Differences In Ca2 Research Articles

Relationship between K+ channel down-regulation and [Ca2+]i in rat ventricular myocytes following myocardial infarction.

1. Cardiac hypertrophy and prolongation of the cardiac action potential are hallmark features of heart disease. We examined the molecular mechanisms and the functional consequences of this action potential prolongation on calcium handling in right ventricular myocytes obtained from rats 8 weeks following ligation of the left anterior descending coronary artery (post-myocardial infarction (MI) myocytes). 2. Compared with myocytes from sham-operated rats (sham myocytes), post-MI myocytes showed significant reductions in transient outward K+ current (Ito) density (sham 19.7 +/- 1.1 pA pF-1 versus post-MI 11.0 +/- 1.3 pA pF-1; means +/- s.e.m.), inward rectifier K+ current density (sham -13.7 +/- 0.6 pA pF-1 versus post-MI -10.3 +/- 0.9 pA pF-1) and resting membrane potential (sham -84.4 +/- 1.3 mV versus post-MI -74.1 +/- 2.6 mV). Depressed Ito amplitude correlated with significant reductions in Kv4.2 and Kv4.3 mRNA and Kv4.2 protein levels. Kv1.4 mRNA and protein levels were increased and coincided with the appearance of a slow component of recovery from inactivation for Ito. 3. In current-clamp recordings, post-MI myocytes showed a significant increase in [Ca2+]i transient amplitude compared with sham myocytes. Using voltage-clamp depolarizations, no intrinsic differences in Ca2+ handling by the sarcoplasmic reticulum or in L-type Ca2+ channel density (ICa,L) were detected between the groups. 4. Stimulation of post-MI myocytes with an action potential derived from a sham myocyte reduced the [Ca2+] transient amplitude to the sham level and vice versa. 5. The net Ca2+ influx per beat via ICa,L was increased about 2-fold in myocytes stimulated with post-MI action potentials compared with sham action potentials. 6. Our findings demonstrate that reductions in K+ channel expression in post-MI myocytes prolong action potential duration resulting in elevated Ca2+ influx and [Ca2+]i transients.

Differential influences of gender and physiological status on calcium dynamics and prolactin gene expression in rat mammotropes.

The rate of prolactin (PRL) secretion is influenced by the gender and physiological state of an animal, but little is known about the mechanisms involved. In the present study, we assessed possible contributions of Ca2+ dynamics and PRL gene expression to these differences. This was accomplished by monitoring spontaneous [Ca2+]i changes and PRL promotor-driven reporter activity in pituitary cultures derived from rats comprising a broad spectrum of PRL secretory capacities: male, cycling female, and lactating rats. We found that Ca2+ oscillatory activity exhibited a rank order of lactating > cycling females > males, consistent with the reported secretory capacities of mammotropes from these sources. Interestingly, we observed that the basal level of PRL promotor-driven reporter activity was the same for all three models, but that mammotropes from males were the most responsive to stimulation of PRL gene expression by elevation of [Ca2+]i. Collectively, our findings reveal gender- and state-specific differences in Ca2+ dynamics and induction of PRL gene expression. These likely contribute to reported differences in secretory capacity.

Ca2+-dependent activity of human DNase I and its hyperactive variants.

We have recently constructed hyperactive human deoxyribonuclease I (DNase I) variants that digest double-stranded DNA more efficiently under physiological saline conditions by introducing positively charged amino acids at eight positions that can interact favorably with the negatively charged DNA phosphates. In this study, we present data from supercoiled DNA nicking, linear DNA digestion, and hyperchromicity assays that distinguish two classes of DNase I hyperactive variants based upon their activity dependence on Ca2+. Class A variants are highly dependent upon Ca2+, having up to 300-fold lower activity in the presence of Mg2+ alone compared to that in the presence of Mg2+ and Ca2+, and include Q9R, H44K, and T205K, in addition to wild-type DNase I. In contrast, the catalytic activity of Class B variants, which comprise the E13R, T14K, N74K, S75K, and N110R hyperactive variants, is relatively Ca2+ independent. A significant proportion of this difference in Ca2+-dependent activity can be attributed to one of the two structural calcium binding sites in DNase I. Compared to wild-type, the removal of Ca2+ binding site 2 by alanine replacements at Asp99, Asp107, and Glu112 decreased activity up to 26-fold in the presence of Mg2+ and Ca2+, but had no effect in the presence of Mg2+ alone. We propose that the rate-enhancing effect of Ca2+ binding at site 2 can be replaced by favorable electrostatic interactions created by proximal positively charged amino acid substitutions such as those found in the Class B variants, thus reducing the dependence on Ca2+.

