Propagated and nonpropagated calcium transients during egg activation in the annelid, Chaetopterus

Transient waves of Ca2+ release cross-fertilizing deuterostome eggs from the point of sperm entry to its antipode and provide much of the activating stimulus for the egg. Based on several indirect lines of experimental evidence, it was proposed that protostome eggs are activated by a prolonged uptake of Ca2+ from the medium due to sperm-induced membrane depolarization and that this uptake then starts an activation wave similar to those in deuterostomes, except that it moves inward from the whole surface rather than through the egg from pole to pole. To test these hypotheses, we microinjected oocytes of the polychaete annelid, Chaetopterus pergamentaceus, with semisynthetic recombinant aequorins and measured light emission in response to both fertilization and artificial activation by excess K+. Both fertilization and K(+)-activation induced multiple, brief Ca2+ transients in the eggs. The first transient did not propagate, but it was followed by a series of globally propagated Ca2+ waves interspersed with additional nonpropagated pulses. The waves traversed the egg at about 30 micrometer/sec. Sequential propagated waves and nonpropagated pulses generally originated at different regions of the egg surface, except the last few, which originated in the same "pacemaker" region. These new data are consistent with the hypothesis that the activation of protostome eggs is initiated by Ca2+ waves. However, the fact that these waves propagated from pole to pole like those in deuterostome eggs refutes the notion that Ca2+ waves in activating protostome eggs move inward from the whole surface.     

Tyrosine hydroxylase expression in primary cultures of olfactory bulb: role of L-type calcium channels

Sensory activity mediates regulation of tyrosine hydroxylase (TH), the first enzyme in the dopamine biosynthetic pathway, in the rodent olfactory bulb. The current studies established for the first time primary cultures of neonatal mouse olfactory bulb expressing TH and tested whether L-type calcium channels mediate the activity-dependent regulation of the dopamine phenotype. After 1 d in vitro (DIV), a small population of TH-immunostained neurons that lacked extensive processes could be demonstrated. After an additional 2 DIV in serum-free medium, the number of TH neurons had doubled, and they exhibited long interdigitating processes. Membrane depolarization for 48 hr with 50 mM KCl produced a further 2.4-fold increase in the number of TH-immunoreactive neurons compared with control cultures. Increased TH neuron number required at least 36 hr of exposure to KCl. Forskolin, which increases intracellular cAMP levels, induced a 1.5- to 1.6-fold increase in the number of TH-immunostained neurons. Combined treatment with KCl and forskolin was not additive. Nifedipine, an L-type calcium channel blocker, completely prevented the depolarization-mediated increase in TH expression but did not block the response to forskolin. Treatment with Bay K8644, an L-type calcium channel agonist, also significantly increased the number of TH-expressing neurons. Depolarization also induced alterations in neuritic outgrowth, resulting in a stellate versus an elongate morphology that, in contrast, was not prevented by nifedipine. These results are the first demonstration that in vitro, as in vivo, depolarization increases TH expression in olfactory bulb and that L-type calcium channels mediate this activity-dependent regulation of the dopamine phenotype.     

Spontaneous activity: functions of calcium transients in neuronal differentiation

Calcium ions play critical roles in neuronal development. Many excitable cells promote calcium influx across their surface membrane during early stages of differentiation, which can trigger further elevation of intracellular calcium by release from stores. Several distinct types of spontaneous elevations of intracellular calcium occur during development of amphibian spinal neurons, both in culture and in the intact spinal cord. Rapid spikes and slow waves originate by different mechanisms and have separate functions. Spikes are required for neurotransmitter expression and channel modulation. Waves occurring in growth cones appear to regulate neurite extension.     

Thromboxane A2 receptor mediation of calcium and calcium transients in rat cardiomyocytes

