Cooperative interaction as the physical basis of the negative stiffness in hair cell stereocilia.

A recent report confirmed that stiffness of the stereocilia can be negative, as predicted by the Howard-Hudspeth model. According to this model, the mechanotransducer channel's gating not only reduces the stereociliary stiffness, but can alter its sign as well. The basic assumptions of this model do not include cooperativity in channel gating. Here we consider two possible explanations for the observed negative stiffness. If the stereocilia have a special structure so that microscopic displacement can be imposed on each channel by controlling the bending of the bundle, negative stiffness can occur without channel cooperativity. If such a microscopic condition cannot be imposed by a macroscopic manipulation, an additional physical process, such as cooperativity in channel gating, is required to explain negative stiffness.

A positive-feedback model for the loss of acetylcholine in Alzheimer's disease.

We describe a two-component positive-feedback system that could account for the large reduction of acetylcholine that is characteristic of patients with Alzheimer's disease (AD). One component is beta-amyloid-induced apoptosis of cholinergic cells, leading to a decrease in acetylcholine. The other component is an increase in the concentration of beta-amyloid in response to a decrease in acetylcholine. We describe each mechanism with a differential equation, and then solve the two equations numerically. The solution provides a description of the time course of the reduction of acetylcholine in AD patients that is consistent with epidemiological data. This model may also provide an explanation for the significant, but lesser, decrease of other neurotransmitters that is characteristic of AD.

The choline-leakage hypothesis for the loss of acetylcholine in Alzheimer's disease.

We present a hypothesis for the loss of acetylcholine in Alzheimer's disease that is based on two recent experimental results: that beta-amyloid causes leakage of choline across cell membranes and that decreased production of acetylcholine increases the production of beta-amyloid. According to the hypothesis, an increase in beta-amyloid concentration caused by proteolysis of the amyloid precursor protein results in an increase in the leakage of choline out of cells. This leads to a reduction in intracellular choline concentration and hence a reduction in acetylcholine production. The reduction in acetylcholine production, in turn, causes an increase in the concentration of beta-amyloid. The resultant positive feedback between decreased acetylcholine and increased beta-amyloid accelerates the loss of acetylcholine. We compare the predictions of the choline-leakage hypothesis with a number of experimental observations. We also approximate it with a pair of ordinary differential equations. The solutions of these equations indicate that the loss of acetylcholine is very sensitive to the initial rate of beta-amyloid production.

Anomalous effects of hormone fragments on the measurement of parathyroid hormone by radioimmunoassay.

One of the basic assumptions underlying the use of radioimmunoassay and other competitive protein-binding assays is the homogeneity of the antigen or ligand. This assumption is not valid for the measurement of parathormone (PTH) because of the presence of fragments. Hence, there is a potential for errors and high variability in the measurement of parathyroid hormone (PTH) by radioimmunoassay. Even though region-specific radioimmunological and immunoradiometric assays for PTH measurement can overcome some of the difficulties caused by the presence of hormone fragments, the possibility for serious measurement errors still remains. We therefore examined experimentally and by modeling the impact of fragments on the estimation of the concentration of a highly purified intact bovine parathyroid hormone by radioimmunoassay. Our experimental results show that the mere presence of fragments can lead to a significant underestimation or overestimation of the amount of the intact hormone. The results have been simulated by a model in which fragments bind to the antibodies, thus competing with the intact hormone, and to the intact hormone as well, thereby reducing the amount of free intact hormone in competition with the radioligand. This work indicates that it may be preferable to consider alternative methods, other than competitive protein-binding assays, for the measurement of secreted PTH.

Role of calcium channels in parathyroid hormone secretion.

