The anatomy and physiology of the Drosophila larval neuromuscular junction were studied. 2. The dependence of muscle resting potentials on [K+]o and [Na+]o follows the Goldman-Hodgkin-Katz equation (PNa/PK=0-23). Chloride ions distribute passively across the membrane. 3. The mean specific membrane resistance of muscle fibres is 4-3 X 10(3) omega cm2, and the mean specific membrane capacitance is 7-1 muF/cm2. The muscle fibre is virtually isopotential. 4. Transmitter release is quantal. Both the miniature excitatory junctional potential and the evoked release follow the Poisson distribution. 5. Transmitter release depends on approximately the fourth power of [Ca2+]o. If Sr2+ replaces Ca2+, it depends on approximately the fourth power of [Sr2+]o. Mg2+ reduces transmitter release without altering the fourth power dependence on [Ca2+]o.

Mutations in SCN5A, the gene encoding the cardiac Na(+) channel, have been identified in 2 distinct diseases associated with sudden death: one form of the long-QT syndrome (LQT(3)) and the Brugada syndrome. We have screened SCN5A in a large 8-generation kindred characterized by a high incidence of nocturnal sudden death, and QT-interval prolongation and the "Brugada ECG" occurring in the same subjects. An insertion of 3 nucleotides (TGA) at position 5537, predicted to cause an insertion of aspartic acid (1795insD) in the C-terminal domain of the protein, was linked to the phenotype and was identified in all electrocardiographically affected family members. ECGs were obtained from 79 adults with a defined genetic status (carriers, n=43; noncarriers, n=36). In affected individuals, PR and QRS durations and QT intervals are prolonged (P<0.0001 for all parameters). ST segment elevation in the right precordial leads is present as well (P<0.0001). Twenty-five family members died suddenly, 16 of them during the night. Expression of wild-type and mutant Na(+) channels in Xenopus oocytes revealed that the 1795insD mutation gives rise to a 7.3-mV negative shift of the steady-state inactivation curve and an 8.1-mV positive shift of the steady-state activation curve. The functional consequence of both shifts is likely to be a reduced Na(+) current during the upstroke of the action potential. LQT(3) and Brugada syndrome are allelic disorders but may also share a common genotype.

Stomatal pores in leaves enable plants to regulate the exchange of gases with their environment. Variations of the pore aperture are mediated by controlled changes of potassium salt concentrations in the surrounding guard cells. The voltage-dependent gating of K(+)-selective channels in the plasma membrane (plasmalemma) of cell-wall-free guard cells (protoplasts) was studied at the molecular level in order to investigate the regulation of K(+) fluxes during stomatal movements. Inward and outward K(+) currents across the plasmalemma of guard cells were identified by using the whole-cell configuration of the patch-clamp technique. Depolarizations of the membrane potential from a holding potential of -60 mV to values more positive than -40 mV produced outward currents that were shown to be carried by K(+). Hyperpolarizations elicited inward K(+) currents. Inward and outward currents were selective for K(+) over Na(+) and could be partially blocked by exposure to extracellular Ba(2+). In cell-attached and excised membrane patches, previously identified K(+)-selective single channels in guard cells were studied. Averaging of single-channel currents during voltage pulses resulted in activation and deactivation kinetics that were similar to corresponding kinetics of inward and outward currents in whole cells, showing that K(+)-selective channels were the molecular pathways for the K(+) currents recorded across the plasmalemma of single guard-cell protoplasts. Estimates demonstrate that K(+) currents through the voltage-gated K(+) channels recorded in whole guard cells can account for physiological K(+) fluxes reported to occur during stomatal movements in leaves.