We have examined the effect of the selective thromboxane A2 (TxA2) receptor agonist U46,619 on intracellular ionized Ca ([Ca2+]i) and the calcium transient rate (CATR) in cultured neonatal rat cardiomyocytes using the Ca-sensitive probe fura 2 and ratiometric microfluoroscopy. U46,619, 10(-6)-10(-8)M, increased basal diastolic Ca fluorescence and 10(-6) and 10(-7) M increased CATR. These effects were completely blocked by the highly selective TxA2 receptor antagonist SQ-29,548 (p > 0.5, n = 4 compared to baseline), confirming this response is a specific receptor-mediated event in the cardiomyocytes. TxA2 blockade did not diminish the Angiotensin (Ang II)-mediated [Ca2+]i and calcium transient rate response from that observed in non-blocked cells (p = 0.18 and 0.21 respectively, n = 4). The TxA2-mediated changes in Ca2+ fluorescence did not exhibit homologous desensitization as does Ang II, they did not exhibit heterologous desensitization, and maximally stimulating concentrations were additive in their effect on peak [Ca2+]i. These data support the hypothesis that TxA2 secretion or release following ischemia or other pathophysiologic events could alter cardiac calcium homeostasis. Although Ang II is reported to stimulate the release of TxA2 in a variety of tissues, including the heart, the Ca2+ and CATR response to Ang II are not diminished when TxA2 receptors are blocked. This study cannot rule out the possibility that Ang II-mediated increases in TxA2 may have an additive effect on Ca homeostasis.     

Serum- and bradykinin-induced calcium transients in familial Alzheimer's fibroblasts

The calcium-sensitive photoprotein, aequorin, was used to examine serum- and bradykinin-induced transient increases in free cytosolic calcium ions in skin fibroblasts from 10 individuals with early onset familial AD (FAD), including four who were biopsied before their clinical symptoms would allow a diagnosis of AD, 2 individuals with late onset FAD, 8 at-risk but nonsymptomatic individuals, and 13 controls. The data show that (a) among controls, the peaks of the calcium transients increase in height as a function of donor age; (b) transients induced by 10% serum, 10 nM bradykinin (BK) or 100 nM BK were generally lower in FAD fibroblasts, including those from donors in the early stages of the disease, than in age-matched control cells; (c) such transients are reduced in cells from a proportion of the nonsymptomatic, at-risk individuals. Thus, serum- and BK-induced calcium transients are reduced in fibroblasts from both early and more advanced stage FAD donors and perhaps even from donors who are presymptomatic carriers of the defective gene. The data also suggest that changes in calcium transients in FAD fibroblasts neither mimic nor exaggerate the effects of normal aging.     

Induced spreading depressions in energy-compromised neocortical tissue: calcium transients and histopathological correlates

Mechanisms causing gradual recruitment of damaged cells in the penumbra zone around the core of a focal ischaemic lesion may encompass irregularly occurring depolarization waves of the spreading depression (SD) type, each leading to transient loading of cells with calcium. It has been speculated that, when elicited in an underperfused or otherwise energy-compromised tissue, such depolarization waves lead to cell damage. We assessed under what conditions the calcium transients during KCl-induced SDs are prolonged, and explored if marked prolongation of the transients leads to brain damage. Cerebral blood flow (CBF) was reduced by marked hypocapnia. Tissue oxygenation was reduced by arterial hypoxia, without or with unilateral carotid artery occlusion, or by occlusion of the carotid arteries in normoxic, anaesthetized rats. In all animals the DC potential and extracellular calcium concentration (Ca2+e) were measured before and during a series of SDs. The animals were recovered for histopathological assessment. Hypoxia alone (Pao2, 32.5 +/- 3.8 mmHg) increased mean and total depolarization times, but repeated SDs elicited over 1.7 (+/-0.4) h failed to induce cell damage. Unilateral carotid artery occlusion further prolonged the SD waves but, in spite of total depolarization times of up to 40 min during 2 h, only two out of seven animals showed damage, localized to caudoputamen and parietal cortex, as well as to the subiculum, CA1 and CA3 sectors of the hippocampus. Bilateral carotid artery occlusion was associated with the most pronounced prolongation of the DC potential shifts and Ca2+ transients, with total depolarization times of up to 70 min. In spite of this, only four out of 13 animals showed brain damage and in two of these the damage was contralateral. The results justify modification of the hypothesis stating that SD-like depolarizations in the perifocal penumbra zone per se is what leads to gradual recruitment of such tissues in the infarction process. It is suggested that additional factors are required, such as a larger reduction in CBF, or the proximity of cells at risk to necrotic tissue.     

Voltage-activated intracellular calcium transients in thalamic relay cells and interneurons

The dynamics of intracellular calcium concentration ([Ca2+]i) following activation of low voltage-activated (LVA) and high voltage-activated (HVA) Ca2+ currents were studied in identified relay neurons and interneurons of the rat dorsal lateral geniculate nucleus (LGNd) in situ using Ca2+ imaging and patch-clamp techniques. In relay neurons, [Ca2+]i transients associated with the LVA Ca2+ current showed a fairly homogeneous somatodendritic distribution, whereas HVA transients significantly decreased to 65% of the somatic value at 60 microns dendritic distance. In interneurons, LVA transients significantly increased to 239% of the somatic value at 60 microns dendritic distance, whereas HVA transients were not significantly different in the soma and dendrites. These results indicate differences in [Ca2+]i dynamics, which may reflect a heterogeneous distribution of Ca2+ channels contributing to subcellular compartmentation in the two types of thalamic neurons.     