Different mechanisms of Ca2+ transport in NMDA and Ca2+-permeable AMPA glutamate receptor channels.

The channel of the glutamate N-methyl-D-aspartate receptor (NMDAR) transports Ca2+ approximately four times more efficiently than that of Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPAR). To investigate the basis of this difference in these glutamate receptors (GluRs), we measured the ratio of Cs+ efflux and Ca2+ influx in recombinant NMDAR and Ca2+-permeable AMPAR channels expressed in human embryonic kidney 293 (HEK 293) cells over a wide voltage range. At any one potential, this biionic flux ratio was measured by quantifying the total charge and the charge carried by Ca2+ using whole-cell currents and fluorometric techniques (dye overload) with Cs+ internally and Ca2+ externally (1.8 or 10 mM) as the only permeant ions. In AMPAR channels, composed of either GluR-A(Q) or GluR-B(Q) subunits, the biionic flux ratio had a biionic flux-ratio exponent of 1, consistent with the prediction of the Goldman-Hodgkin-Katz current equation. In contrast, for NMDAR channels composed of NR1 and NR2A subunits, the biionic flux-ratio exponent was approximately 2, indicating a deviation from Goldman-Hodgkin-Katz. Consistent with these results, in NMDAR channels under biionic conditions with high external Ca2+ and Cs+ as the reference ions, Ca2+ permeability (PCa/PCs) was concentration dependent, being highest around physiological concentrations (1-1.8 mM; PCa/PCs approximately 6.1) and reduced at both higher (110 mM; PCa/PCs approximately 2.6) and lower (0.18 mM; PCa/PCs approximately 2.2) concentrations. PCa/PCs in AMPAR channels was not concentration dependent, being around 1.65 in 0.3-110 mM Ca2+. In AMPAR and NMDAR channels, the Q/R/N site is a critical determinant of Ca2+ permeability. However, mutant AMPAR channels, which had an asparagine substituted at the Q/R site, also showed a biionic flux-ratio exponent of 1 and concentration-independent permeability ratios, indicating that the difference in Ca2+ transport is not due to the amino acid residue located at the Q/R/N site. We suggest that the difference in Ca2+ transport properties between the glutamate receptor subtypes reflects that the pore of NMDAR channels has multiple sites for Ca2+, whereas that of AMPAR channels only a single site.

Age and gender differences in excitation-contraction coupling of the rat ventricle.