Na(+)/K(+)-ATPase as an energy transducing ion pump has been studied extensively since its discovery in 1957. Although early findings suggested a role for Na(+)/K(+)-ATPase in regulation of cell growth and expression of various genes, only in recent years the mechanisms through which this plasma membrane enzyme communicates with the nucleus have been studied. This research, carried out mostly on cardiac myocytes, shows that in addition to pumping ions, Na(+)/K+-ATPase interacts with neighboring membrane proteins and organized cytosolic cascades of signaling proteins to send messages to the intracellular organelles. The signaling pathways that are rapidly elicited by the interaction of ouabain with Na(+)/K(+)-ATPase, and are independent of changes in intracellular Na(+) and K(+) concentrations, include activation of Src kinase, transactivation of the epidermal growth factor receptor by Src, activation of Ras and p42/44 mitogen-activated protein kinases, and increased generation of reactive oxygen species by mitochondria. In cardiac myocytes, the resulting downstream events include the induction of some early response proto-oncogenes, activation of the transcription factors, activator protein-1 and nuclear factor kappa-B, regulation of a number of cardiac growth-related genes, and stimulation of protein synthesis and myocyte hypertrophy. For these downstream events, the induced reactive oxygen species and rise in intracellular Ca(2+) are essential second messengers. In cells other than cardiac myocytes, the proximal pathways linked to Na(+)/K(+)-ATPase through protein-protein interactions are similar to those reported in myocytes, but the downstream events and consequences may be significantly different. The likely extracellular physiological stimuli for the signal transducing function of Na+/K+-ATPase are the endogenous ouabain-like hormones, and changes in extracellular K+ concentration.

GABA is the principal inhibitory neurotransmitter in the mature brain, but during early postnatal development the elevated [Cl(-)](i) in immature neocortical neurones causes GABA(A) receptor activation to be depolarizing. The molecular mechanisms underlying this intracellular Cl(-) accumulation remain controversial. Therefore, the GABA reversal potential (E(GABA)) or [Cl(-)](i) in early postnatal rat neocortical neurones was measured by the gramicidin-perforated patch-clamp method, and the relative expression levels of the cation-Cl(-) cotransporter mRNAs (in the same cells) were examined by semiquantitative single-cell multiplex RT-PCR to look for statistical correlations with [Cl(-)](i). The mRNA expression levels were positively (the Cl(-) accumulating Na(+),K(+)-2Cl(-) cotransporter NKCC1) or negatively (the Cl(-) extruding K(+)-Cl(-) cotransporter KCC2) correlated with [Cl(-)](i). NKCC1 mRNA expression was high in early postnatal days, but decreased during postnatal development, whereas KCC2 mRNA expression displayed the opposite pattern. [Cl(-)](i) and NKCC1 mRNA expression were each higher in cortical plate (CP) neurones than in the presumably older layer V/VI pyramidal neurones in a given slice. The pharmacological effects of bumetanide on E(GABA) were consistent with the different expression levels of NKCC1 mRNA. These data suggest that NKCC1 may play a pivotal role in the generation of GABA-mediated depolarization in immature CP cells, while KCC2 promotes the later maturation of GABAergic inhibition in the rat neocortex.

The rates of different ATP-consuming reactions were measured in concanavalin A-stimulated thymocytes, a model system in which more than 80% of the ATP consumption can be accounted for. There was a clear hierarchy of the responses of different energy-consuming reactions to changes in energy supply: pathways of macromolecule biosynthesis (protein synthesis and RNA/DNA synthesis) were most sensitive to energy supply, followed by sodium cycling and then calcium cycling across the plasma membrane. Mitochondrial proton leak was the least sensitive to energy supply. Control analysis was used to quantify the relative control over ATP production exerted by the individual groups of ATP-consuming reactions. Control was widely shared; no block of reactions had more than one-third of the control. A fuller control analysis showed that there appeared to be a hierarchy of control over the flux through ATP: protein synthesis>> RNA/DNA synthesis and substrate oxidation>> Na+ cycling and Ca2+ cycling>> other ATP consumers and mitochondrial proton leak. Control analysis also indicated that there was significant control over the rates of individual ATP consumers by energy supply. Each ATP consumer had strong control over its own rate but very little control over the rates of the other ATP consumers.

