Calcium imaging of motoneuron activity in the en-bloc spinal cord preparation of the neonatal rat

1. This paper describes the use of calcium imaging to monitor patterns of activity in neonatal rat motoneurons retrogradely labeled with the calcium-sensitive dye, calcium green-dextran. 2. Pressure ejection of calcium green-dextran into ventral roots and into the surgically peeled ventrolateral funiculi (VLF) at the lumbar cord labeled spinal motoneurons and interneurons. The back labeled motoneurons often formed two or three discrete clusters of cells. 3. Fluorescent changes (10-20%) could be detected in labeled motoneurons after a single antidromic stimulus of the segmental ventral root. These changes progressively increased in amplitude during stimulus trains (1-5 s) at frequencies from 5 to 50 Hz, presumably reflecting a frequency-dependent increase in free intracellular calcium. 4. Stimulation of the ipsilateral VLF at the caudal lumbar level (L6), elicited frequency-dependent, synaptically induced motoneuronal discharge. Frequency-dependent fluorescent changes could be detected in calcium green-labeled motoneurons during the VLF-induced synaptic activation. 5. The spatial spread of synaptic activity among calcium green-labeled clusters of motoneurons could be resolved after dorsal root stimulation. Low-intensity stimulation of the roots produced fluorescence changes restricted to the lateral clusters of motoneurons. With increasing stimulation intensity the fluorescence change increased in the lateral cells and could spread into the medial motoneuronal group. After a single supramaximal stimulus a similar pattern was observed with activity beginning laterally and spreading medially. 6. Substantial changes in fluorescence of calcium green-labeled motoneurons were also observed during motoneuron bursting induced by bath application of the glycine receptor antagonist strychnine or the potassium channel blocker 4-aminopyridine (4-AP). 7. Our results show that membrane-impermeant fluorescent calcium indicators can be used as a tool to study the activity of specific populations of spinal neurons during execution of motor functions in the developing mammalian spinal cord. They also suggest that lateral clusters of motoneurons in the developing spinal cord of the rat are more recruitable or excitable than more medial clusters. Further understanding of these findings requires identification of these clusters.     

Apolipoprotein A-I stimulates human placental lactogen release by activation of MAP kinase

Cytoplasmatic calcium concentrations are elevated two to three fold during cerebral ischemia. In order to determine the role of calcium-release from intracellular stores vs. calcium entry from the extracellular space, intracellular stores were depleted by the use of thapsigargin and calcium was removed from the incubation fluid prior to energy deprivation (ED). CA 1 pyramidal neurons in hippocampal rat slices were filled with a 1:2 mixture of Fluo-3 and Fura Red by intracellular injection. The neurons were visualized in a Confocal Laser Scanning Microscope (CLSM) and the fluorescence ratio from the probe mixture was used to quantify the calcium concentration. Intracellular calcium concentration was monitored before and during ED. The intracellular calcium concentration was 55 nM prior to ED and increased to 25 microM during ED. The resting levels were the same in the experimental groups, but the increase during ED was significantly lower in the intervention groups. The increase in the calcium free group was to 1 microM and in the thapsigargin group to 5 microM. In the last experimental group, thapsigargin treatment and removal of extracellular calcium, the intracellular calcium increased to 630 nM. These results demonstrate that the increased intracellular calcium seen during ED originates from several sources. Calcium-release from intracellular stores may be of major importance in calcium-related neuronal injury during cerebral ischemia.     

Involvement of calcium and L-type channels in nicotine-induced antinociception

The nature of the signaling process activated by neuronal nicotinic receptors has not been fully defined; however, several recent studies have implicated the involvement of calcium ion fluxes in the response to nicotine on a cellular level. Alteration of nicotine-induced antinociception in mice after systemic administration was therefore investigated in the presence of several drugs that increase intracellular calcium. Calcium, (+/-)-BAYK 8644, thapsigargin, glyburide and A23187 administered intrathecally (i.t.) were found to enhance nicotine-induced antinociception by shifting its dose-response curve to the left. Conversely, i.t. administration of agents which decrease intracellular calcium, such as EGTA and alpha-calcitonin gene-related peptide, blocked nicotine-induced antinociception. These findings support a role for spinal intracellular calcium in the pharmacological effects of nicotine. Additionally, blockade of antinociception by nimodipine and nifedipine indicates that a L-type calcium channel is involved in nicotine's effect. However, nicotine did not compete for [3H] nitrendipine binding. Intrathecal administration of mecamylamine, a nicotinic antagonist, resulted in a blockade of antinociception produced by the i.t. injection of thapsigargin, A23187, calcium and (+/-)-BAYK 8644. The mechanism of mecamylamine's antagonism of nicotine is uncertain. However, these results suggest that mecamylamine blocks the effects of drugs which increase intracellular calcium by either a modulation of intracellular calcium-dependent mechanisms or a blockade of calcium channels. Thus, mecamylamine could modulate a calcium signaling process secondary to receptor activation resulting in blockade of antinociception produced by diverse agents.     