1. The objective of this study was to determine potential post-pubertal gender-specific differences in the contractility of papillary muscles, the electrophysiological properties and Ca2+ transients of freshly dissociated ventricular myocytes from the rat heart. 2. The contractions of rat papillary muscles from 2- to 14-month-old male and female rats were studied under isometric and isotonic conditions (29 degrees C). While the hearts of young (2-4 months) male and female rats displayed a similar contractile profile, papillary muscles of female rats aged 6 months and older exhibited smaller isometric and isotonic contractions, smaller maximal rates of tension and shortening development and decline (+/-DT/dt and +/-DL/dt) velocities during both the onset and relaxation phases, and shorter contractions than age-matched males. 3. To explore the possible cellular basis accounting for these differences, action potentials and macroscopic currents were recorded from freshly dissociated myocytes using the whole-cell patch clamp technique (35 C). Action potentials from male and female myocytes of 3- and 9-month-old rats did not vary as a function of age or gender. Consistent with these results, the magnitude (expressed in pA pF-1), voltage-dependence and kinetics of the inward rectifier (IK1), transient outward (Ito) and sustained (IK) K+ currents displayed little, if any dependence on age or gender. 4. L-type Ca2+ current (ICa(L)) measured in caesium-loaded myocytes (35 C) from male and female rats of 3, 6 and 9 months of age exhibited similar characteristics. In contrast, while Ca2+ transients measured with indo-1 were similar between 3-month-old male and female rat myocytes, Ca2+ transients of 10-month-old female myocytes were significantly reduced and showed a diminished rate of relaxation in comparison with those recorded in male rats of similar age. 5. These results suggest that important gender-related changes in excitation-contraction coupling occur following puberty, probably due to differences in Ca2+ handling capabilities at the level of the sarcoplasmic reticulum.

Activity-Dependent Development of Calcium Regulation in Growing Motor Axons

In cultured nerve cord explants from the crayfish (Procambarus clarkii), the normal impulse activity levels of growing motor axons determine their response to Ca2+ influx. During depolarization or Ca2+ ionophore application, normally active tonic motor axons continue to grow, whereas inactive phasic motor axons retract and often degenerate. To determine the role of Ca2+ regulation in this difference, we measured the intracellular free Ca2+ concentration ([Ca2+]i) with fura-2. Growth cones from tonic axons normally had a higher [Ca2+]i than those from phasic axons. When depolarized with 60 mM K+, growth cones and neurites from phasic axons had a [Ca2+]i three to four times higher than did those from tonic axons. This difference in Ca2+ regulation includes greater Ca2+-handling capacity for growing tonic axons; the increase in [Ca2+]i produced by the Ca2+ ionophore 4-bromo-A23187 (0.25 microM) is four to five times greater in phasic than in tonic axons, and the decline in [Ca2+]i at the end of a depolarizing pulse is three to four times faster in tonic axons than phasic ones. Blocking impulses in growing tonic axons for 2-3 d with tetrodotoxin reduces their capacity to regulate [Ca2+]i. Thus, growing tonic and phasic axons have differences in Ca2+ regulation that develop as a result of their different activity levels. These activity-dependent differences in Ca2+ regulation influence axon growth and degeneration and probably influence other neuronal processes that are mediated by changes in [Ca2+]i.

Regulation of Na+/Ca2+ exchange activity by cytosolic Ca2+ in transfected Chinese hamster ovary cells.

Transfected Chinese hamster ovary cells stably expressing the bovine cardiac Na+/Ca2+ exchanger (CK1.4 cells) were used to determine the range of cytosolic Ca2+ concentrations ([Ca2+]i) that activate Na+/Ca2+ exchange activity. Ba2+ influx was measured in fura 2-loaded, ionomycin-treated cells under conditions in which the intracellular Na+ concentration was clamped with gramicidin at approximately 20 mM. [Ca2+]i was varied by preincubating ionomycin-treated cells with either the acetoxymethyl ester of EGTA or medium containing 0-1 mM added CaCl2. The rate of Ba2+ influx increased in a saturable manner with [Ca2+]i, with the half-maximal activation value of 44 nM and a Hill coefficient of 1.6. When identical experiments were carried out with cells expressing a Ca2+-insensitive mutant of the exchanger, Ba2+ influx did not vary with [Ca2+]i. The concentration for activation of exchange activity was similar to that reported for whole cardiac myocytes but approximately an order of magnitude lower than that reported for excised, giant patches. The reason for the difference in Ca2+ regulation between whole cells and membrane patches is unknown.

Single-channel properties of the sarcoplasmic reticulum calcium-release channel in slow- and fast-twitch muscles of Rhesus monkeys.