Recent breakthroughs in the store-operated calcium (Ca(2+)) entry (SOCE) pathway have identified Stim1 as the endoplasmic reticulum Ca(2+) sensor and Orai1 as the pore forming subunit of the highly Ca(2+)-selective CRAC channel expressed in hematopoietic cells. Previous studies, however, have suggested that endothelial cell (EC) SOCE is mediated by the nonselective canonical transient receptor potential channel (TRPC) family, TRPC1 or TRPC4. Here, we show that passive store depletion by thapsigargin or receptor activation by either thrombin or the vascular endothelial growth factor activates the same pathway in primary ECs with classical SOCE pharmacological features. ECs possess the archetypical Ca(2+) release-activated Ca(2+) current (I(CRAC)), albeit of a very small amplitude. Using a maneuver that amplifies currents in divalent-free bath solutions, we show that EC CRAC has similar characteristics to that recorded from rat basophilic leukemia cells, namely a similar time course of activation, sensitivity to 2-aminoethoxydiphenyl borate, and low concentrations of lanthanides, and large Na(+) currents displaying the typical depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOCE and I(CRAC) in ECs, which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, knockdown of either TRPC1 or TRPC4 proteins had no effect on SOCE and I(CRAC). Ectopic expression of Stim1 in ECs increased their I(CRAC) to a size comparable to that in rat basophilic leukemia cells. Knockdown of Stim1, Stim2, or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim proteins. These results are first to our knowledge to establish the requirement of Stim1/Orai1 in the endothelial SOCE pathway.

BACKGROUND

We investigated whether catheter-based, intramyocardial transplantation of autologous endothelial progenitor cells can enhance neovascularization in myocardial ischemia.

RESULTS

Myocardial ischemia was induced by placement of an ameroid constrictor around swine left circumflex artery. Four weeks after constrictor placement, CD31+ mononuclear cells (MNCs) were freshly isolated from the peripheral blood of each animal. After overnight incubation of CD31+ MNCs in noncoated plates, nonadhesive cells (NA/CD31+ MNCs) were harvested as the endothelial progenitor cell-enriched fraction. Nonadhesive CD31- cells (NA/CD31- MNCs) were also prepared. Autologous transplantation of 10(7) NA/CD31+ MNCs, 10(7) NA/CD31- MNCs, or PBS was performed with a NOGA mapping injection catheter to target ischemic myocardium. In a parallel study, 10(5) human CD34+ MNCs, 10(5) human CD34- MNCs, or PBS was transplanted into ischemic myocardium of nude rats 10 minutes after ligation of the left anterior descending coronary artery. In the swine study, ischemic area by NOGA mapping, Rentrop grade angiographic collateral development, and echocardiographic left ventricular ejection fraction improved significantly 4 weeks after transplantation of NA/CD31+ MNCs but not after injection of NA/CD31- MNCs or PBS. Capillary density in ischemic myocardium 4 weeks after transplantation was significantly greater in the NA/CD31+ MNC group than the control groups. In the rat study, echocardiographic left ventricular systolic function and capillary density were significantly better preserved in the CD34+ MNC group than in the control groups 4 weeks after myocardial ischemia.

CONCLUSIONS

These favorable outcomes encourage future clinical trials of catheter-based, intramyocardial transplantation of autologous CD34+ MNCs in the setting of chronic myocardial ischemia.

1. Using the whole-cell recording mode of the patch-clamp technique, we have investigated kinetic and selectivity properties of a low-voltage-activated (l.v.a.) Ca2+ current in chick and rat dorsal root ganglion (d.r.g.) neurones. 2. L.v.a currents were activated at about -50 mV and reached maximum amplitudes between -30 and -20 mV with averages of -0.16 nA in chick and -0.3 nA in rat d.r.g. cells with 5 mM-extracellular Ca2+. Between -60 and -20 mV, the time to peak, tp, of this current decreased with increasing membrane depolarizations. An e-fold change of tp required a 14 mV potential change in chick and a 17 mV change in rat d.r.g. cells at 22 degrees C. 3. Between -50 and +20 mV inactivation of this current was fast, single exponential and voltage dependent. In rat, the time constant of inactivation, tau h, was smaller and less voltage dependent than in chick. 4. The amplitude of these currents increased by a factor of 5-10, when the extracellular Ca2+ concentration was changed from 1 to 95 mM. Amplitudes and kinetic parameters of the currents showed typical shifts along the voltage axis. No correlation between Ca2+ current amplitudes and activation-inactivation kinetics was found, suggesting that the reaction rates which control these processes are not dependent on Ca2+ entry. 5. Recovery from inactivation was voltage dependent and developed with a time constant, tau r, in the order of 1 s. tau r was nearly halved by changing the potential from -80 to -120 mV. 6. Tail currents associated with membrane repolarization were also voltage dependent and developed exponentially. Their time constant decreased by a factor of 3 when the potential was changed from -60 to -100 mV. 7. A second and more prominent Ca2+ current was activated at potentials positive to -20 mV (high-voltage-activated Ca2+ currents, h.v.a.), masking the time course of l.v.a. currents. Between -20 and 0 mV, time to peak of the entire current increased by a factor of 2 but decreased again at higher membrane potentials. Inactivation also became significantly slower in this potential range. 8. The contribution of the h.v.a. component to the total membrane current was markedly reduced using a high intracellular Ca2+ concentration, [Ca2+]i, or internal fluoride salts. This made it possible to study the kinetic parameters and the I-V characteristics of the l.v.a. current more precisely over a wider potential range (-50 to +30 mV).(ABSTRACT TRUNCATED AT 400 WORDS)

