nm) was influenced by one or more variables in addition to cell volume.     

Radial propagation of muscle action potential along the tubular system examined by potential-sensitive dyes

Isolated single (Xenopus) muscle fibers were stained with a non-permeant potential-probing dye, merocyanine rhodanine (WW375) or merocyanine oxazolone (NK2367). When the fiber was massively stimulated, an absorption change (wave a), which seemed to reflect the action potential, occurred. Simultaneous recording of optical changes and intracellular action potentials revealed that the time-course of wave a was slower than the action potential: the peak of wave a was attained at 1 ms, and the peak of action potential was reached at 0.5 ms after the stimulation. This difference suggests that wave a represents the potential changes of the whole tubular membrane and the surface membrane, whereas the action potential represents a surface potential change. This idea was substantiated by recording absorption signals preferentially from the surface membrane by recording the absorption changes at the edge of the fiber. Wave a obtained by this method was as quick as the intracellular action potential. The value of radial conduction velocity of action potential along the T system, calculated by comparing the action potential with wave a, was 6.4 cm/s at 24.5 degrees C, in fair agreement with González-Serratos (1971. J. Physiol. [Lond.]. 212:777-799). The shape of wave a suggests the existence of an access delay (a conduction delay at the orifice of the T system) of 130 microseconds.     

Rapid mechanical and thermal changes in the garfish olfactory nerve associated with a propagated impulse.

Mechanical and thermal changes associated with a propagated nerve impulse were determined using the garfish olfactory nerve. Production of an action potential was found to be accompanied by swelling of the nerve fibers. The swelling starts nearly at the onset of the action potential and reaches its peak at the peak of the action potential. There is a decrease in the length of the fibers while an impulse travels along the fibers. The time-course of the initial heat was determined at room temperature using heat-sensors with a response-time of 2-3 ms. Positive heat production was found to start and reach its peak nearly simultaneously with the action potential. The rise in temperature of the nerve was shown to be 23 (+/- 4) mu degrees C. In the range between 10 degrees and 20 degrees C, the temperature coefficient of heat production is negative, primarily due to prolongation of the period of positive heat production at low temperatures. The amount of heat absorbed during the negative phase varies widely between 45 and 85% of the heat evolved during the positive phase. It is suggested that both mechanical and thermal changes in the nerve fibers are associated with the release and re-binding of Ca-ions in the nerve associated with action potential production.     

Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature.

We have established stably transfected HEK 293 cell lines expressing high levels of functional human ether-a go-go-related gene (HERG) channels. We used these cells to study biochemical characteristics of HERG protein, and to study electrophysiological and pharmacological properties of HERG channel current at 35 degrees C. HERG-transfected cells expressed an mRNA band at 4.0 kb. Western blot analysis showed two protein bands (155 and 135 kDa) slightly larger than the predicted molecular mass (127 kDa). Treatment with N-glycosidase F converted both bands to smaller molecular mass, suggesting that both are glycosylated, but at different levels. HERG current activated at voltages positive to -50 mV, maximum current was reached with depolarizing steps to -10 mV, and the current amplitude declined at more positive voltages, similar to HERG channel current expressed in other heterologous systems. Current density at 35 degrees C, compared with 23 degrees C, was increased by more than twofold to a maximum of 53.4 +/- 6.5 pA/pF. Activation, inactivation, recovery from inactivation, and deactivation kinetics were rapid at 35 degrees C, and more closely resemble values reported for the rapidly activating delayed rectifier K+ current (I(Kr)) at physiological temperatures. HERG channels were highly selective for K+. When we used an action potential clamp technique, HERG current activation began shortly after the upstroke of the action potential waveform. HERG current increased during repolarization to reach a maximum amplitude during phases 2 and 3 of the cardiac action potential. HERG contributed current throughout the return of the membrane to the resting potential, and deactivation of HERG current could participate in phase 4 depolarization. HERG current was blocked by low concentrations of E-4031 (IC50 7.7 nM), a value close to that reported for I(Kr) in native cardiac myocytes. Our data support the postulate that HERG encodes a major constituent of I(Kr) and suggest that at physiological temperatures HERG contributes current throughout most of the action potential and into the postrepolarization period.     