Synchronization of neuronal activity promotes survival of individual rat neocortical neurons in early development

Neural activity is thought to play a significant role during the development of the cerebral cortex. In this study, we examined the effects of global activity block or enhancement and the effects of patterned firing on the ability of cultured rat neocortical neurons to survive during the second week in vitro, beyond the beginning of synaptogenesis. Blockade of neuronal activity by adding tetrodotoxin (TTX) and increasing magnesium concentration in the medium strongly reduced the survival of cortical cells. Increasing neuronal activity by raising the external potassium concentration significantly improved the survival of cortical neurons. We postulated that in a developing neuronal network the survival of nerve cells is regulated by synaptically mediated events that involve changes in the intracellular calcium concentration. To examine this question further, we monitored the activity of the developing network by optically recording the intracellular calcium signals of many neurons simultaneously. These recordings show that in low magnesium neocortical neurons express synchronized oscillation of their intracellular calcium concentration. The ability of a network to synchronize the changes in intracellular calcium of multiple cells appeared gradually during the second week in culture, paralleled by both an increase in the synaptic density and a decline in the number of surviving neurons. By examining the fate of identified cells several days after a recording session, we found that those nerve cells that were co-activated with other neurons had a significantly higher chance to survive than cells that did not participate in synchronized events. These experiments demonstrate that during early cortical network development cortical neurons show synchronized firing activity and that the survival of neurons is at least partially dependent on this pattern of neuronal activity.     

Dissociation between the development of myocardial contracture and heart damage in the "calcium paradox"

The interrelationship between contracture development and heart damage during the calcium paradox under different sodium concentration in Ca-free media was studied on isolated rat hearts. It had been shown that calcium paradox development accompanied contracture development, intensive membrane disruption and alteration of tissue energy state. We had not found relation between contracture magnitude and degree of myocardial alterations in calcium paradox. Our dates confirm so-called intracellular hypothesis of calcium paradox. The experiments had shown close correlation between transmembrane sodium gradient in Ca-free media and degree of cellular damage and energy state alterations during calcium readmission in solution.     

Jejunal myenteric denervation induced by benzalkonium chloride

We examined the effect of a toxic concentration of allyl alcohol (0.5 mM) on intracellular calcium concentrations in isolated rat hepatocytes. An increase in phosphorylase a activity was evident in the hepatocytes after 30 min of incubation with allyl alcohol, suggesting that the toxicant may produce an early rise in cytosolic free calcium. The increase in phosphorylase a activity was not reversed by the addition of dithiothreitol (DTT), a sulfhydryl compound that reverses the events that initiate cell killing by allyl alcohol. When intracellular calcium concentrations were measured directly, using fura-2 as the calcium indicator, there was no effect of allyl alcohol on cytosolic free calcium during the first 60 min of exposure, a critical period for development of irreversible damage. Incubation with allyl alcohol did not interfere with the measurement of intracellular calcium. The increases in cytosolic free calcium produced by phenylephrine or ATP were similar to those reported by others and not affected by the presence of allyl alcohol. The results from this study demonstrate that increased cytosolic free calcium is not essential for allyl alcohol-induced cytotoxicity to isolated rat hepatocytes.     