Adenovirus type 2 (Ad2) binds the coxsackie B virus Ad receptor and is endocytosed upon activation of the alphav integrin coreceptors. Here, we demonstrate that expression of dominant negative clathrin hub, eps15, or K44A-dynamin (dyn) inhibited Ad2 uptake into epithelial cells, indicating clathrin-dependent viral endocytosis. Surprisingly, Ad strongly stimulated the endocytic uptake of fluid phase tracers, coincident with virus internalization but without affecting receptor-mediated transferrin uptake. A large amount of the stimulated endocytic activity was macropinocytosis. Macropinocytosis depended on alphav integrins, PKC, F-actin, and the amiloride-sensitive Na+/H+ exchanger, which are all required for Ad escape from endosomes and infection. Macropinocytosis stimulation was not a consequence of viral escape, since it occurred in K44A-dyn-expressing cells. Surprisingly, 30-50% of the endosomal contents were released into the cytosol of control and also K44A-dyn-expressing cells, and the number of fluid phase-positive endosomes dropped below the levels of noninfected cells, indicating macropinosomal lysis. The release of macropinosomal contents was Ad dose dependent, but the presence of Ad particles on macropinosomal membranes was not sufficient for contents release. We conclude that Ad signaling from the cell surface controls the induction of macropinosome formation and leakage, and this correlates with viral exit to the cytosol and infection.

To begin to determine which genes are essential for salt tolerance in higher plants, we identified four salt-hypersensitive mutants of Arabidopsis by using a root-bending assay on NaCl-containing agar plates. These mutants (sos1-1, sos1-2, sos1-3, and sos1-4) are allelic to each other and were caused by single recessive nuclear mutations. The SOS1 gene was mapped to chromosome 2 at 29.5 [plusmn] 6.1 centimorgans. The mutants showed no phenotypic changes except that their growth was >20 times more sensitive to inhibition by NaCl. Salt hypersensitivity is a basic cellular trait exhibited by the mutants at all developmental stages. The sos1 mutants are specifically hypersensitive to Na+ and Li+. The mutants were unable to grow on media containing low levels (below ~1 mM) of potassium. Uptake experiments using 86Rb showed that sos1 mutants are defective in high-affinity potassium uptake. sos1 plants became deficient in potassium when treated with NaCl. The results demonstrate that potassium acquisition is a critical process for salt tolerance in glycophytic plants.

Amino acid, polyamine, and organocation (APC) transporters are secondary transporters that play essential roles in nutrient uptake, neurotransmitter recycling, ionic homeostasis, and regulation of cell volume. Here, we present the crystal structure of apo-ApcT, a proton-coupled broad-specificity amino acid transporter, at 2.35 angstrom resolution. The structure contains 12 transmembrane helices, with the first 10 consisting of an inverted structural repeat of 5 transmembrane helices like the leucine transporter LeuT. The ApcT structure reveals an inward-facing, apo state and an amine moiety of lysine-158 located in a position equivalent to the sodium ion site Na2 of LeuT. We propose that lysine-158 is central to proton-coupled transport and that the amine group serves the same functional role as the Na2 ion in LeuT, thus demonstrating common principles among proton- and sodium-coupled transporters.