Effect of ouabain on mechanical and electrical properties of rat and guinea pig hearts at 180 C.

The effects of ouabain 10(-6) M on rat and guinea pig hearts have been studied at 18 degrees C, in order to reduce almost fully both the Na+, K+-dependent ATPase activity and the ouabain induced inhibition of this enzyme. In isolated guinea pig hearts the positive inotropic response to ouabain obtained at 32 degrees C disappeared at 18 degrees C. On the contrary, the contractile strength of rat hearts was slightly reduced by ouabain and in the same manner at both temperatures. Current and voltage clamp experiments carried out at 18 degrees C in ventricular fibres revealed that ouabain 10(-6) M decreased both the action potential overshoot and the fast sodium current in rat and guinea pig, by reduction of the membrane sodium conductance. Ouabain did not change the calcium current in guinea pig preparations, whereas in rat heart muscle this current was reduced. The effects of ouabain on both the action potential plateau and outward repolarizing current indicated some inconsistencies from preparation to preparation and cannot therefore be considered as significant. The persistence of the ouabain induced alterations of g Na (in rat and guinea pig) and calcium current (in rat) at 18 degrees C supports the hypothesis of two ouabain cell receptors in heart muscle.     

Antagonist effect of DMSO on the cryoprotection ability of glycerol during cryopreservation of buffalo sperm

The objective of the present study was to investigate the synergistic effect of DMSO and glycerol added at various temperatures on the post-thaw quality of buffalo sperm. Pooled ejaculates from four Nili-Ravi buffalo bulls were divided into 18 aliquots and extended (1:10) in Tris-citric acid extender differing in glycerol:DMSO ratios (0:0, 0:1.5, 0:3; 3:0, 3:1.5, 3:3; and 6:0, 6:1.5, 6:3, respectively; %, v:v) either at 37 or 4 degrees C. Semen was packaged in 0.5 mL French straws and frozen in a programmable cell freezer. Thawing was performed at 37 degrees C for 50s. Post-thaw motion characteristics, plasma membrane integrity and acrosome morphology of buffalo sperm were determined using computer-assisted semen analyzer (CASA), hypoosmotic swelling (HOS) assay and phase-contrast microscopy, respectively. Glycerol (6%) in extender yielded better post-thaw sperm motility, velocities (straight-line and average path), plasma membrane integrity, and normal acrosomes (P<0.05). Post-thaw sperm motility and plasma membrane integrity declined in the presence of DMSO (P<0.01). The addition of glycerol (6%) at 37 degrees C yielded better post-thaw sperm motility, plasma membrane integrity and velocities than addition at 4 degrees C (P<0.05). In conclusion, glycerol is still an essential cryoprotectant for buffalo sperm. The addition of DMSO antagonized the cryoprotection ability of glycerol and reduced the post-thaw quality of buffalo sperm. Furthermore, 6% glycerol added at 37 degrees C, provided better cryoprotection to the motility apparatus and plasma membrane integrity of buffalo sperm.     

Survival of mint shoot tips after exposure to cryoprotectant solution components

Many plant species can be cryopreserved by treating shoot tips with complex cryoprotectant solutions before rapidly cooling them to liquid nitrogen temperatures. Plant vitrification solution 2 (PVS2), a commonly selected cryoprotectant, can be lethal with extended exposure times. To determine potentially toxic combinations, we have exposed mint shoot tips to one-, two-, three-, and four-component solutions of PVS2 chemicals (30% glycerol, 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.4 M sucrose) at 0 and 22 degrees C. Overall, solution exposures at 22 degrees C were more damaging than exposures at 0 degree C. Solutions with glycerol, particularly in combination with ethylene glycol and dimethyl sulfoxide, were also damaging. Cryoprotectant solutions PGluD (10% PEG8000, 10% glucose, and 10% dimethyl sulfoxide) and PVS3 (50% glycerol, 50% sucrose) were less damaging than PVS2 at 22 degrees C. When plant cryoprotectants are characterized on a toxicological and biophysical basis, less damaging cryoprotectant solutions could be developed.     

