Twenty-Seventh Annual Meeting February 13–16, 1983 Town & Country Hotel, San Diego, California

1983 Biophysical Journal  
We are using the dye arsenazo III to study the changes in internal Ca +, [Cail] in the neurosecretory bag cells of Aplysia. The bag cells undergo a period of spontaneous discharge (and secretion of egg laying hormone in the intact system) when treated with cyclic AMP analogs. A similar spontaneous discharge can be obtained with the potassium channel blocker TEA. We have pressure injected arsenazo III into isolated bag cells grown on culture dishes. The cells are transilluminated with white
more » ... in an inverted microscope. The light is picked up by a fiber optic probe and separated into its component wavelengths with a grating monochromator similar to one designed by Steve Smith (Yale University). The change in the %ifference in light transmitted at 70Q+15 nm and that at 660+15 nm gives a measure of change in [Ca)]. We have detected changes in [Ca) during a train of stimulated action potentials (A.P.s). Five A.P.s produce a differe+tial absorbance change of as much as 2.2 x 10 . Such measuremepts show that there is significant Ca influx during an A.P. and that the2e is potentiation of the Ca influx if the spacing of the A.P.s is less than about one second. [Ca +] usually ret f ns to its previous level within about 12 to 20 sec showing that the cell is able to buffer small Ca changes. The change in internal Ca during an A.P. can be greahly enhanced with TEA. 50 mM TEA allows a change in differential absorbance of as mucheas 3.8 x 10o with a single stimulated (much lengthened) A.P. We have also been able to detect Ca influx during spontaneous didcharges induced by N -n-butyl-8-(benzylthio)-cAMP and TEA or by TEA alone. Sizeable changes in [Cai] are evident during membrane depolarizations (both long and short). RF radiation, amplitude modulated at ELF, has been shown to cause changes in the efflux of calcium ions from brain tissue, in vitro. These responses occur in the absence of temperature rise in the samples, and occur only at certain modulation frequencies. We have examined the effects of ELF signals alone on calcium ion efflux in the following manner: the entire forebrain is removed from newly-hatched chickens and incubated for 30 minutes in a buffer containing 45Cal. After rinsing to remove any loosely-bound 45Ca++, the tissues are placed in test tubes containing unlabeled buffer and treated for 20 minutes either to an ELF signal or to a sham exposure. The buffer is then assayed for the presence of 45Ca++. Compared to the sham exposures, specific frequencies and intensities of ELF signals were able to cause enhanced efflux of 45Ca++ during the treatment period. We recently reported that 16-Hz signals within two intensity ranges, 5-7.5 and 35-50 Vp-p/m, could cause enhanced efflux while intensities outside these ranges, as well as 1-Hz and 30-Hz signals at 40 V/m, would not affect the efflux. We now demonstrate that 45-Hz and 50-Hz signals at intensities between 45 and 50 V/m can also induce enhanced efflux. The underlying mechanism and the physiological significance of the phenomenon are unknown. Membrane currents in immature (stage V and VI) Xenopus oocytes were studied using the two microelectrode voltage clamp technique. Oocytes were clamped to their approximate resting voltage (-70 or -80 mV). During step depolarizations, total membrane current showed a transient outward peak between -20 and +30 mV, with a maximum amplitude near 0 mV. At 0 mV, 210C and the normal external Ca concentration (1 mM), the peak occurred at about 0.3 seconds, and the steady state was reached after about 3 seconds. Removal of external Cl reversed the outward peak to a transient inward surge without affecting the shape of the steady state V-I relation. Tail currents recorded after the peak showed a component not present in those recorded at the steady state, and which inverted at about -25 to -30 mV. This was the approximate Eci in the normal external solution (87.5 mM Cl) as determined with Cl-selective microelectrodes. The reversal potential for tail current changed with the external Cl concentration as predicted by the Nernst relation. The transient outward peak seen in records of total membrane current thus appeared to be due to a Cl current. Removal of external Ca (Mg, Sr or Ba substitution), or addition of low concentrations of Ni, blocked the appearance of the peak outward current, and increasing