Cortical neurons are sensitive to the timing of their synaptic inputs. They can synchronize their firing on a millisecond time scale and follow rapid stimulus fluctuations with high temporal precision. These findings suggest that cortical neurons have an enhanced sensitivity to synchronous synaptic inputs that lead to rapid rates of depolarization. The voltage-gated currents underlying action potential generation may provide one mechanism to amplify rapid depolarizations. We have tested this hypothesis by analyzing the relations between membrane potential fluctuations and spike threshold in cat visual cortical neurons recorded intracellularly in vivo. We find that visual stimuli evoke broad variations in spike threshold that are caused in large part by an inverse relation between spike threshold and the rate of membrane depolarization preceding a spike. We also find that spike threshold is inversely related to the rate of rise of the action potential upstroke, suggesting that increases in spike threshold result from a decrease in the availability of Na(+) channels. By using a simple neuronal model, we show that voltage-gated Na(+) and K(+) conductances endow cortical neurons with an enhanced sensitivity to rapid depolarizations that arise from synchronous excitatory synaptic inputs. Thus, the basic mechanism responsible for action potential generation also enhances the sensitivity of cortical neurons to coincident synaptic inputs.

Cardiac glycosides are a diverse family of naturally derived compounds that bind to and inhibit Na+/K+-ATPase. Members of this family have been in clinical use for many years for the treatment of heart failure and atrial arrhythmia, and the mechanism of their positive inotropic effect is well characterized. Exciting recent findings have suggested additional signalling modes of action of Na+/K+-ATPase, implicating cardiac glycosides in the regulation of several important cellular processes and highlighting potential new therapeutic roles for these compounds in various diseases. Perhaps most notably, the increased susceptibility of cancer cells to these compounds supports their potential use as cancer therapies, and the first generation of glycoside-based anticancer drugs are currently in clinical trials.

Endogenous cardiotonic steroids (CTS), also called digitalis-like factors, have been postulated to play important roles in health and disease for nearly half a century. Recent discoveries, which include the specific identification of endogenous cardenolide (endogenous ouabain) and bufadienolide (marinobufagenin) CTS in humans along with the delineation of an alternative mechanism by which CTS can signal through the Na(+)/K(+)-ATPase, have increased the interest in this field substantially. Although CTS were first considered important in the regulation of renal sodium transport and arterial pressure, more recent work implicates these hormones in the regulation of cell growth, differentiation, apoptosis, and fibrosis, the modulation of immunity and of carbohydrate metabolism, and the control of various central nervous functions and even behavior. This review focuses on the physiological interactions between CTS and other regulatory systems that may be important in the pathophysiology of essential hypertension, preeclampsia, end-stage renal disease, congestive heart failure, and diabetes mellitus. Based on our increasing understanding of the regulation of CTS as well as the molecular mechanisms of these hormone increases, we also discuss potential therapeutic strategies.

Coherent network oscillations in the brain are correlated with different behavioural states. Intrinsic resonance properties of neurons provide a basis for such oscillations. In the hippocampus, CA1 pyramidal neurons show resonance at theta (theta) frequencies (2-7 Hz). To study the mechanisms underlying theta-resonance, we performed whole-cell recordings from CA1 pyramidal cells (n = 73) in rat hippocampal slices. Oscillating current injections at different frequencies (ZAP protocol), revealed clear resonance with peak impedance at 2-5 Hz at approximately 33 degrees C (increasing to approximately 7 Hz at approximately 38 degrees C). The theta-resonance showed a U-shaped voltage dependence, being strong at subthreshold, depolarized (approximately -60 mV) and hyperpolarized (approximately -80 mV) potentials, but weaker near the resting potential (-72 mV). Voltage clamp experiments revealed three non-inactivating currents operating in the subthreshold voltage range: (1) M-current (I(M)), which activated positive to -65 mV and was blocked by the M/KCNQ channel blocker XE991 (10 microM); (2) h-current (I(h)), which activated negative to -65 mV and was blocked by the h/HCN channel blocker ZD7288 (10 microM); and (3) a persistent Na(+) current (I(NaP)), which activated positive to -65 mV and was blocked by tetrodotoxin (TTX, 1 microM). In current clamp, XE991 or TTX suppressed the resonance at depolarized, but not hyperpolarized membrane potentials, whereas ZD7288 abolished the resonance only at hyperpolarized potentials. We conclude that these cells show two forms of theta-resonance: "M-resonance" generated by the M-current and persistent Na(+) current in depolarized cells, and "H-resonance" generated by the h-current in hyperpolarized cells. Computer simulations supported this interpretation. These results suggest a novel function for M/KCNQ channels in the brain: to facilitate neuronal resonance and network oscillations in cortical neurons, thus providing a basis for an oscillation-based neural code.