RyR1 is the main isoform of ryanodine receptor expressed in fast- and slow-twitch mammalian skeletal muscles although differences in Ca2+-release kinetics and properties have been reported. Single-channel measurements reveal that a large proportion (82%) of Ca2+-release channels measured in slow-twitch muscle preparations have properties similar to those of the Ca2+-release channels of fast-twitch preparations, i.e. the same conductance, an identical sensitivity to caffeine and a bell-shaped Ca2+ activation curve for pCa (-log10[Ca2+]) 7 to 3. A low proportion (18%) of Ca2+-release channels observed in preparations from slow-twitch muscles were characterized by a very high activity level. These channels were not inhibited at a millimolar concentration of Ca2+. Our data suggest that the different properties of Ca2+ release in slow- and fast-twitch muscles might not be related to intrinsic properties of the Ca2+-release channels of each type of muscle but rather to the co-expression of two isoforms of ryanodine receptor and the lower amount of Ca2+-release channels expressed in slow- than in fast-twitch muscles.

Cation sensitivity and kinetics of guard-cell potassium channels differ among species

ward rectifying uard ell + hannel, GCKC1in, from three major crop plants Solanum tuberosum L., Nicotiana tabacum L., and Vicia faba L. Selecting guard cells for our analyses we aimed to test whether K+ channels of the same cell type differ among species. The channels shared basic features including voltage-dependence, selectivity and single-channel conductance. They activated at hyperpolarization (V 1/2 ≈ −164 mV) with single channels of 7 pS underlying the whole-cell current. The channel density in S. tuberosum was higher than in V. faba and N. tabacum while the activation and deactivation kinetics were faster in the latter two species. Among different monovalent cations the K+ channels discriminated strongly against Na+, Li+, and Cs+. The sensitivity to Cs+ was similar for the three species. Extracellular Ca2+ blocked the V.␣faba K+ channel at concentrations ≥1 mM but only affected its functional homologs in S. tuberosum and N.␣tabacum at higher concentrations and more-negative membrane potentials. Like the differences in Ca2+-sensitivity, protoplasts from the three species differed remarkably in their response towards extracellular pH changes. Whereas protons neither altered the open probability nor the kinetic parameters of the V. faba GCKC1in, in S. tuberosum and N. tabacum this cation affected the voltage-dependent properties strongly. An increase in proton concentration from pH 8.5 to 4.5 shifted the potential of half-maximal open probability to less-negative values with a maximum effect around pH 6.2. The pH modulation of the K+ channels could be described assuming a two-state model where the open and closed channel can be protonated. The observed differences in cation-sensitivity and voltage-dependent kinetics between K+ channels reflect the diversification of guard-cell channels that may contribute to species-specific variations in the control of stomatal aperture.

Ca2+ permeability of mouse and chick nicotinic acetylcholine receptors expressed in transiently transfected human cells.

1. Combinations of cDNAs encoding mouse and chick nicotinic acetylcholine receptor (nAChR) subunits were transiently transfected into human BOSC 23 cells, and the expressed receptors were studied by simultaneously recording transmembrane currents and fluorescence transients using the whole-cell patch-clamp technique, and confocal microscopy with the Ca2+ indicator dye fluo-3. 2. The fractional Ca2+ current, Pf, of nAChRs was evaluated as the normalized ratio of nicotine-evoked fluorescence transient over total charge entering the cell (F/Q ratio). Mouse fetal muscle nAChR channels had a Pf, alphabetagammadelta value of 2.1 %. The substitution of the gamma subunit with the epsilon subunit resulted in a 2-fold increase in Pf (4.2 %). The difference in Ca2+ permeability was confirmed by determination of Ca2+/Cs+ permeability ratios. 3. Among the chick neuronal nAChRs tested, Pf,alpha3beta4 was 4.6 %, while Pf, alpha4beta4 and Pf,alpha4beta2 were 3.0 % and 2.9 %, respectively. 4. The amplitude of the current elicited by the activation of alpha3beta4 nAChRs increased as the external Ca2+ concentration was raised from 2 to 110 mM, whereas currents flowing through all other nAChRs tested were reduced to various extents. 5. Our findings indicate that the adult-type muscle nAChR (alphabetaepsilondelta) is more permeable to Ca2+ than the fetal-type (alphabetagammadelta), while ganglionic-like alpha3beta4 nAChR is more permeable to Ca2+ than the examined alpha4-containing nAChRs. The functional significance is discussed.