Strong activation of parallel fibers produces localized calcium transients and a form of LTD that spreads to distant synapses

The temporal and spatial changes in intracellular calcium levels during separate activation of parallel fiber (PF) and climbing fiber (CF) inputs to cerebellar Purkinje cells were studied. PF stimulation (1 Hz), at relatively high stimulus strengths, led to accumulations of calcium that were similar in peak levels to those following CF stimulation but that remained spatially localized. Such stimuli consistently induced a durable depression of PF synaptic transmission that partially occluded further depression by conventional conjunctive stimuli and that was independent of nitric oxide. This depression was accompanied by a reduction of synaptic efficacy in spatially isolated PF inputs to the same cell that was independent of postsynaptic calcium but that was mediated by nitric oxide. These data indicate that LTD comprises at least two separate processes and that parameters of PF stimulation that are capable of raising calcium levels in Purkinje cell dendrites are also able to induce long-term changes in synaptic efficacy.     

Human cytochrome P4502B6: interindividual hepatic expression, substrate specificity, and role in procarcinogen activation

In situ hybridization was combined with serotonin (5-hydroxytryptamine, 5-HT) or tyrosine hydroxylase immunocytochemistry and with Fluoro-Gold retrograde labeling of bulbo-spinal pathways in order to investigate the expression of GAP-43 mRNA in monoamine cell groups of the adult rat brain stem. Consistent with previous reports, GAP-43 mRNA was observed in serotonin and dopamine cell groups in the pons. In addition, GAP-43 expressing cells were observed in all the major monoamine cell groups in the medulla. Thus the B1, B2 and B3 serotonin cell groups all showed high GAP-43 expression in all contained many GAP-43 expressing serotonin cells with spinal cord projections. The A1, A2, A5 and A6 noradrenaline cell groups also showed high GAP-43 expression, although cells with spinal cord projections were largely restricted to the A5 group and A6 subcoeruleus region. In all areas, GAP-43 expressing cells with spinal cord projections were also observed which were not serotonergic or noradrenergic.     

Signal transduction: specificity of growth factors explained by parallel distributed processing

The effects of noxious surgical stimulation on the electroencephalogram (EEG) in 15 horses anesthetized with isoflurane were evaluated during orthopedic (group 1) and soft tissue (group 2) procedures. The quantitative EEG variables theta/delta ratio (T/D), alpha/delta ratio (A/D), beta/delta ratio (B/D), median power frequency (MED), and 80% spectral edge frequency (SEF 80) recorded during Surgeries at 1.7% end-tidal concentration of isoflurane (ET(iso)) were compared with values from five nonstimulated control horses anesthetized at 1,7% ET(iso). The EEG variables T/D, A/D, MED, and SEF 80 from surgically stimulated horses were significantly higher compared with controls. These differences in measured EEG variables were accompanied by a significantly lower relative power in the delta frequency band and a concomitant significantly higher alpha activity. Because the A/D ratio, MED, and SEF 80 in surgically stimulated horses were significantly higher than in nonstimulated control horses these measured EEG variables may provide a valuable tool for identification of nociceptive transmission in isoflurane anesthetized horses.     

Endothelin: receptor subtypes, signal transduction, regulation of Ca2+ transients and contractility in rabbit ventricular myocardium