The sigma-1 receptor is widely distributed in the central nervous system and periphery. Originally mischaracterized as an opioid receptor, the sigma-1 receptor binds a vast number of synthetic compounds but does not bind opioid peptides; it is currently considered an orphan receptor. The sigma-1 receptor pharmacophore includes an alkylamine core, also found in the endogenous compound N,N-dimethyltryptamine (DMT). DMT acts as a hallucinogen, but its receptor target has been unclear. DMT bound to sigma-1 receptors and inhibited voltage-gated sodium ion (Na+) channels in both native cardiac myocytes and heterologous cells that express sigma-1 receptors. DMT induced hypermobility in wild-type mice but not in sigma-1 receptor knockout mice. These biochemical, physiological, and behavioral experiments indicate that DMT is an endogenous agonist for the sigma-1 receptor.

Human TWIK-1, which has been cloned recently, is a new structural type of weak inward rectifier K+ channel. Here we report the structural and functional properties of TREK-1, a mammalian TWIK-1-related K+ channel. Despite a low amino acid identity between TWIK-1 and TREK-1 (approximately 28%), both channel proteins share the same overall structural arrangement consisting of two pore-forming domains and four transmembrane segments (TMS). This structural similarity does not give rise to a functional analogy. K+ currents generated by TWIK-1 are inwardly rectifying while K+ currents generated by TREK-1 are outwardly rectifying. These channels have a conductance of 14 pS. TREK-1 currents are insensitive to pharmacological agents that block TWIK-1 activity such as quinine and quinidine. Extensive inhibitions of TREK-1 activity are observed after activation of protein kinases A and C. TREK-1 currents are sensitive to extracellular K+ and Na+. TREK-1 mRNA is expressed in most tissues and is particularly abundant in the lung and in the brain. Its localization in this latter tissue has been studied by in situ hybridization. TREK-1 expression is high in the olfactory bulb, hippocampus and cerebellum. These results provide the first evidence for the existence of a K+ channel family with four TMS and two pore domains in the nervous system of mammals. They also show that different members in this structural family can have totally different functional properties.

A long-standing controversy is whether autophagy is a bona fide cause of mammalian cell death. We used a cell-penetrating autophagy-inducing peptide, Tat-Beclin 1, derived from the autophagy protein Beclin 1, to investigate whether high levels of autophagy result in cell death by autophagy. Here we show that Tat-Beclin 1 induces dose-dependent death that is blocked by pharmacological or genetic inhibition of autophagy, but not of apoptosis or necroptosis. This death, termed "autosis," has unique morphological features, including increased autophagosomes/autolysosomes and nuclear convolution at early stages, and focal swelling of the perinuclear space at late stages. We also observed autotic death in cells during stress conditions, including in a subpopulation of nutrient-starved cells in vitro and in hippocampal neurons of neonatal rats subjected to cerebral hypoxia-ischemia in vivo. A chemical screen of ~5,000 known bioactive compounds revealed that cardiac glycosides, antagonists of Na(+),K(+)-ATPase, inhibit autotic cell death in vitro and in vivo. Furthermore, genetic knockdown of the Na(+),K(+)-ATPase α1 subunit blocks peptide and starvation-induced autosis in vitro. Thus, we have identified a unique form of autophagy-dependent cell death, a Food and Drug Administration-approved class of compounds that inhibit such death, and a crucial role for Na(+),K(+)-ATPase in its regulation. These findings have implications for understanding how cells die during certain stress conditions and how such cell death might be prevented.