Changes of intracellular calcium regulation in Alzheimer's disease and vascular dementia.

Free intracellular calcium ([Ca2+]i) represents probably the most important intracellular messenger for many signal transduction pathways. Due to this crucial role of [Ca2+]i, it has been assumed that alterations of [Ca2+]i are critically involved in brain aging and in the pathophysiology of Alzheimer's disease (AD). This hypothesis is corroborated by several studies demonstrating changes of [Ca2+]i in peripheral cells from AD patients. However, the findings are still controversial. Using blood lymphocytes and neutrophils as two different peripheral model systems, we evaluated several parameters of intracellular Ca2+ regulation in a very large group of AD patients and non-demented controls. We found no major difference in Ca2+ homeostasis, since neither the basal [Ca2+]i, nor the activation-induced Ca2+ responses differed among neutrophils or lymphocytes from aged controls and AD patients. However, we observed a delayed Ca2+ response of AD lymphocytes after phytohemagglutinin (PHA) stimulation indicating an impaired function of Ca2+ influx-controlling mechanisms. Furthermore, we studied whether differences exist in Ca2+ regulation between lymphocytes from patients with vascular dementia and AD patients, to define AD-specific alterations and to distinguish between the two dementia groups and non-demented control subjects respectively. First evidences indicate that Ca2+ mobilization in lymphocytes is specifically impaired in lymphocytes from patients with vascular dementia.

Contribution of thromboxane and endomembrane Ca2+-ATPases to variability in Ca2+ signalling of platelets from healthy volunteers

Inter-individual variability in Ca2+ signal generation was studied in platelets from 15 healthy volunteers. The possible involvement of variation in thromboxane A production and variation in sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) was investigated by using platelets isolated before and after intake of 500 mg aspirin, and by measuring the expression levels of two main SERCA isoforms (SERCA-2b and PL/IM 430-recognizable SERCA). Considerable difference in Ca2+ responses were detected after platelet stimulation with thrombin, collagen or the SERCA-2b inhibitor, thapsigargin (TG), with inter-individual coefficients of variance of 22-43% in the absence and 15-41% in the presence of aspirin. Differences in thromboxane A2 generation and SERCA expression contributed to this variability in various ways. In the absence of aspirin, the amount of formed thromboxane A2 partially explains the level of the Ca2+ response induced by TG. On the other hand, in the absence of thromboxane-dependent effects, the expression levels of SERCA-2b and SERCA PL/IM 430 were inversely related to the responses evoked by collagen and TG, respectively. None of these factors were related to the level of the thrombin-evoked Ca2+ signal.

Ca(2+)-sensitivity of portal vein circular muscle from normotensive and hypertensive rats.

Contractile sensitivity to Ca2+ was studied in rat portal vein circular muscle isolated from normotensive Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). There was no difference in resting membrane potential between WKY and SHRSP. Elevation of extracellular K+ concentration induced similar increase in tension in preparations from WKY and SHRSP. High K(+)-depolarized preparation developed tension increase when extracellular Ca2+ concentration was increased. The sensitivity to extracellular Ca2+ was similar in preparations from WKY and SHRSP. Permeabilized preparation contracted when free Ca2+ concentration was increased. Sensitivity of permeabilized preparation to Ca2+ was similar in preparations from WKY and SHRSP. These results indicate that regulation of contraction by Ca2+ was similar between WKY and SHRSP. Since there was no change in high K-induced contraction in intact preparations or Ca-induced contraction in permeabilized preparations, there seemed to be no difference in Ca2+ influx via voltage-dependent channels in portal vein circular muscle from WKY and SHRSP.