Temperature-dependent inotropic action of A23187 on guinea-pig ventricular muscles

The effects of the Ca2+ ionophore, A23187, on the contraction and membrane action potential of the isolated guinea-pig papillary muscle were examined at various temperatures (30-16 degrees C) and compared to those of isoprenaline and a high calcium medium. A23187 caused a marked positive inotropic effect with a significant prolongation of the action potential duration at an early repolarization phase but not a late repolarization phase at normal temperature (30 degrees C). Such an inotropic effect was completely abolished at low temperature (16 degrees C) where a marked positive inotropic effect of isoprenaline (5 X 10(-8) M) and a high calcium medium (6.2 mM) still remained. These results suggest that the cardiac responsiveness to A23187 was sensitive to a low temperature at which a membrane lipid phase transition may occur.     

Sodium channel inactivation in the crayfish giant axon. Must channels open before inactivating?

Experiments on sodium channel inactivation kinetics were performed on voltage-clamped crayfish giant axons. The primary goal was to investigate whether channels must open before inactivating. Voltage-clamp artifacts were minimized by the use of low-sodium solutions and full series resistance compensation, and the spatial uniformity of the currents was checked with a closely spaced pair of electrodes used to measure local current densities. For membrane potentials between -40 and +40 mV, sodium currents decay to zero with a single exponential time-course. The time constant for decay is a steep function of membrane potential. The time-course of inactivation measured with the double-pulse method is very similar to the decay of current at the same potential. Steady-state inactivation curves measured with different test pulses are identical. The time-course of double pulse inactivation shows a lag that roughly correlates with the opening of sodium channels, but detailed comparisons with the time course of the prepulse current suggest that it is not strictly necessary for channels to open before inactivating. Measurements of the potential dependence of the integral of sodium conductance area also inconsistent with the simplest cases of models in which channels must open before inactivating.     

On the mechanism of injury to slowly frozen erythrocytes.

When cells are frozen slowly in aqueous suspensions, the solutes in the suspending solution concentrate as the amount of ice increases; the cells undergo osmotic dehydration and are sequestered in ever-narrowing liquid-filled channels. Cryoprotective solutes, such as glycerol, reduce the amount of ice that forms at any specified subzero temperature, thereby controlling the buildup in concentration of those other solutes present, as well as increasing the volume of the channels that remain to accommodate the cells. It has generally been thought that freezing injury is mediated by the increase in electrolyte concentration in the milieu surrounding the cells, rather than reduction of temperature or any direct action of ice. In this study we have frozen human erythrocytes in isotonic solutions of sodium chloride and glycerol and have demonstrated a correlation between the extent of damage at specific subzero temperatures, and that caused by the action at 0 degrees C of solutions having the same composition as those produced by freezing. The cell lysis observed increased directly with glycerol concentration, both in the freezing experiments and when the cells were exposed to corresponding solutions at 0 degrees C, showing that the concentration of sodium chloride alone is not sufficient to account quantitatively for the damage observed. We then studied the effect of freezing in anisotonic solutions to break the fixed relationship between solute concentration and the volume of the unfrozen fraction, as described by Mazur, P., W. F. Rall, and N. Rigopoulos (1981. Biophys. J. 653-675). We confirmed their experimental findings, but we explain them differently. We ascribe the apparently dominant effect of the unfrozen fraction to the fact that the cells were frozen in, and returned to, anisotonic solutions in which their volume was either less than, or greater than, their physiological volume. When similar cell suspensions were subjected to a similar cycle of increase and then decrease in solution strength, but in the absence of ice (at 20 degrees C), a similar pattern of hemolysis was observed. We conclude that freezing injury to human erythrocytes is due solely to changes that occur in the composition of their surrounding milieu, and is most probably mediated by a temporary leak in the plasma membrane that occurs during the thawing (reexpansion) phase.     

Effects of cryoprotective agents and freezing on abdominal ganglia of aplysia.