Endothelin (ET) isopeptides, ET-1, ET-2 and ET-3, elicit a positive inotropic effect (PIE) in association with a negative lusitropic effect, essentially with identical efficacies and potencies in the isolated rabbit papillary muscle, but with different concentration-dependent properties. Pharmacological analysis indicates that the PIE of ET-1 is mediated by an ETA2 subtype that is less sensitive to BQ-123 and FR139317, whereas the PIE of ET-3 is mediated by an ETA1 subtype that is highly sensitive to these ETA antagonists. ETs increased the amplitude of intracellular Ca2+ transient (CaT) in indo-1 loaded rabbit ventricular myocytes, but the increase was much smaller than that produced by elevation of [Ca2+]o or isoproterenol for a given extent of PIE, an indication of increased myofibrillar Ca2+ sensitivity. ETs stimulate phosphoinositide (PI) hydrolysis, which leads to production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Evidence for the role of IP3-induced Ca2+ release in cardiac E-C coupling is tenuous. Generation of IP3 induced by ET-1 was transient and returned to the baseline level when the PIE reached an elevated steady level. Protein kinase C (PKC) that is activated by DAG and also via other pathways triggered by ETs stimulates Na+-H+ exchanger to lead to an increased [Na+]i and alkalinization. The former may contribute to an increase in the amplitude of CaT through Na+-Ca2+ exchanger, and the latter, to an increase in myofibrillar Ca2+ sensitivity. A number of PKC inhibitors, such as staurosporine, H-7, calphostin C and chelerythrine, consistently and selectively inhibited the PIE of ET-3 without affecting the PIE of isoproterenol and Bay k 8644. The maximum inhibition was 20-30% of the total response. A Na+-H+ exchange inhibitor, [5-(N-ethyl-N-isopropyl) amiloride (EIPA)] or a Ca2+ antagonist, verapamil, could not completely inhibit the PIE of ET-3, but the combination of both inhibitors totally abolished the PIE of ET-3. These findings indicate that activation of PKC and subsequent activation of Na+-H+ exchanger and/or L-type Ca2+ channels may play a crucial role in the cardiac action of ET isopeptides in the rabbit ventricular myocardium.     

Receptor-mediated activation of Gsalpha: evidence for intramolecular signal transduction

To investigate the mechanism by which cell surface receptors activate heterotrimeric G proteins, we applied a scanning mutagenesis approach to the carboxyl-terminal 40% of alphas (residues 236-394) to identify residues that play a role in receptor-mediated activation. We identified four regions of sequence in which mutations significantly impaired receptor-dependent stimulation of cAMP synthesis in transiently transfected cyc- S49 lymphoma cells, which lack endogenous alphas. Residues at the carboxyl terminus are likely to be receptor contact sites. Buried residues near the bound GDP are connected to the carboxyl terminus by an alpha helix and may regulate GDP affinity. Residues in two adjacent loops of the GTPase domain at the interface with the helical domain, one of which includes a region, switch III, that changes conformation on GTP binding, are positioned to relay the receptor-initiated signal across the domain interface to facilitate GDP release. Consistent with this hypothesis, replacing the helical domain of alphas with that of alphai2 in an alphas/alphai2/alphas chimera corrects the defect in receptor-mediated activation caused by alphai2 substitutions on the GTPase side of the interface. Thus, complementary interactions between residues across the domain interface seem to play a role in receptor-catalyzed activation.     

Integrins: a role for adhesion molecules in olfactory memory

A gene required for short-term memory in Drosophila, Volado, encodes an alpha integrin and is preferentially expressed in the mushroom bodies of the adult brain. Adhesion molecules of this kind may play a role in olfactory memory by altering the strength of synaptic connections in an experience-dependent manner.     

In vivo regulation of axon extension and pathfinding by growth-cone calcium transients

Growth cones at the tips of extending neurites migrate through complex environments in the developing nervous system and guide axons to appropriate target regions using local cues. The intracellular calcium concentration ([Ca2+]i) of growth cones correlates with motility in vitro, but the physiological links between environmental cues and axon growth in vivo are unknown. Here we report that growth cones generate transient elevations of [Ca2+]i as they migrate within the embryonic spinal cord and that the rate of axon outgrowth is inversely proportional to the frequency of transients. Suppressing Ca2+ transients by photorelease of a Ca2+ chelator accelerates axon extension, whereas mimicking transients with photorelease of Ca2+ slows otherwise rapid axonal growth. The frequency of Ca2+ transients is cell-type specific and depends on the position of growth cones along their pathway. Furthermore, growth-cone stalling and axon retraction, which are two important aspects of pathfinding, are associated with high frequencies of Ca2+ transients. Our results indicate that environmentally regulated growth-cone Ca2+ transients control axon growth in the developing spinal cord.     