Imaging of calcium in Drosophila larval motor nerve terminals.

Calcium measurements in the presynaptic terminal are essential in the investigation of mechanisms underlying neurotransmitter release. To enhance the genetic analysis of secretory mechanisms, we have developed Ca2+ imaging techniques for Drosophila larval motor nerve terminals. We studied Ca2+ signals in "big" (type Ib) and "small" (type Is) boutons that innervate ventral longitudinal muscles 6 and 7 in each abdominal segment of Canton-S (CS)-strain 3rd instar larvae. The indicator fluo-3 in conjunction with confocal microscopy was used to detect stimulus-dependent changes in [Ca2+]i. The Ca2+ signals were reliable and reproducible, and the resting fluorescence remained constant throughout the experiments. The Ca2+ signals increased with stimulus frequency from 5 to 20 Hz for both bouton types. No significant differences in the Ca2+ signals were seen between the two bouton types at 5 and 20 Hz, but there was a difference at 10 Hz. The decay of the Ca2+ signal was more prolonged after 20-Hz stimulation than after 5 and 10 Hz. At the single-synapse level, the secretory efficacy of Is synapses is greater than that of Ib synapses, but our data show that factors other than differences in Ca2+ entry may govern the strength of synaptic transmission.

The High Affinity Calcium-binding Sites in the Epidermal Growth Factor Module Region of Vitamin K-dependent Protein S

Vitamin K-dependent protein S, a cofactor of the anticoagulant enzyme-activated protein C, has four epidermal growth factor (EGF)-like modules, all of which have one partially hydroxylated Asp (EGF 1; beta-hydroxyaspartic acid) or Asn (EGF 2, 3, and 4; beta-hydroxyasparagine) residue. The three C-terminal modules have a typical Ca2+ binding sequence motif that is usually present in EGF modules with hydroxylated Asp/Asn residues. Using the chromophoric Ca2+ chelators Quin 2 and 5,5'-Br2BAPTA, we have now determined the Ca2+ affinity of recombinant fragments containing EGF modules 1-3, 1-4, 2-3, and 2-4. EGF modules 1-4 and 2-4 each contains two very high affinity Ca2+-binding sites, i.e. with dissociation constants ranging from 10(-10) to 10(-8) M in the absence of salt and from 10(-8) to 10(-6) M in the presence of 0.15 M NaCl. In contrast, in EGF 1-3 and EGF 2-3, the Ca2+ affinity is 2-4 orders of magnitude lower. EGF 4 thus appears to have the highest Ca2+ affinity, and furthermore it seems to influence the Ca2+ affinity of its immediate N-terminal neighbor EGF 3 by a factor of approximately 230. In addition, EGF 4 seems to influence the Ca2+ affinity of EGF 2 by a factor of approximately 25. The Ca2+ affinity of the binding sites in EGF modules 3 and 4 in fragments EGF 1-4 and EGF 2-4 is 10(3)-10(5)-fold higher than in the corresponding isolated modules, implying important contributions to the Ca2+ affinity of each module from interactions with neighboring modules. This difference is much higher than the approximately 10-fold difference previously found in similar comparisons of EGF modules from fibrillin. However, the modules studied in protein S and fibrillin appear to have the similar Ca2+ ligands. The structural basis for the difference in Ca2+ affinity is not yet understood.

Circular Dichroism and 1H NMR Studies on the Structures of Peptides Derived from the Calmodulin-binding Domains of Inducible and Endothelial Nitric-oxide Synthase in Solution and in Complex with Calmodulin: NASCENT α-HELICAL STRUCTURES ARE STABILIZED BY CALMODULIN BOTH IN THE PRESENCE AND ABSENCE OF Ca2+