the external Ca concentration increased its amplitude. These treatments did not affect the amplitude of the steady state currents. These experiments suggested that the activation of the Cl current was dependent on Ca entry. This Ca-dependent Cl current could be related to the activation potential of amphibian oocytes, which is Cl-sensitive and which can be elicited by Ca injection. (Supported by N.I.H. grant NS09012 to S. Hagiwara, and fellowship GM08676). Ca++ CHANNELS 60a Calcium current, ICa' was isolated under voltage clamp in axotomized Aplysia neurons and dialyzed Helix neurons. Intracellular TEA and/or Cs plus extracellular TEA and 4-aminopyridine blocked virtually all outward current at membrane potentials up to 0 mV. ICa exhibited currentdependent decay with an initial rapid, Ti, and a subsequent slow, T2, time constant toward a steady inward current, I>. Factors that reduce the current amplitude (i.e., partial Cd2+ block, previous Ca influx, reduced Vm) preferentially reduced the early peak ICa' while having less effect in reducing the late phases. These kinetics were accurately fitted with an equation based on a sixstate model that includes H-H m2 activation plus inactivation that depends on elevation of intracellular free Ca2+, Cai, resulting from the influx of Ca2+ carrying the current: ICa = gCa(Vm -ECa) rmm -(mOm )et/Tm]2 1/(1+K.S) in which K = efficacy of Ca2+ in inactivating Ca channels; S = .tt-PB)ICadt; and PB probability that Ca2+ entering will be lost to diffusion or buffering. The inactivation term 1/(1+K.S) represents the kinetics of Ca interacting with a receptor site to cause the inactivation of the channel. In this model the two phases of inactivation, Ti and T2, result from successive increments in Cai producing progressively smaller increments of inactivation, and from the reduction in current due to inactivation decreasing progressively the rate of increase of Cai with time. The disproportionate loss of the initial phase of current with diminished current flow results from the slower increase in Cai and thus slower, more uniform increase in the Ca-mediated inactivation. Cruralis tonic and twitch fibers from Rana Pipiens were exposed to a saline containing (mM): TEA methanesulphonate (MeSO3), 120; Ca MeSO3 10; and 3,4-diaminopyridine 5 (pH 7.3). In this solution gNa and gCl were eliminated and gK was greatly reduced. Contraction was blocked by 350 mM-sucrose. Experiments were performed at 18'C. Muscle fibers were current clamped with standard two microelectrode technique using 1 sec pulses. Membrane potential was held at -80 mV. Tonic and twitch fibers were identified by their passive electrical properties. Tonic fibers had a very large effective resistance (V./I.) of 54.8+ 7 MQ (X+S.E. n=21) resulting in a very large membrane time constant (Tm) of 440+70 sec n=8. In contrast, in twitch fibers V./I,=3.5+0.8 MQ n=10 and Tm=56+4 sec n=6. Tonic fibers elicited a slow action potential when depolarizing pulses were applied. This response had a threshold of about -40 mV reaching a peak amplitude of -10 mV at 0.5 sec and had a plateau phase of variable duration (1-10 sec) after termination of the pulse. It was blocked by 2 mM Cd++ and 5 mM Co-l. The three microelectrode voltage technique was used to study the underlying currents. The holding potential was -100 mV and 8 sec pulses of variable amplitude were delivered. Slow inward currents were detected at -60 mV reaching a maximum value at 0 mV and having a reversal potential of about 40 mV. In three fibers the main peak current measured at 0 mV was 25 pA/cm2. It had a peak time of 1.2 sec and decayed with a time constant of 3.3 sec. These currents were blocked by 2 mM Cd++. Ca currents in tonic fibers are much smaller than in twitch fibers. Sperelakis and Pappano (1969) showed that veratridine depolarized cultured heart cells to -5 mV and that this effect was prevented by TTX. In Na-free solution, veratridine hyperpolarized and lowered R , consistent with an increase in gK. Catterall and Luzdunski and their associates have confirmeW these observations, but attributed their results to the presence of silent fast Na channels, i.e. they assumed that veratridine can only act on the fast Na channels. To test this possibility, we have studied the effect of veratridine on Ca uptake into cultured heart cell monolayers. Veratridine (10 -10-4 M) stimulated Ca uptake into the heart cells in a dose-dependent manner. The half-maximal dose was 37 pM. The veratridine stimulation was independent of the age of the chick embryos from which the heart cells were taken, and was inhibited by TTX (3 vM). Since voung embryonic hearts have few or no functional fast Na channels (Sperelakis, N., Cardiol. Toxicol. 