Experimental evidence exists for the presence of parathyroid cell membrane calcium channels that respond to plasma calcium. In previous reports, the effects of various calcium channel agents on PTH secretion have revealed conflicting results. To resolve some of these inconsistencies, we have compared the pure calcium channel agonist, (+)202-791, and its antagonistic enantiomer (-)202-791 with other calcium channel agents--verapamil, nifedipine, and (+)Bay-K-8644. The agonist (+)202-791 enhanced 45Ca+2 uptake and decreased PTH secretion, while the antagonist (-)202-791 decreased 45Ca+2 uptake and increased PTH secretion. The calcium channel appears coupled to a G-protein as indicated by pertussis toxin treatment of the cells. The enantiomers (+/-)202-791 had little effect on intracellular cAMP production suggesting that the calcium channel may not be responsible for the previously observed calcium-mediated changes in cAMP. The antagonist (-)202-791 increased the phosphorylation of a 60-kd protein. The enantiomers (+/-)202-791 did not alter the effect of depolarizing concentrations of potassium on PTH secretion. Our results suggest that calcium channels provide a pathway for the movement of calcium across the plasma membrane and that this pool of calcium regulates, at least in part, PTH secretion.

beta-Amyloid polypeptide increases calcium-uptake in PC12 cells: a possible mechanism for its cellular toxicity in Alzheimer's disease.

Calcium-uptake into PC12 cells was measured by incubation with 45Ca after the cells were exposed for 24 h to beta-amyloid peptide(1-40) at concentrations between 0 and 46 microM. The rate of influx of 45Ca into PC12 cells was constant for the first 10 min. For 46 microM beta-amyloid peptide(1-40), the rate of influx was about 1,300 ions/s/microns 2 and the number of cells decreased significantly. There was no significant decrease in cell number when cells were exposed to beta-amyloid in calcium-free medium. These results indicate that beta-amyloid increases calcium uptake into PC12 cells, and suggest that the increased uptake is responsible for the toxicity of beta-amyloid in PC12 cells.

beta-Amyloid increases choline conductance of PC12 cells: possible mechanism of toxicity in Alzheimer's disease.

When beta-amyloid-(1-40) is added to PC12 cells, there is an increase in choline conductance that is proportional to the beta-amyloid concentration. If a similar effect occurs in cholinergic brain cells of Alzheimer's disease patients, the intracellular choline concentration would be reduced, leading to a decrease in the production of acetylcholine. This could explain the reduced level of acetylcholine that has been found in post-mortem brain tissue of Alzheimer's disease patients.

Regulation of parathyroid hormone secretion.

Calcium is the most important physiological regulator of PTH secretion. Peak PTH secretion occurs at an intracellular calcium concentration of about 200 nM, regardless of the extracellular calcium concentration. We suggest, therefore, that intracellular calcium concentration is a regulator of PTH secretion that maintains calcium homeostasis. Other factors may be responsible for modulation of the intracellular calcium concentration, ultimately modulating PTH secretion. The "paradoxical" nature of the dependence of PTH secretion on the calcium concentration may be explained by considering PTH secretion to be unusual in a quantitative, rather than a qualitative, fashion. A possible mechanism for the control of PTH secretion by intracellular calcium, which involves calcium-activated potassium channels, is proposed. The parathyroid cell plasma membrane contains several sensors or channels by means of which the cell senses extracellular calcium. It is not clear whether these entities are coupled to each other or whether they function independently. Guanine nucleotide regulatory proteins are transducers of extracellular signals, including calcium. Several other second messengers that influence PTH secretion have also been described, but possible interactions between these messengers have not yet been determined.

High concentration of inositol 1,4,5-trisphosphate in sea urchin sperm.

We measured inositol 1,4,5-trisphosphate (InsP3) content of sea urchin gametes by using a specific protein binding assay, and found that a spermatozoon contains 4 x 10(-19) to 1 x 10(-18) moles of InsP3 before the acrosome reaction. Since the acrosome reaction has previously been shown to increase the InsP3 content of sperm severalfold, our measurement indicates that a spermatozoon contains at least 2 x 10(-18) moles of InsP3 at fertilization, corresponding to a concentration in the spermatozoon of about 1 mM. The threshold for activation of eggs by injection of InsP3 dissolved in a much larger volume of solution has been found to be about 3 x 10(-18) moles, corresponding to a concentration in the injectate of 1 microM. This suggests that sea urchin sperm may contain enough InsP3 to activate eggs. With an electroporation method, we also showed that sperm extract acts on eggs only from inside, consistent with a primary messenger role for InsP3.

The biphasic calcium dose-response curve for parathyroid hormone secretion in electropermeabilized adult bovine parathyroid cells.

