Magnitude Of Ca2 Research Articles

Heterogeneity in low voltage‐activated Ca2+ channel‐evoked Ca2+ responses within neurons of the thalamic paraventricular nucleus

Low voltage-activated Ca2+ channels (LVA or T-type Ca2+ channels) are crucial to burst firing and oscillations in thalamocortical relay cells and are exhibited by neurons in the paraventricular nucleus of thalamus (PVT), a dorsal midline nucleus deemed important in the neural representation of motivational behaviours. We used a functional approach (whole-cell patch-clamp electrophysiology combined with confocal laser scanning microscopy) to analyse the spatial distribution of LVA Ca2+ channel-evoked Ca2+ transients in PVT neurons. We observed that the magnitude of LVA Ca2+ channel-evoked Ca2+ transients was significantly greater in proximal dendrites (located up to 20 microm from the soma) than in the soma. In addition, the magnitudes of these Ca2+ transients varied significantly not only among different dendrites of the same cell but also within individual dendrites. These findings suggest that LVA Ca2+ channels are expressed (i) predominantly on the proximal dendrites and (ii) heterogeneously within individual dendrites of PVT neurons. The spatial characteristics of dendritic LVA Ca2+ channels in PVT neurons suggest that these channels may regulate burst firing by modulating dendritic afferent inputs.

Coordinated control of renal Ca2+ handling

Ca2+ homeostasis is an important factor, which is underlined by the numerous clinical symptoms that involve Ca2+ deficiencies. The overall Ca2+ balance is maintained by the concerted action of Ca2+ absorption in the intestine, reabsorption in the kidney, and exchange from bone, which are all under the control of the calciotropic hormones that are released upon a demand for Ca2+. In the kidney, these calciotropic hormones affect active Ca2+ reabsorption, which consists of TRPV5 as the apical entry gate for Ca2+ influx, calbindin-D28K as an intracellular ferry for Ca2+ and, NCX1 and PMCA1b for extrusion of Ca2+ across the basolateral membrane. This review highlights the action of hormones on renal Ca2+ handling and focuses on the coordinated control of the renal Ca2+ transport proteins. Parathyroid hormone stimulates renal Ca2+ handling by regulating active Ca2+ reabsorption on both the genomic and non-genomic level. Estrogens harbor calciotropic hormone characteristics positively regulating the expression of TRPV5, independently of vitamin D. Besides having a strong regulatory effect on the expression of the intestinal Ca2+ transport proteins, vitamin D contributes to the overall Ca2+ balance by enhancing the expression of the Ca2+ transport machinery in the kidney. Dietary Ca2+ is involved in regulating its own handling by controlling the expression of the renal Ca2+ transport proteins. Thus, the magnitude of Ca2+ entry via TRPV5 controls the expression of the other Ca2+ transport proteins underlining the gatekeeper function of this Ca2+ channel in the renal Ca2+ handling.

Cell specificity of the cytoplasmic Ca2+ response to tolbutamide is impaired in β-cells from hyperglycemic mice

We recently reported that the timing and magnitude of the nutrient-induced Ca(2+) response are specific and reproducible for each isolated beta-cell. We have now used tolbutamide and arginine to test if the cell specificity exists also for the response to non-nutrient stimulation of beta-cells and if so, whether it is disturbed in beta-cells from hyperglycemic ob/ob and db/db mice. Zn(2+) outflow measurements were used to study the correlation between Ca(2+) response and insulin secretion in individual beta-cells. Tolbutamide and arginine induced cell-specific Ca(2+) responses in lean mouse beta-cells both with regard to lag times for [Ca(2+)](i) rise and peak [Ca(2+)](i) heights. beta-Cells within intact islets also showed cell-specific timing of their Ca(2+) responses to tolbutamide. However, in tolbutamide- and arginine-stimulated single beta-cells from ob/ob and db/db mice only the magnitude of Ca(2+) response was cell-specific, not the timing. The lag time of tolbutamide-induced insulin secretion was cell-specific in lean mouse beta-cells but not in ob/ob mouse cells. Therefore, cell specificity seems to be a robust mechanism, and probably important for an adequate beta-cell function. The loss of temporal cell specificity for the response to tolbutamide in single beta-cells from hyperglycemic mice may be a sign of K(ATP)- or voltage-dependent calcium channel dysfunction.