Contractile behaviour and intracellular calcium during afterloaded contraction in mitral valve disease

It was the aim of the present study to analyze left-ventricular contractile behaviour (force development, shortening) and intracellular calcium handling using afterloaded contractions of papillary muscle fibres from patients operated upon for mitral valve stenosis (MVS, n = 12) or mitral valve incompetence (MVI, n = 15). Isometric force development and passive resting tension at Lmax were similar in MVI and MVS (n.s.). Isotonic shortening amplitudes were reduced in MVI (p < 0.0001) compared to MVS. The peak intracellular calcium transient (ICT) preceeded the maximum force- and shortening amplitude in MVI and MVS. The amplitude of the ICT rose with decreasing afterload, became broader during shortening and presented a prolongation of the diastolic decay. Those differences were much more pronounced in MVI. The calcium-time integral (CTI) at minimal load (isotonic contraction) was 119 +/- 5% in MVS and 165 +/- 14% in MVI (p < 0.0001). The data reveal a severe diastolic calcium overload during shortening in left-ventricular MVI myocardium. An increased dissociation rate of calcium from the contractile proteins during shortening, a depressed calcium re-uptake into the sarcoplasmic reticulum during shortening, or altered mechanosensitive ion channels in MVI may be involved.     

Staircase in frog ventricular muscle. Its dependence on membrane excitation and extracellular ionic composition

Staircase was studied in frog ventricle strip preparations where it was possible to alter extracellular ionic composition extremely rapidly in the diastolic interval between beats. Several findings strongly indicate that staircase in this tissue is a result of progressively increasing calcium influx per beat, rather than a beat-by-beat augmentation of an intracellular calcium pool which contributes to activation. After a steady state of force development, the very next beat could be graded, from approximately zero force to the steady state value attained during the staircase progression, by grading the calcium concentration of a new Ringer's solution switched to perfuse the muscle in the diastolic period immediately before that beat. Also, action potentials, elicited during the "quiescent" period in the virtual absence of contraction (in 0.025 mm calcium Ringer's solution), markedly increase force development and accelerate the staircase seen upon return to normal Ringer's solution. Staircase is augmented and accelerated by prior exposure of the muscle, during the quiescent period, to calcium-poor media and markedly suppressed by prior exposure to sodium-poor media. Tetrodotoxin, in a dose that markedly slows the action potential upstroke, has no effect on staircase. Finally, staircase is seen to occur during a train of depolarizations (by voltage clamp) to inside positive levels greater than the equilibrium potential for sodium. It is concluded that changes in intracellular sodium concentration will alter the staircase response and may contribute to its genesis, but that this cannot be the sole cause of staircase.     

Suppression of inflammatory neurotoxins by highly active antiretroviral therapy in human immunodeficiency virus-associated dementia

The development in culture of 1-cell hamster embryos prior to the completion of fertilization is not well understood. In this study it was observed that culture for only 6 h of these early 1-cell embryos collected before pronuclei formation (3 h post-egg activation; PEA) significantly increased intracellular free calcium levels (194.3 +/- 3.1 nM) compared to levels in similarly aged 1-cell embryos collected from the oviduct at 9 h PEA, after pronuclei formation is complete (134.2 +/- 6.8 nM). Not only was the developmental competence of cultured 3-h PEA embryos with elevated intracellular free calcium levels compromised as compared with that of embryos collected from the oviduct at 9 h PEA; these embryos also had impaired cytoplasmic mitochondrial distribution (ratio of 0.62 +/- 0. 06 for cultured embryos compared to 0.44 +/- 0.04 for in vivo-developed embryos) and decreased lactate metabolism (2.93 +/- 0. 22 pmol/embryo per 3 h for cultured embryos compared to 5.37 +/- 0. 36 for in vivo-developed embryos). This impairment in mitochondrial distribution and function and reduced development in culture by 3-h PEA embryos appears related to the ability to regulate intracellular calcium homeostasis. Intracellular free calcium levels were reduced by culture with increased medium magnesium concentrations, calcium channel inhibitors nifedipine or verapamil, or an intracellular calcium chelator. All of these treatments also stimulated development of 3-h PEA embryos to the morula/blastocyst stages and prevented impairment in mitochondrial organization and function. Conversely, culture with low medium magnesium and high calcium concentrations that increased intracellular free calcium levels resulted in low development and reduced mitochondrial function. Therefore, it appears that removal of the early embryo from the oviduct results in an inability to regulate intracellular calcium levels. As increased magnesium concentrations, nifedipine, and verapamil inhibit L-gated calcium channels, it may be a loss of regulation of these channels that alters calcium homeostasis resulting in impaired developmental competence.     