Isolated Aplysia depilans abdominal ganglia were exposed to 10 and 20% dimethylsulphoxide (Me₂SO) or glycerol at room temperature. Results indicate that Me₂SO induced an irreversible depression of extracellularly recorded ganglionic spontaneous spike generation while glycerol proved to be non-toxic. Intracellular recordings of individual nerve cell spontaneous activity during exposure to the cryoprotective agents were obtained in a few preliminary experiments. Both Me₂SO and glycerol induced a decrement in the nerve cell membrane potential. The main difference between the action of the two cryoprotectants was in the rate and the amount of depolarization, both being higher in the case of Me₂SO exposure.The Aplysia abdominal ganglia were frozen to ?20 °C and to ?196 °C. In all but one ganglia frozen to ?20 °C, including the preparations frozen in the absence of any cryoprotective agent, functional recovery was obtained after thawing. However, only the application of 20% glycerol improved the recovery of the preparations to a significant extent. In ganglia protected with 20% glycerol a full recovery of the action potential amplitude and frequency was obtained. In ganglia protected with 20% glycerol intracellular recordings of individual nerve cells demonstrated spontaneous spike activities before freezing and after thawing.No functional recovery was observed in ganglia frozen to ?196 °C in the absence of a cryoprotective agent. While in most preparations frozen with a cryoprotectant spontaneously generated spikes were recorded after thawing. However, the action potential frequency and amplitude were significantly depressed. It is concluded that further investigation is required to improve the freezing technique so that Aplysia ganglia may be preserved at low temperatures. It is suggested that intracellular exploration of the effects of cryoprotectants and freezing on identified nerve cell membrane may prove to be useful in future investigations.     

Multicopy suppressors of phenotypes resulting from the absence of yeast VDAC encode a VDAC-like protein.

The permeability of the outer mitochondrial membrane to most metabolites is believed to be based in an outer membrane, channel-forming protein known as VDAC (voltage-dependent anion channel). Although multiple isoforms of VDAC have been identified in multicellular organisms, the yeast Saccharomyces cerevisiae has been thought to contain a single VDAC gene, designated POR1. However, cells missing the POR1 gene (delta por1) were able to grow on yeast media containing a nonfermentable carbon source (glycerol) but not on such media at elevated temperature (37 degrees C). If VDAC normally provides the pathway for metabolites to pass through the outer membrane, some other protein(s) must be able to partially substitute for that function. To identify proteins that could functionally substitute for POR1, we have screened a yeast genomic library for genes which, when overexpressed, can correct the growth defect of delta por1 yeast grown on glycerol at 37 degrees C. This screen identified a second yeast VDAC gene, POR2, encoding a protein (YVDAC2) with 49% amino acid sequence identity to the previously identified yeast VDAC protein (YVDAC1). YVDAC2 can functionally complement defects present in delta por1 strains only when it is overexpressed. Deletion of the POR2 gene alone had no detectable phenotype, while yeasts with deletions of both the POR1 and POR2 genes were viable and able to grow on glycerol at 30 degrees C, albeit more slowly than delta por1 single mutants. Like delta por1 single mutants, they could not grow on glycerol at 37 degrees C. Subcellular fractionation studies with antibodies which distinguish YVDAC1 and YVDAC2 indicate that YVDAC2 is normally present in the outer mitochondrial membrane. However, no YVDAC2 channels were detected electrophysiologically in reconstituted systems. Therefore, mitochondrial membranes made from wild-type cells, delta por1 cells, delta por1 delta por2 cells, and delta por1 cells overexpressing YVDAC2 were incorporated into liposomes and the permeability of resulting liposomes to nonelectrolytes of different sizes was determined. The results indicate that YVDAC2 does not confer any additional permeability to these liposomes, suggesting that it may not normally form a channel. In contrast, when the VDAC gene from Drosophila melanogaster was expressed in delta por1 yeast cells, VDAC-like channels could be detected in the mitochondria by both bilayer and liposome techniques, yet the cells failed to grow on glycerol at 37 degrees C. Thus, channel-forming activity does not seem to be either necessary or sufficient to restore growth on nonfermentable carbon sources, indicating that VDAC mediates cellular functions that do not depend on the ability to form channels.     