Functional adult myocardium in the absence of Na+-Ca2+ exchange: cardiac-specific knockout of NCX1.

The excitation-contraction coupling cycle in cardiac muscle is initiated by an influx of Ca2+ through voltage-dependent Ca2+ channels. Ca2+ influx induces a release of Ca2+ from the sarcoplasmic reticulum and myocyte contraction. To maintain Ca2+ homeostasis, Ca2+ entry is balanced by efflux mediated by the sarcolemmal Na+-Ca2+ exchanger. In the absence of Na+-Ca2+ exchange, it would be expected that cardiac myocytes would overload with Ca2+. Using Cre/loxP technology, we generated mice with a cardiac-specific knockout of the Na+-Ca2+ exchanger, NCX1. The exchanger is completely ablated in 80% to 90% of the cardiomyocytes as determined by immunoblot, immunofluorescence, and exchange function. Surprisingly, the NCX1 knockout mice live to adulthood with only modestly reduced cardiac function as assessed by echocardiography. At 7.5 weeks of age, measures of contractility are decreased by 20% to 30%. We detect no adaptation of the myocardium to the absence of the Na+-Ca2+ exchanger as measured by both immunoblots and microarray analysis. Ca2+ transients of isolated myocytes from knockout mice display normal magnitudes and relaxation kinetics and normal responses to isoproterenol. Under voltage clamp conditions, the current through L-type Ca2+ channels is reduced by 50%, although the number of channels is unchanged. An abbreviated action potential may further reduce Ca2+ influx. Rather than upregulate other Ca2+ efflux mechanisms, the myocardium appears to functionally adapt to the absence of the Na+-Ca2+ exchanger by limiting Ca2+ influx. The magnitude of Ca2+ transients appears to be maintained by an increased gain of sarcoplasmic reticular Ca2+ release. The myocardium of the NCX1 knockout mice undergoes a remarkable adaptation to maintain near normal cardiac function.

CHARACTERIZATION OF THE CONTROL OF INTRACELLULAR [CA 2+] AND THE CONTRACTILE PHENOTYPE OF CULTURED HUMAN DETRUSOR SMOOTH MUSCLE CELLS

We measured the functional properties of cultured human detrusor myocytes with respect to their ability to regulate their intracellular [Ca2+] and generate force in collagen matrices. Human detrusor biopsies were dissociated into single cells by collagenase treatment and used immediately or cultured in D-valine medium and subsequently used after culture trypsinization. Intracellular [Ca2+] was measured in Fura-2 loaded myocytes. Cell force development was measured by incorporating cells into a collagen gel and attaching it to an isometric strain gauge. Carbachol was equally effective in generating Ca transients in freshly isolated and cultured cells. Carbachol potency (pEC50) and the magnitude of Ca2+ transients were similar. Adenosine triphosphate potency was decreased in cultured cells and Ca2+ transients showed properties consistent with a purinoceptor shift from a purinergic subtype. Temporal restitution of Ca2+ transients was similar in the 2 groups, indicative of retained intracellular Ca2+ stores in cultured cells. Cultured cells (approximately 10(6)) embedded in collagen gel generated a force about 10 times greater than that generated by gel alone. The cell dependent force could be further increased by adding carbachol. Cultured cells retain the ability to generate agonist induced intracellular Ca2+ transients. There was no evidence that the cell culture altered the properties of muscarinic receptors, although purinoceptor mediated properties were altered. Restitution experiments indicated that functional intracellular Ca2+ stores were retained in cultured cells. Cultured cells also retained a contractile phenotype, especially in response to carbachol. The magnitude of force was attenuated, which may be a function of the biomechanical properties of the gel used to embed the cells.

Modulation of plasma membrane calcium-ATPase activity by local calcium microdomains near CRAC channels in human T cells.