Intracellular calcium parallels motoneuron degeneration in SOD-1 mutant mice

Transgenic mice with Cu,Zn superoxide dismutase (SOD-1) mutations provide a unique model to examine altered Ca homeostasis in selectively vulnerable and resistant motoneurons. In degenerating spinal motoneurons of G93 A SOD-1 mice, developing vacuoles were filled with calcium, while calcium was gradually depleted from the cytoplasm and intact mitochondria. In oculomotor neurons, no degenerative changes, vacuolization, or increased calcium were noted. Motor axon terminals of interosseus muscle gradually degenerated and intracellular calcium was depleted. Oculomotor terminals of mutant SOD-1 mice were smaller and exhibited no degenerative changes, but did exhibit unique membrane-enclosed organelles containing calcium. Spinal motoneurons of SOD-1 mice were shown to have fewer calcium binding proteins, such as parvalbumin, compared with oculomotor neurons. These data suggest that the SOD-1 mutation is associated with impaired calcium homeostasis in motoneurons in vivo, with increased likelihood of degeneration associated with higher levels of intracellular calcium and lower to absent levels of calbindin-D28K and/or parvalbumin, and decreased likelihood of degeneration associated with minimally changed calcium and ample calbindin-D28K and/or parvalbumin.     

Regulation of neural cell adhesion molecule polysialylation: evidence for nontranscriptional control and sensitivity to an intracellular pool of calcium

The up- and downregulation of polysialic acid-neural cell adhesion molecule (PSA-NCAM) expression on motorneurons during development is associated respectively with target innervation and synaptogenesis, and is regulated at the level of PSA enzymatic biosynthesis involving specific polysialyltransferase activity. The purpose of this study has been to describe the cellular mechanisms by which that regulation might occur. It has been found that developmental regulation of PSA synthesis by ciliary ganglion motorneurons is not reflected in the levels of polysialyltransferase-1 (PST) or sialyltransferase-X (STX) mRNA. On the other hand, PSA synthesis in both the ciliary ganglion and the developing tectum appears to be coupled to the concentration of calcium in intracellular compartments. This study documents a calcium dependence of polysialyltransferase activity in a cell-free assay over the range of 0.1-1 mM, and a rapid sensitivity of new PSA synthesis, as measured in a pulse-chase analysis of tissue explants, to calcium ionophore perturbation of intracellular calcium levels. Moreover, the relevant calcium pool appears to be within a specific intracellular compartment that is sensitive to thapsigargin and does not directly reflect the level of cytosolic calcium. Perturbation of other major second messenger systems, such as cAMP and protein kinase-dependent pathways, did not affect polysialylation in the pulse chase analysis. These results suggest that the shuttling of calcium to different pools within the cell can result in the rapid regulation of PSA synthesis in developing tissues.     

Bacq and Alexander Award lecture--chemical radioprotection: past, present, and future prospects

The intracellular magnesium and calcium ion concentrations of in vivo-developed 2-cell hamster embryos were measured using ratiometric fluorometry. Intracellular magnesium and calcium ion concentrations were found to be 0.369 +/- 0.011 mM and 129.3 +/- 7.5 nM respectively. Culture of 1-cell hamster embryos for 24 hr to the 2-cell stage in control medium containing 0.5 mM magnesium and 2.0 mM calcium resulted in approximately a threefold increase to 343.5 +/- 8.0 nM in intracellular calcium ion concentration, while magnesium ion levels were not altered (0.355 +/- 0.007 mM). Increasing medium magnesium concentrations to 2.0 mM significantly increased intracellular magnesium ion concentrations of cultured 2-cell embryos with a concomitant reduction in intracellular calcium ion concentrations. Furthermore, increasing the medium magnesium concentration to 2.0 mM significantly increased development of 1-cell embryos collected at either 3 or 9 hr post-egg activation to the morula/blastocyst and blastocyst stages. Resultant blastocysts had an increased total cell number and increased development of the inner cell mass. Most important, however, culture with 2.0 mM magnesium increased the fetal potential of cultured 1-cells twofold. Therefore, because highest rates of development were observed in a medium that resulted in reduced intracellular calcium ion concentrations, it appears that altered calcium homeostasis is associated with impaired developmental competence of 1-cell embryos in culture.     