What's new in ras genes? Physiological role of ras genes in signal transduction and significance of ras gene activation in tumorigenesis

Ras gene mutations have been found with variable prevalence in different tumor types. While during the past decade a lot of information has been accumulated on the frequency of ras oncogene activation in tumors, the last two years brought considerable progress in elucidating molecular mechanisms of signal transduction for which cellular ras proteins are key elements. They transmit signals from upstream tyrosine kinases to downstream serine/threonine kinases ultimately leading to changes of gene expression cytoskeletal architecture, cell-to-cell interactions and metabolism. These signalling pathways are of interest for the physiological regulation of proliferation and differentiation in normal, as well as in cancer tissue. Mutational activation of cellular ras genes to transforming oncogenes is thought to promote cell growth even in the absence of extracellular stimuli, and may thereby contribute to the initiation and/or progression of tumors.     

The effects of low density lipoprotein on calcium transients in isolated rabbit cardiomyocytes

The purpose of this study was to examine the effects of low density lipoprotein (LDL) on Ca2+ transients of isolated rabbit cardiomyocytes. Incubation of cardiomyocytes with > or = 1 mg of LDL cholesterol/ml of perfusion medium induced a slow (> or = 30 min) but significant increase (2-fold) in the cellular Ca2+ transient. The time course for the effect was similar to that observed for the accumulation of cholesterol in the cells. Using Dil- labeled LDL as a fluorescent marker for LDL interaction with the cardiomyocytes, it was concluded that LDL interacted via a receptor-mediated event, but probably this was not the primary mechanism whereby the lipid entered the cell. LDL-treated cells were resistant to the depressant actions for ryanodine, nicardipine, and dichlorobenzamil on the cellular Ca2+ transient. Lowering the extracellular Ca2+ concentration removed the stimulatory effect of LDL on the Ca2+ transient. It is concluded that LDL can induce an increase in the magnitude of the Ca2+ transient in isolated cardiomyocytes. This is a relatively slow process. The mechanism appears to involve a stimulation of a transsarcolemmal Ca2+ transport pathway. These findings have important implications for cardiac contractile function in hypercholesterolemic and drug-treated hypercholesterolemic subjects.     

Multitasking in the olfactory system: context-dependent responses to odors reveal dual GABA-regulated coding mechanisms in single olfactory projection neurons

Studies of olfaction have focused mainly on neural processing of information about the chemistry of odors, but olfactory stimuli have other properties that also affect central responses and thus influence behavior. In moths, continuous and intermittent stimulation with the same odor evokes two distinct flight behaviors, but the neural basis of this differential response is unknown. Here we show that certain projection neurons (PNs) in the primary olfactory center in the brain give context-dependent responses to a specific odor blend, and these responses are shaped in several ways by a bicuculline-sensitive GABA receptor. Pharmacological dissection of PN responses reveals that bicuculline blocks GABAA-type receptors/chloride channels in PNs, and that these receptors play a critical role in shaping the responses of these glomerular output neurons. The firing patterns of PNs are not odor-specific but are strongly modulated by the temporal pattern of the odor stimulus. Brief repetitive odor pulses evoke fast inhibitory potentials, followed by discrete bursts of action potentials that are phase-locked to the pulses. In contrast, the response to a single prolonged stimulus with the same odor is a series of slow oscillations underlying irregular firing. Bicuculline disrupts the timing of both types of responses, suggesting that GABAA-like receptors underlie both coding mechanisms. These results suggest that glomerular output neurons could use more than one coding scheme to represent a single olfactory stimulus. Moreover, these context-dependent odor responses encode information about both the chemical composition and the temporal pattern of the odor signal. Together with behavioral evidence, these findings suggest that context-dependent odor responses evoke different perceptions in the brain that provide the animal with important information about the spatiotemporal variations that occur in natural odor plumes.     

Encoding specificity revisited: The role of semantics.

Three experiments with undergraduates examined the effects of semantic characteristics of word pairs on memory using the encoding specificity paradigm. The paradigm involved four phases: (a) an encoding phase to relate cues and targets, (b) a phase in which words were generated to new cues, (c) a phase for recognition of generated targets, and (d) a cued-recall phase using the original encoding cues. Encoding pairs were classified a priori as either semantically similar (e.g., alluring PRETTY), semantically contrasting (e.g., drab-PRETTY) or semantically unrelated (e.g., sore-PRETTY) Generation pairs were classified a priori as either semantically similar (e.g., beautiful-PRETTY) or semantically contrasting (e.g., ugly-PRETTY). For recall, the results showed that both the semantic relation between the encoding cue and target and the reprovision of the encoding cue at retrieval were important factors. In the case of recognition, however, both the semantic congruence between the encoding and generation contexts and the amount of semantic elaboration provided by the encoding context were important factors. (PsycINFO Database Record (c) 2010 APA, all rights reserved)