1: 39-108, 1981), the present results indicate that the action of veratridine is not dependent solely on fast Na channels. For example, veratridine may increase the resting Na permeability and/or open the slow channels. The increased Ca influx could then be a consequence of Cao:Nai exchange reation. In addition, veratridine stimulated Ca uptake into cat ileal smooth muscle, which possess only slow channels. It is concluded that veratridine does not act solely on fast Na channels, but also affects other types of ion channels. It is even possible that veratridine opens the voltage-independent, Ca-operated, non-specific Na-K channel (gNa K(Ca)), since this can account for its effects on gNA and gK. (Supported by HL-18711 and American Heart Association, Virginia affiliate). '. CHANT. Biophysical Journal vol. 41, 1983 Thyrotropin-releasing hormone or depolarizing concentrations of K+ have been shown to stimulate the release of prolactin and growth hormone from neoplastic GH3 pituitary cells, in the presence of extracellular calcium (Ca2+). GH3 cells like normal anterior pituitary cells are capable of generating Ca2+ action potentials in culture. In this study, the fluorescent properties of terbium (Tb3+) were used to detect and monitor the Ca2+ binding sites on GH3 cells. The Tb3+ fluorescence emission was enhanced with the binding of GH3 cells, accompanied by a red shift in its excitation maximum to resemble the excitation peak of the native cell fluorescence. The fluorescence enhancement of Tb3+ increased with increasing concentrations of GH3 cells. Scatchard plots of a fluorometric titration procedure revealed at least two classes of binding sites. The low and high affinity sites have apparent dissociation constants equal to 0.56 mM and 11 pm, respectively. The high affinity Tb3+ binding was displaced by Ca2+, but the more abundant low affinity site was not sensitive to Ca2 The data suggests that GH3 cells have a specific Ca2+ binding receptor on their plasma membrane. ila Ca++ CHANNELS r,I mV ([KC1] :143mM, [KCl].:400mM). Current noise was significantly greater when channels were open, particularly at negative potentials. Replacing KCI. with NaCl, tetraethylammonium chloride, or NH4Cl, or adding 1 mM 4-aminopyridine to the internal solution had little effect on channel properties. DNDS -inhibitable chloride net efflux from gramicidin-treated red cells can be divided into two components: one component that decreases hyperbolically with increasing Cl0, and a C10-independent component. Previously we have tentatively assigned the C10-inhibitable component to slippage and the C10-independent component to tunneling. Slippage refers to net transport mediated by the return conformational change of the unloaded transport site subsequent to translocation of the loaded transporter, and tunneling refers to the movement of the anion through the transport protein without a conformation change of the protein. Bromide and nitrate net efflux also exhibited anioninhibitable and -independent components. Both components were lqrger than for chloride net efflux: 230 and 300, respectively, compared to 50 mmoles (kg Hgb * min)'for anion-inhibitable efflux, and 20 and 50, respectively, compared to 10 mmoles (kg Hgbmin)1 for the independent component. Since slippage in this system is limited by the return reaction of the empty transporter, it is independent of the nature of the transported anion. The data, therefore, suggest that even at low extracellular anion concentrations net efflux is mainly accomplished by tunneling and not by slippage as previously assumed. This is also supported by experiments in which chloride net efflux was measured as function of Cli. At CIO=O net efflux was proportional to Cli between Cli=25 -200 mMM and became saturating above 300 mM. If net efflux under these conditions had been mostly by slippage one should have observed a nearly Cli-independent rate of chloride net efflux. It therefore appears that the proposed tunneling mechanism can account for a major portion of the anion net transport and that the slippage process is only a minor component.(Supported by NIH grant HL-28674)
doi:10.1016/s0006-3495(83)84425-7 fatcat:5fnwzrl5nvatplzs3kobya4wqe