Gibberellin-repressible gene expression in the barley aleurone layer

Enamel cells handle large amounts of calcium, particularly during the developmental phase (termed maturation) when dental enamel is hypermineralized. The extent of intracellular calcium burden, and the nature of calcium homeostasis machinery used to accommodate it, are largely unknown. Here, the calcium-binding capacity of enamel cell cytosol was found to increase during development, in parallel with the putative transcellular flux of calcium. At maturation, the abundance of calcium-binding proteins in enamel cells exceeded that in brain and other established calcium-oriented tissues, which implies a large calcium burden. A search for likely cytosolic calcium transporters revealed only one high-affinity calcium-binding protein (12 kDa, distinguished from alpha-parvalbumin) that was up-regulated during maturation, but its low abundance (0.02% of soluble protein) precluded a major calcium transport or cytoprotective role. Two low-affinity calcium-binding proteins up-regulated during maturation (by 1.8-fold and 2.1-fold respectively) were identified as calreticulin and endoplasmin, both residents of the endoplasmic reticulum. Together, calreticulin and endoplasmin constituted an exceptionally high proportion (5%) of soluble protein during maturation, which gives an inferred calcium capacity 67-fold higher than that of the principal cytosolic calcium-binding protein. 28-kDa calbindin. Evidence that endoplasmin expression varied inversely with serum calcium concentration, and that the inositol trisphosphate receptor also was highly expressed during maturation, supported the novel hypothesis that non-mitochondrial calcium stores play a major role in transcellular calcium transport. In conclusion: (a) enamel cells contain a general high abundance of calcium homeostasis proteins, consistent with a heavy intracellular calcium burden; (b) the expression pattern (phenotype) of calcium-binding proteins varies with enamel cell function; (c) enamel cells appear to contain unusually large non-mitochondrial calcium stores; (d) contrary to the prevailing view that calcium passes mainly through the cytosol of calcium-transporting cells, the findings imply a route through the endoplasmic reticulum. This study gives novel information about how a highly calcium-oriented tissue avoids calcium toxicity, and provides a new focus for investigations into the mechanisms of transcellular calcium transport.     

Regulation of intracellular calcium concentrations by calcium and magnesium in cardioplegic solutions protects rat neonatal myocytes from simulated ischemia

The effects of calcium and magnesium ions in cardioplegic solutions on cardioprotection and intracellular calcium ion handling during ischemia and reoxygenation were investigated in cultured neonatal rat myocardial cells. Myocytes were subjected to simulated ischemia for 60 min at 37 degrees C in hyperkalemic cardioplegic solutions containing various concentrations of calcium and magnesium ions, followed by 30 min of reoxygenation. For each Ca2+ concentration (0.1, 0.6, 1.2, or 2.4 mM), the Mg2+ concentration was either 0, 1.2, 8, or 16 mM. The increase in intracellular Ca2+ concentration during ischemia and reoxygenation was suppressed by the addition of magnesium ion, independent of cardioplegic Ca2+ concentration. The recovery of spontaneous contraction rate and enzyme leakage (creatine phosphokinase and lactate dehydrogenase) during both ischemia and reoxygenation correlated with the degree of inhibition of intracellular Ca2+ accumulation. However, in the 0.1 mM Ca2+ groups in which the Mg2+ concentration was greater than 8 mM, the intracellular Ca2+ concentration increased during reoxygenation in a dose-dependent fashion of Mg2+, and was associated with increased enzyme leakage. The findings suggest that in immature cardiac myocytes, the concentrations of Ca2+ and Mg2+ present in cardioplegic solutions control the intracellular Ca2+ concentration during ischemia and reoxygenation, which, in turn, influences the cardioprotective effect of the cardioplegic solution.     

Triiodothyronine reverses depressed contractile performance after excessive catecholamine stimulation