Effect of amino acids on goat cauda epididymal sperm cryopreservation using a chemically defined model system

Studies were carried out to analyze the cryoprotecting efficacy of several amino acids by use of a chemically defined synthetic medium (modified Ringer's solution) and goat cauda epididymal sperm as the model system. Motile goat cauda sperm dispersed in the synthetic medium were subjected to a freezing protocol in a computer-controlled bio-freezer, cooling 0.25 degrees C x min(-1) to 5 degrees C, 5 degrees C x min(-1) to -20 degrees C, and 20 degrees C x min(-1) to -100 degrees C, prior to being plunged into liquid nitrogen. In the absence of amino acids, sperm cells completely lost their flagellar motility. Of all the amino acids tested, l-alanine showed maximal cryoprotection potential. l-Alanine at 135 mM offered optimum cryoprotection potential: recovery of sperm forward motility and total motility were 14 +/- 2% and 19 +/- 2%, respectively. l-Glutamine, l-proline, and glycine at optimum concentration (100-150 mM) cryopreserved approx. 11-17% total motility of the sperm cells, whereas amino acids such as l-arginine, l-lysine, and l-histidine offered little cryoprotection (0-5%) to the cells. Increasing the amino acid concentration beyond the optimum level sharply decreased the recovery of the sperm motility, which therefore showed a biphasic cryoprotection profile. Addition of amino acids enhanced (approx. 7-10%) the cryoprotection efficacy of the well-known cryoprotectants glycerol and a combination of glycerol and dimethyl sulfoxide. The presence of glycerol caused a marked reduction (from 100-150 mM to 20-70 mM levels) in the optimal cryoprotective concentration of the amino acids. The combined cryoprotecting action of glycerol, dimethyl sulfoxide, and amino acids provided motility recovery as high as 52%. The observation that amino acids and dimethyl sulfoxide had an additive effect in augmenting the cryoprotecting potential of glycerol suggests that the mechanism of their action is different from that of glycerol. This cocktail of cryoprotectants may be useful for cryopreservation of semen of various species.     

The initial inward current in spherical clusters of chick embryonic heart cells

The rapid inward sodium current in spherical clusters of 11-d-old embryonic chick heart cells, ranging in size between 65 and 90 micron diameter, was studied using the two-microelectrode voltage-clamp technique. Using these preparations, it was possible to resolve the activation phase of the rapid inward current for potentials negative to -25 mV at 37 degrees C. The rapid inward current exhibited a voltage and time dependence similar to that observed in other excitable tissues. It was initiated at potential steps more positive than -45 mV. The magnitude of the current reached its maximum value at a potential of approximately -20 mV. The measured reversal potential was that predicted by the Nernst equation for sodium ions. The falling phase of the current followed a single exponential time-course with a time constant of inactivation, tau h, ranging between 2.14 ms at -40 mV and 0.18 ms at -5 mV. The time constant of inactivation, tau h, determined by a single voltage-step protocol was compared to the constant, tau c, determined by a double voltage-step protocol and no significant different between the two constants of inactivation was found. Furthermore, the time constants of inactivation and reactivation at the same potential in the same preparation were similar. The results of this study demonstrate that the sodium current of heart cells recorded at 37 degrees C can be described by Hodgkin-Huxley kinetics with speeds approximately four times faster than the squid giant axon at 15 degrees C.     

Effects of cooling and warming rate to and from -70 degrees C, and effect of further cooling from -70 to -196 degrees C on the motility of mouse spermatozoa