There exist two types of nitric-oxide synthase (NOS); constitutive isozymes that are activated by binding calmodulin in response to elevated Ca2+ and an inducible isozyme that binds calmodulin regardless of Ca2+. To study the structural basis of the difference in Ca2+ sensitivity, we have designed synthetic peptides of minimal lengths derived from the calmodulin-binding domain of endothelial NOS (eNOS) and that of macrophage NOS (iNOS). A peptide, KRREIPLKVLVKAVLFACMLMRK, derived from human iNOS sequence, retained the ability to bind to calmodulin both in the presence and absence of Ca2+, while a peptide derived from human eNOS sequence, RKKTFKEVANAVKISASLMG, bound to calmodulin only in the presence of Ca2+. Circular dichroism and two-dimensional 1H nuclear magnetic resonance studies suggested that both peptides assume nascent alpha-helical structures in aqueous solution. When mixed with calmodulin, both peptides showed circular dichroism spectra characteristic for alpha-helix. In contrast to other target proteins, the addition of iNOS peptide to calmodulin did not affect the Ca2+ binding of calmodulin appreciably. The peptide derived from the calmodulin-binding domain of iNOS, therefore, binds in alpha-helical structures both to Ca2+-calmodulin and apo-calmodulin, which is unique among various target proteins of calmodulin.

Feedback Regulation of ATP-induced Ca2+ Signaling in HL-60 Cells Is Mediated by Protein Kinase A- and C-mediated Changes in Capacitative Ca2+ Entry

Extracellular ATP increases intracellular Ca2+ ([Ca2+]i) in HL-60 cells. When cells are stimulated with supramaximal concentrations of ATP, although the initial [Ca2+]i increase is similar over a range of 30, 100, and 300 microM ATP, the rate of the return to basal [Ca2+]i level is faster in cells treated with higher concentrations of ATP. This probably results from differences in Ca2+ influx rather than Ca2+ release, since the influx of the unidirectional Ca2+ surrogates Ba2+ and Mn2+ also exhibit similar responses. Furthermore, while 300 microM ATP had an inhibitory effect on the thapsigargin-induced capacitative Ca2+ entry, 30 microM ATP potentiated the response. However, the inhibitory action of 300 microM ATP was blocked by protein kinase C (PKC) inhibitors, such as GF 109203X and chelerythrine, and the potentiating action of 30 microM ATP was blocked by protein kinase A (PKA) inhibitors H89 and Rp-cAMPS. The PKC inhibitors also slowed the decay rate of the Ca2+ response induced by 300 microM ATP, and the PKA inhibitors increased it when induced by 30 microM ATP. In the measurements of PKA and PKC activity, 30 microM ATP activates only PKA, while 300 microM ATP activates both kinases. Taken together, these data suggest that the changes in the ATP-induced Ca2+ response result from differential modulation of ATP-induced capacitative Ca2+ entry by PKC and PKA in HL-60 cells.

Differential effects of depolarization on the growth of crayfish tonic and phasic motor axons in culture

Previous studies have demonstrated neuron-specific differences in the inhibitory effects of depolarization upon neurite outgrowth. We examined whether there is a relationship between the normal impulse activity level of an axon and the effect of depolarization upon its growth. Inactive phasic motor axons and active tonic motor axons grow from crayfish abdominal nerve cord explants in culture. Depolarization of these axons with high K solutions produced greater inhibition of advancing growth cones from the phasic axons than from the tonic axons. During the period 20-40 min after the beginning of depolarization, tonic axon growth cones continued to advance, whereas phasic axon growth cones retracted. During chronic depolarization, all of the phasic axons retracted during the first day and approximately half of the phasic axons had degenerated after 4 days of depolarization. The majority of tonic axons continue to grow after 3 days of depolarization, and all of the tonic axon growth survived the 4 days of depolarization. The different responses of the growing phasic and tonic axons to depolarization appear to be Ca2+ dependent. The inhibitory effects of depolarization upon phasic axon growth were reduced by the Ca2+ channel blockers La3+ and Mg2+. Application of a Ca2+ ionophore, A23187, produces greater inhibition of phasic axon growth than tonic axon growth. This study demonstrates that depolarization produces greater inhibition of growth from inactive motor axons than from active motor axons. This is likely due to differences in Ca2+ regulation and/or sensitivity to intracellular Ca2+.