BACKGROUND: Conflicting results have been reported regarding the acute effects of triiodothyronine (T3) on myocardial contractile performance. The present study analyzes the role of T3 in reversing the depressant effect of excessive catecholamine stimulation in isolated porcine left ventricular myocardium. METHODS: Thirty-six left ventricular trabeculae (0.4 x 6.0 mm) obtained from 6 pigs were used for measurements of isometric force development, isotonic shortening, and intracellular calcium in three experimental series (measurement conditions: 37 degrees C; optimal length; supramaximal electrical stimulation, 1 Hz; calcium measurement, fura-2 ratio method; frequency, 225 Hz). In series 1, isometric force development was measured before and after a 60-minute incubation with 10(-7) mol/L epinephrine in preparations with (n = 6) and without (n = 6) preceding fura-2 loading for calcium measurements. In series 2, the acute effects of a 30-minute administration of T3 (10(-9) mol/L) on isometric force and intracellular calcium were analyzed (n = 6). In series 3, after simultaneous fura-2 loading and a 6-hour 10(-7) mol/L epinephrine exposure the effects of T3 (10(-9) mol/L, 30 minutes) on force development, shortening, and intracellular calcium transient were analyzed. RESULTS: Long-term and high-dose epinephrine exposure induced a severe contractile depression with a significant reduction of isometric force development (p < 0.05) and increased diastolic (p < 0.001) and systolic calcium (p < 0.001). In normal porcine myocardium T3 had no effect on the extent of isometric force generation but accelerated the time course of force development (p < 0.05) and increased the calcium transient (p < 0.001). After induction of myocardial depression by epinephrine exposure T3 accelerated the intracellular calcium transients and reduced diastolic calcium. Triiodothyronine increased the shortening amplitude and the force amplitude (p < 0.01). CONCLUSIONS: Triiodothyronine reverses depressed contractile performance after preceding high-dose epinephrine exposure in isolated porcine myocardium. Increased force amplitudes and unaltered or even reduced intracellular calcium transients argue in favor of a resensitization of the contractile apparatus for calcium by T3. The study supports a potential role for T3 treatment in depressed myocardium after previous excessive catecholamine exposure (eg, brain death, catecholamine treatment, ischemia).     

Tyrphostin A9 inhibits calcium release-dependent phosphorylations and calcium entry via calcium release-activated channel in Jurkat T cells

The mechanism by which calcium-depleted intracellular stores may trigger an external calcium influx through a calcium release-activated channel was investigated by analyzing the effects of several protein tyrosine kinase inhibitors on calcium movements in Jurkat T cells. Tyrphostin A9, an inhibitor of the kinase activity of the platelet-derived growth factor (PDGF) receptor, dramatically impaired the sustained elevation of cytosolic calcium concentration, induced by either CD3 mAbs, thapsigargin, ionomycin at low (10(-7) M) concentration, or passive depletion of intracellular stores; other tested tyrphostins, lavendustin, genistein, and compound 5 lacked significant effect. Tyrphostin A9, added during the plateau phase, was able to return cytosolic calcium to resting concentration. Likewise, it abrogated manganese entry in cells stimulated by CD3 or thapsigargin, measured by the quenching of the fluorescence of Indo-1. However, it did not measurably modify kinetics of intracellular calcium releases monitored in the absence of extracellular calcium, nor did it reverse the inhibition of phosphatidylserine that occurs as a consequence of emptying intracellular stores. Analyses of tyrosine phosphorylations demonstrated that A9 inhibited the phosphorylation of proteins, which occurred every time that internal calcium stores were depleted. These phosphorylations were not impaired by chelation of external Ca2+, nor by La3+ that inhibits calcium release-induced calcium entry. We concluded that their inhibition was not a consequence, but may be a cause, of the blockade of calcium release-activated channel by tyrphostin A9.     

Three-dimensional modeling of renal glomerular capillary networks

Using whole cell patch clamp recordings on unfertilized eggs of the ascidian Ciona intestinalis, we are able to detect ryanodine receptors within the oocytes. Our approach is based on measurements of the voltage-activated inward calcium currents. Two types of Ca2+ currents have been described on the oocyte membrane of Ciona: a low threshold slowly activating current, and a high threshold faster one. We show here that caffeine induces a decrease in the intensity of the Ca2+ currents, when applied either externally or internally from the mouth of a patch pipette. Caffeine application mimics fertilization which transiently decreases the high threshold Ca2+ current density during density during the first meiotic cycle. Ryanodine (> 1 nM) has an effect similar to caffeine. This partial decrease in Ca2+ current density elicited by caffeine or ryanodine is prevented by intracellular application of the calcium chelator BAPTA, then imputable to calcium release. In summary, the depolarization-induced Ca2+ current intensity allows monitoring of an intracellular calcium store which is sensitive to low concentrations of ryanodine in Ciona oocytes. Further identification of a ryanodine receptor was obtained by immunological staining with antibodies against mammalian skeletal muscle ryanodine receptor. Ryanodine receptors were asymmetrically localized in the cortex of Ciona eggs. We discuss the methodological relevance of our patch-clamp approach, in connection with the possible biological role of such a ryanodine receptor in the early stages of development.     