1. A slow inward current in ventricular preparations of the dog heart can be measured by the voltage clamp method without interference from the initial rapid sodium current if the sodium system is inactivated by conditioning depolarization.2. The slow inward current is very sensitive to variation in [Ca](o). It occurs above the equilibrium potential of I(Na) immediately after changing the bathing fluid to a sodium-free solution and persists under this condition for a long time without much alteration, while I(Na) is rapidly abolished. Tetrodotoxin and [Mg](o) have no effect on this current component. These results strongly support the view that the slow inward current in cardiac tissue is carried by calcium ions.3. The threshold for initiation of the calcium current is around -35 mV in Tyrode solution and is shifted to more negative potentials by either increasing [Ca](o) or reducing [Na](o).4. Calcium sensitive inward current tails associated with repolarization are assumed to represent a proportional measure of calcium conductance activated during the preceding depolarization. Calcium conductance declines rapidly with time in the inside negative potential range and slowly at positive potentials. The time constants for this ;inactivation' process vary between 40 and 700 msec in the potential range -35 to +50 mV.5. By using instantaneous current-voltage relations the reversal potential of calcium current was estimated to be about +60 mV in normal Tyrode solution. As shown in the Appendix, however, the calcium equilibrium potential cannot be considered to be constant.6. The importance of the calcium current for the plateau of the cardiac action potential is discussed.

In Saccharomyces cerevisiae, the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin, is activated by specific environmental conditions, including exposure to Ca(2+) and Na(+), and induces gene expression by regulating the Crz1p/Tcn1p transcription factor. We used DNA microarrays to perform a comprehensive analysis of calcineurin/Crz1p-dependent gene expression following addition of Ca(2+) (200 mm) or Na(+) (0.8 m) to yeast. 163 genes exhibited increased expression that was reduced 50% or more by calcineurin inhibition. These calcineurin-dependent genes function in signaling pathways, ion/small molecule transport, cell wall maintenance, and vesicular transport, and include many open reading frames of previously unknown function. Three distinct gene classes were defined as follows: 28 genes displayed calcineurin-dependent induction in response to Ca(2+) and Na(+), 125 showed calcineurin-dependent expression following Ca(2+) but not Na(+) addition, and 10 were regulated by calcineurin in response to Na(+) but not Ca(2+). Analysis of crz1Delta cells established Crz1p as the major effector of calcineurin-regulated gene expression in yeast. We identified the Crz1p-binding site as 5'-GNGGC(G/T)CA-3' by in vitro site selection. A similar sequence, 5'-GAGGCTG-3', was identified as a common sequence motif in the upstream regions of calcineurin/ Crz1p-dependent genes. This finding is consistent with direct regulation of these genes by Crz1p.

Single-cell suspensions obtained from sequential enzymatic digestions of fetal rat calvaria were grown in long-term culture in the presence of ascorbic acid, Na beta-glycerophosphate, and dexamethasone to determine the capacity of these populations to form mineralized bone. In cultures of osteoblastlike cells grown in the presence of ascorbic acid and beta-glycerophosphate or ascorbic acid alone, three-dimensional nodules (approximately 75 micron thick) covered by polygonal cells resembling osteoblasts could be detected 3 days after confluency. The nodules became macroscopic (up to 3 mm in diameter) after a further 3-4 days. Only in the presence of organic phosphate did they mineralize. Nodules did not develop without ascorbic acid in the medium. Dexamethasone caused a significant increase in the number of nodules. Histologically, nodules resembled woven bone and the cells covering the nodules stained strongly for alkaline phosphatase. Immunolabeling with specific antibodies demonstrated intense staining for type I collagen that was mineral-associated, a weaker staining for type III collagen and osteonectin, and undetectable staining for type II collagen. Nodules did not develop from population I and the number of nodules formed by populations II-V bore a linear relationship to the number of cells plated (r = .99). The results indicate that enzymatically released calvaria cells can form mineralized bone nodules in vitro in the presence of ascorbic acid and organic phosphate.