We have previously reported high survival in mouse sperm frozen at 21 degrees C/min to -70 degrees C in a solution containing 18% raffinose in 0.25 x PBS (400 mOsm) and then warmed rapidly at approximately 2000 degrees C/min, especially under lowered oxygen tensions induced by Oxyrase, a bacterial membrane preparation. The best survival rates were obtained in the absence of glycerol. The first concern of the present study was to determine the effects of the cooling rate on the survival of sperm suspended in this medium. The sperm were cooled to -70 degrees C at rates ranging from 0.3 to 530 degrees C/min. The survival curve was an inverted "U" shape, with the highest motility occurring between 27 and 130 degrees C/min. Survival decreased precipitously at higher cooling rates. Decreasing the warming rate, however, decreased survivals at all cooling rates. The motility depression with slow warming was especially evident in sperm cooled at the optimal rates. This fact is consistent with our current view that the frozen medium surrounding sperm cells is in a metastable state, perhaps partly vitrified as a result of the high concentrations of sugar. The decimation of sperm cooled more rapidly than optimum (>130 degrees C/min), even with rapid warming, is consistent with the induction of considerable quantities of intracellular ice at these rates. When glycerol was added to the above medium, motilities were also dependent on the cooling rate, but they tended to be substantially lower than those obtained in the absence of glycerol. The minimum temperature in the above experiments was -70 degrees C. When sperm were frozen to -70 degrees C at optimum rates, lowering the temperature to -196 degrees C had no adverse effect.     

Effect of temperature on the electrical and mechanical activity of lizard ventricular myocardium

Intracellular action potentials and isometric twitches were recorded from lizard ventricles electrically driven at 20 and 4 beats/min and submitted to temperatures changes between 10.5 and 21 degrees C. It was found that cooling induced a depolarization of the diastolic membrane potential ER, which below 15 degrees C exceeded that predictable for a diffusion potential; on the contrary, during the recovery from hypothermia ER underwent a transitory hyperpolarization. Other effects of the low temperature were a decrease of the maximum rate of depolarization, a lengthening of both the action potential duration and the time to peak contraction, an increase of the strength of contraction, in the hearts driven at 20/min it became apparent also an increase of the action potential overshoot. The hypothesis is discussed that the positive inotropic effect of low temperatures may be due not only to a slowing down of the repolarization of the action potential, but also to an increase of the slow inward current intensity.     

Interactions of cooling rate, warming rate, glycerol concentration, and dilution procedure on the viability of frozen-thawed human granulocytes

Difficulties in the successful freezing of human granulocytes could lie at two levels. One is that critical cryobiological variables have not yet been identified, the other is that the inconsistent results may be due to unusual biological aspects of the cell. This paper is concerned with the former. A prerequisite for the successful freezing of mammalian cells is the ability of the cell to tolerate cryoprotective levels of additive. The additive studied here was glycerol. Based on fluorescent staining with fluorescein diacetate, we found that 1 and 2 M concentrations are in fact chemically toxic at 22 degrees C. Superimposed on this toxicity is some osmotic sensitivity to the removal of the additive by other than slow dilution. The dilution procedure was selected on the basis of computer modeling of the osmotic response of the cells. The model requires a value for the permeability coefficient for glycerol. The value (4 X 10(-5) cm/min) was obtained by measuring the rate of increase of the volume of cells in hyperosmotic glycerol. The response of human granulocytes to freezing to -196 degrees C and thawing in 1 or 2 M glycerol was not unusual. The optimum cooling rate was 1-3 degrees C/min, and cooling at 10 degrees C/min or faster was especially deleterious if warming was slow (1 degree C/min) rather than rapid (188 degrees C/min). The FDA assay showed that some 75% of the cells survived freezing and thawing at optimum rates in 1 or 2 M glycerol; and some 50-60% remained viable after the glycerol had been removed, provided that the cells remained at 0 degrees C. However, granulocytes normally function at 37 degrees C. Because chemotaxis is considered a good assay of normal function, we developed a modified procedure capable of discriminating among random migration, enhanced random migration (chemokinesis), and directed cell migration (true chemotaxis). When frozen-thawed-diluted cells were incubated for 60 min at 37 degrees C, their survival, based both on the FDA assay and on the chemotaxis assay, was zero. In fact, a prior exposure of the cells to 2 M glycerol at 0 degrees C, even in the absence of freezing, resulted in a rapid loss in FDA viability when the cells were subsequently held at 37 degrees C for up to 60 min. Survivals based on FDA are usually reported to be considerably higher than survivals based on functional assays such as chemotaxis or phagocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)