Length dependence of calcium- and force-transients in normal and failing human myocardium

Two questions were analysed: (1) Is the Frank-Starling mechanism operative in failing human myocardium? (2) Are length-dependent changes in force accompanied by length-dependent changes in intracellular calcium transients in human myocardium? METHODS: (I) in electrically stimulated left-ventricular trabeculae [normal donor heart (NDH), n = 8; end stage dilated cardiomyopathy (DCM), n = 11], isometric force development was analysed as a function of muscle length (37 degrees C, oxygenated Krebs-Henseleit solution, supramaximal electrical stimulation, frequency: 1 Hz). (II) Myocardium from the same patients were loaded with the fluorescent dye FURA-2/AM for simultaneous measurements of intracellular calcium transient (ICT) and force development at different muscle lengths. Muscle length, resting force, developed force and intracellular Calcium ("ratio method") were monitored continuously. RESULTS: (I) developed force increased up to an optimum as a function of muscle length in NDH- and DCM-myocardium. The slope of this increase was flatter in DCM-myocardium (P < 0.01). (II) In NDH- and DCM-myocardium, diastolic and systolic calcium increased significantly with muscle length. With decreasing muscle lengths the ICT became broader, the diastolic decay was retarded and the peak of the ICT was flatter. At Lmax the calcium amplitude was 23% smaller in DCM than in NDH (P < 0.04). CONCLUSION: there is a clear length dependence of active force in DCM-myocardium. The length dependence of force development is associated with length-dependent modulations of the ICT. The flatter slope of the length-force curve in DCM may be partly explained by altered intracellular calcium handling in failing myocardium.     

Calcium oxalate crystal attachment to cultured kidney epithelial cell lines

Lamprey spinal neurons exhibit a fast afterhyperpolarization and a late afterhyperpolarization (AHP) which is due to the activation of apamin-sensitive SK Ca2+-dependent K+ channels (KCa) activated by calcium influx through voltage-dependent channels during the action potential (Hill et al. 1992, Neuroreport, 3, 943-945). In this study we have investigated which calcium channel subtypes are responsible for the activation of the KCa channels underlying the AHP. The effects of applying specific calcium channel blockers and agonists were analysed with regard to their effects on the AHP. Blockade of N-type calcium channels by omega-conotoxin GVIA resulted in a significant decrease in the amplitude of the AHP by 76.2+/-14.9% (mean +/- SD). Application of the P/Q-type calcium channel blocker omega-agatoxin IVA reduced the amplitude of the AHP by 20.3+/-10.4%. The amplitude of the AHP was unchanged during application of the L-type calcium channel antagonist nimodipine or the agonist (+/-)-BAY K 8644, as was the compound afterhyperpolarization after a train of 10 spikes at 100 Hz. The effects of calcium channel blockers were also tested on the spike frequency adaptation during a train of action potentials induced by a 100-200 ms depolarizing pulse. The N- and P/Q-type calcium channel antagonists decreased the spike frequency adaptation, whereas blockade of L-type channels had no effect. Thus in lamprey spinal cord motor- and interneurons, apamin-sensitive KCa channels underlying the AHP are activated primarily by calcium entering through N-type channels, and to a lesser extent through P/Q-type channels.     

Engagement of human PECAM-1 (CD31) on human endothelial cells increases intracellular calcium ion concentration and stimulates prostacyclin release

Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a member of the immunoglobulin superfamily that plays a role in a number of endothelial cell (EC) functions including migration, angiogenesis, and transmigration of leukocytes across endothelium. We postulated that one way PECAM-1 might exert its effects was by regulating intracellular EC levels of calcium. Using single-cell fluorometry, we found that engagement of PECAM-1 by mAbs induced a slow but sustained increase in intracellular calcium, both in EC and in an adherent PECAM-1-transfected cell line that models endothelium. Generation of this signal was specific for certain anti-PECAM-1 antibodies, required the presence of the cytoplasmic domain, depended on extracellular calcium and on tyrosine phosphorylation, but did not require cross-linking; in fact, calcium increases were stimulated by certain Fab fragments. Activation of EC by PECAM-1 also caused a time-dependent increase in prostacyclin release. Given the importance of intracellular calcium and prostacyclin release as signaling molecules, engagement of PECAM-1 during cell-cell interactions may alter a number of EC functions including secretion of vasoactive mediators.