We have used the method of electropermeabilization to measure the dependence of PTH secretion on internal calcium concentration in adult bovine parathyroid cells. The dose-response curve is biphasic, with a peak at 10(-7) M calcium. This result differs significantly from the dose-response curves that have been determined by this method for many other secretory systems, where secretion requires much more calcium and the dependence of secretion on calcium is monotonic. Guanine nucleotide analogs did not modify the calcium dose-response curve of PTH secretion in the electropermeabilized parathyroid cells. The unique properties of adult parathyroid cell secretion are analogous to the unique properties of parathyroid calcium-activated potassium channels, which differ from calcium-activated potassium channels of other cells in that they open at unusually low calcium concentrations and have a peak open probability at about 10(-7) M calcium. We suggest that the opening of these channels in secretory vesicles is required for secretion.

Potassium Channels in Motor Cells of Samanea saman: A Patch-Clamp Study.

Leaflet movements in Samanea saman are driven by the shrinking and swelling of cells in opposing (extensor and flexor) regions of the motor organ (pulvinus). Changes in cell volume, in turn, depend upon large changes in motor cell content of K(+), Cl(-) and other ions. We performed patch-clamp experiments on extensor and flexor protoplasts, to determine whether their plasma membranes contain channels capable of carrying the large K(+) currents that flow during leaflet movement. Recordings in the "whole-cell" mode reveal depolarization-activated K(+) currents in extensor and flexor cells that increase slowly (t((1/2)) = ca. 2 seconds) and remain active for minutes. Recordings from excised patches reveal a single channel conductance of ca. 20 picosiemens in both cell types. The magnitude of the K(+) currents is adequate to account quantitatively for K(+) loss, previously measured in vivo during cell shrinkage. The K(+) channel blockers tetraethylammonium (5 millimolar) or quinine (1 millimolar) blocked channel opening and decreased light- and dark-promoted movements of excised leaflets. These results provide evidence for the role of potassium channels in leaflet movement.

Unusual calcium-activated potassium channels of bovine parathyroid cells.

Parathyroid cells have unusual responses to an increase in the external calcium concentration--a reduction in secretion and a depolarization. In an attempt to explain this behavior, we have studied voltage-clamped inside-out patches from bovine parathyroid cells. We found a potassium-selective channel that requires internal calcium to open and has a 175-pS conductance. This channel differs from calcium-activated potassium channels that have been found in other cells in that it closes when the internal calcium concentration is increased above about 160 nM. This channel can account for the depolarization that occurs in parathyroid cells in high-calcium solutions. It may also account for the reduced secretion in high-calcium solutions.

Evidence for anion channels in secretory vesicles.

Secretory vesicles from bovine neurohypophysis were reconstituted into lipid bilayers. Electrical measurements on the lipid bilayers under voltage clamp demonstrated the presence of channels that are permeable to chloride ions, are blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and are slightly voltage dependent. When several different membrane fractions were used for the reconstitution, the probability of finding channels correlated with the fraction of secretory vesicle membrane in the membrane fraction, indicating that the secretory vesicles are the source of the channels. The observed anion channel can provide a pathway for the anion transport that has previously been described for secretory vesicles. The secretory vesicle anion channel may play a role in calcium-induced secretion.

Evidence for interactions between batrachotoxin-modified channels in hybrid neuroblastoma cells.

Current records from voltage-clamped membrane patches containing two batrachotoxin-modified sodium channels were analyzed to determine whether these channels are identical and independent. In most two-channel patches, the experimentally observed probabilities that zero, one, or two channels are open differ from the binomial distribution, demonstrating that the two channels are nonidentical or nonindependent or both. From the same current records, we also determined the rate for the transition from two open channels to one open channel and for the transition from one open channel to zero open channels. These data are consistent with closing rates for the two channels that are equal and independent. Both probability and closing rate data can be fit by a model wherein the channels are identical, the closing rates are independent, and the opening rate is greater when the other channel is closed than when it is open. The implications of this model for analyzing noise spectra and current variance are examined.

A model for exocytosis based on the opening of calcium-activated potassium channels in vesicles.