Effects of Age on Ca 2+ Currents in Small Mesenteric Artery Myocytes From Wistar-Kyoto and Spontaneously Hypertensive Rats

The purpose of this study was to test the hypothesis that differences in voltage-gated Ca2+ channels increase with age during the development of sustained hypertension in the spontaneously hypertensive rat (SHR). Using patch-clamp methods, we measured whole-cell Ca2+ currents in freshly isolated myocytes from small mesenteric arteries of juvenile (5 to 7 weeks), young (10 to 12 weeks), and mature (19 to 23 weeks) Wistar-Kyoto rats (WKY) and SHR. Indirect tail artery systolic pressure increased progressively with age in SHR (from 125 +/- 5 to 159 +/- 5 to 192 +/- 5 mm Hg) but only in the younger WKY (from 107 +/- 6 to 130 +/- 4 to 136 +/- 4 mm Hg). Peak Ca2+ current density (current per cell capacitance) was larger in SHR than WKY myocytes at all ages (at 6 weeks, 3.5 +/- 0.4 versus 2.3 +/- 0.2 pA/pF; at 12 weeks, 3.8 +/- 0.2 versus 3.1 +/- 0.2; at 20 weeks. 4.9 +/- 0.4 versus 3.3 +/- 0.4). Cell capacitance (surface area) was significantly smaller in 12-week-old SHR than WKY (25.2 +/- 1.1 versus 31.8 +/- 1.6 pF), but no differences were found in the 6- or 20-week-old groups. There were significant differences in Ca2+ current with strain, age, and voltage but no significant age-strain interactions. The ratio of peak Ca2+ current for SHR to that of WKY declined linearly with voltage at all ages suggesting differences in the voltage dependence of Ca2+ current activation. The voltage dependence of Ca2+ current was shifted to the left in SHR compared with WKY at all ages. Activation curves were shifted significantly in the negative-voltage direction only in 20-week-old SHR myocytes. We have found differences with age in Ca2+ current density and its voltage dependence in SHR compared with WKY during the phase of development in which blood pressure becomes established in the SHR. The net effect of these differences predicts a larger Ca2+ current in SHR at voltages in the physiological range of membrane potential.

Alterations in Calcium Stores in Aortic Myocytes From Spontaneously Hypertensive Rats

The aim of the present work was to further characterize intracellular calcium stores released by angiotensin II (Ang II) in spontaneously hypertensive rat (SHR) and Wistar-Kyoto rat (WKY) vascular smooth muscle cells (VSMCs) and to study their alterations associated with proliferation. Intracellular Ca2+ concentration was monitored by image analysis in aortic myocytes loaded with fura 2. In the presence of extracellular Ca2+, sensitivity to Ang II in proliferating VSMCs was not different in the two strains, but it increased 10-fold in confluent VSMCs from SHR-compared with those from WKY. In Ca(2)+-free medium, Ca2+ release induced by thapsigargin (10 mumol/L) was significantly greater (about twofold) in SHR than WKY, in both proliferating and confluent cultures, with responses during proliferation being 0.7-fold smaller. Responses to Ang II were abolished after exposure of the cells to thapsigargin. In proliferating cultures, ryanodine (10 mumol/L) did not modify the rises in intracellular Ca2+ concentration induced by Ang II in VSMCs from both strains. Conversely, in confluent cultures, ryanodine reduced Ang II (100 nmol/L)-induced Ca2+ release to the same level as in proliferating cultures, and it suppressed the difference between SHR and WKY. These results show that the ryanodine-sensitive Ca2+ release induced by Ang II is enhanced in VSMCs from SHR at confluence and is impaired during proliferation. Thus, they suggest that differences in Ca2+(-)induced Ca2+ release from the sarcoplasmic reticulum may participate in increased responsiveness of VSMCs to Ang II in SHR and in phenotypic modulation of vascular myocytes during proliferation.