It is proposed that the role of calcium in calcium-induced exocytosis is to open Ca-activated K channels present in vesicle membranes. The opening of these channels coupled with anion transport across the vesicle membranes would result in an influx of K and anions, increasing the osmotic pressure of the vesicles. For those vesicles situated very close to the cell plasma membrane, this would lead to fusion with the membrane and exocytosis of the vesicle contents. This model can account for facilitation and other key properties of transmitter release. In addition, the model predicts that vesicles with a higher transmitter content, and hence higher initial osmotic pressure, would be preferentially discharged. The model also predicts that a faster response can be obtained for small vesicles than for large vesicles, providing a rationale as to why neurotransmitters, which must be released quickly, are packaged in small vesicles.

Injection of a soluble sperm fraction into sea-urchin eggs triggers the cortical reaction.

Fertilization membranes form around unfertilized sea-urchin eggs after microinjection of a soluble spermatozoa fraction isosmotic with seawater. This demonstrates that the spermatozoon contains a chemical that triggers an increase in cytosolic calcium, leading to exocytosis of cortical granules. It also demonstrates that the triggering mechanism does not require an externally-activated egg-membrane process. Further experiments show that the chemical trigger is not calcium.

Ion channels in plasmalemma of wheat protoplasts.

The patch-clamp technique was used to study passive movements of ions through the plasmalemma of wheat leaf protoplasts. This method overcomes the problems inherent in conventional electrophysiological study of plant cells. Changes in conductance were recorded in patches excised from the plasmalemma. Two types of patches were observed: (i) regions of low channel density, where discrete single-channel currents could be resolved and conductance ranged from 10 to 200 picosiemens and (ii) regions of high channel density, where single-channel currents could not be resolved and conductance was on the order of a few nanosiemens. The results indicate a striking similarity between animal and plant cell membranes in the basic phenomena of transport. Moreover, the approach used constitutes a new degree of refinement in the study of processes of regulation, pathology, and toxicity in plants.

Gating kinetics of batrachotoxin-modified sodium channels in neuroblastoma cells determined from single-channel measurements.

We have observed the opening and closing of single batrachotoxin (BTX)-modified sodium channels in neuroblastoma cells using the patch-clamp method. The conductance of a single BTX-modified channel is approximately 10 pS. At a given membrane potential, the channels are open longer than are normal sodium channels. As is the case for normal sodium channels, the open dwell times become longer as the membrane is depolarized. For membrane potentials more negative than about -70 mV, histograms of both open-state dwell times and closed-state dwell times could be fit by single exponentials. For more depolarized potentials, although the open-state histograms could still be fit by single exponentials, the closed-state histograms required two exponentials. This data together with macroscopic voltage clamp data on the same system could be accounted for by a three-state closed-closed-open model with transition rates between these states that are exponential functions of membrane potential. One of the implications of this model, in agreement with experiment, is that there are always some closed BTX-modified sodium channels, regardless of membrane potential.

Maternal effects and temperature-sensitive period of mutations affecting embryogenesis in Caenorhabditis elegans.

We have used standard tests to investigate the nature of gene expression of a new set of temperature-sensitive mutants defining 30 emb genes (essential for embryogenesis) in the nematode Caenorhabditis elegans. The mode of gene expression as determined by progeny tests for parental effects divides the genes into four classes. For 18 genes maternal gene expression is necessary and sufficient for normal embryogenesis; for 2 genes zygotic expression is necessary and sufficient; for 7 genes either maternal or zygotic expression is sufficient; for 3 genes both maternal and zygotic expression are necessary. One mutant displayed partial paternal sufficiency. The results of temperature-shift experiments define two "execution stages," corresponding to the limits of the temperature-sensitive period (TSP), and indicate the nature and the time of action or synthesis of the gene products. Most of the maternally expressed genes have very early execution stages indicating translation before fertilization, but some are temperature sensitive late in embryogenesis. Early execution stages for 2 zygotically necessary genes demonstrate that the zygotic genome can be active in the earliest stages of embryogenesis. All taken together, the mode of gene expression, TSP, and arrest stage (terminal phenotype) allow us to classify functionally and begin to order the genes essential for embryogenesis. The results indicate a preeminent role for maternal genes and gene products in embryogenesis, in agreement with the results of others.