The spatial distribution of Ca(2+) signalling molecules is critical for establishing specific interactions that control Ca(2+) signal generation and transduction. In many cells, close physical coupling of Ca(2+) channels and their targets enables precise and robust activation of effector molecules through local [Ca(2+)](i) elevation in microdomains. In T cells, the plasma membrane Ca(2+)-ATPase (PMCA) is a major target of Ca(2+) influx through Ca(2+) release-activated Ca(2+) (CRAC) channels. Elevation of [Ca(2+)](i) slowly modulates pump activity to ensure the stability and enhance the dynamic nature of Ca(2+) signals. In this study we probed the functional organization of PMCA and CRAC channels in T cells by manipulating Ca(2+) microdomains near CRAC channels and measuring the resultant modulation of PMCAs. The amplitude and spatial extent of microdomains was increased by elevating the rate of Ca(2+) entry, either by raising extracellular [Ca(2+)], by increasing the activity of CRAC channels with 2-aminoethoxyborane (2-APB), or by hyperpolarizing the plasma membrane. Surprisingly, doubling the rate of Ca(2+) influx does not further increase global [Ca(2+)](i) in a substantial fraction of cells, due to a compensatory increase in PMCA activity. The enhancement of PMCA activity without changes in global [Ca(2+)](i) suggests that local [Ca(2+)](i) microdomains near CRAC channels effectively promote PMCA modulation. These results reveal an intimate functional association between CRAC channels and Ca(2+) pumps in the plasma membrane which may play an important role in governing the time course and magnitude of Ca(2+) signals in T cells.

Spatial Distribution of Calcium Entry Evoked by Single Action Potentials within the Presynaptic Active Zone.

The nature of presynaptic calcium (Ca(2+)) signals that initiate neurotransmitter release makes these signals difficult to study, in part because of the small size of specialized active zones within most nerve terminals. Using the frog motor nerve terminal, which contains especially large active zones, we show that increases in intracellular Ca(2+) concentration within 1 msec of action potential invasion are attributable to Ca(2+) entry through N-type Ca(2+) channels and are not uniformly distributed throughout active zone regions. Furthermore, changes in the location and magnitude of Ca(2+) signals recorded before and after experimental manipulations (omega-conotoxin GVIA, diaminopyridine, and lowered extracellular Ca(2+)) support the hypothesis that there is a remarkably low probability of a single Ca(2+) channel opening within an active zone after an action potential. The trial-to-trial variability observed in the spatial distribution of presynaptic Ca(2+) entry also supports this conclusion, which differs from the conclusions of previous work in other synapses.

ROS are required for rapid reactivation of Na+/Ca2+exchanger in hypoxic reoxygenated guinea pig ventricular myocytes

The cardiac Na(+)/Ca(2+) exchanger (NCX) contributes to cellular injury during hypoxia, as its altered function is largely responsible for a rise in cytosolic Ca(2+) concentration ([Ca(2+)](i)). In addition, the NCX in guinea pig ventricular myocytes undergoes profound inhibition during hypoxia and rapid reactivation during reoxygenation. The mechanisms underlying these changes in NCX activity are likely complex due to the participation of multiple inhibitory factors including altered cytosolic Na(+) concentration, pH, and ATP. Our main hypothesis is that oxidative stress is an essential trigger for rapid NCX reactivation in guinea pig ventricular myocytes and is thus a critical factor in determining the timing and magnitude of Ca(2+) overload. This hypothesis was evaluated in cardiac myocytes using fluorescent indicators to measure [Ca(2+)](i) and oxidative stress. An NCX antisense oligonucleotide was used to decrease NCX protein expression in some experiments. Our results indicate that NCX activity is profoundly inhibited in hypoxic guinea pig ventricular myocytes but is reactivated within 1-2 min of reoxygenation at a time of rising oxidative stress. We also found that several interventions to decrease oxidative stress including antioxidants and diazoxide prevented NCX reactivation and Ca(2+) overload during reoxygenation. Furthermore, application of exogenous H(2)O(2) was sufficient by itself to reactivate the NCX during sustained hypoxia and could reverse the suppression of reoxygenation-mediated NCX reactivation by diazoxide. These data suggest that elevated oxidative stress in reoxygenated guinea pig ventricular myocytes is required for rapid NCX reactivation, and thus reactivation should be viewed as an active process rather than being due to the simple decline of NCX inhibition.

Ca2+ responses in Chinese hamster ovary-K1 cells demonstrate an atypical pattern of ligand-induced 5-HT1A receptor activation.

Little experimental evidence has been reported for diverse signaling via 5-hydroxytryptamine (5-HT)1A receptors despite the fact that agonists seem to be more efficacious at dorsal raphe somatodendritic 5-HT1A autoreceptors than at postsynaptic 5-HT1A receptors. The present study investigated Ca2+ responses in Chinese hamster ovary (CHO)-K1 cells expressing a human 5-HT1A receptor by 5-HT, prototypical 5-HT1A agonists, N-(3-chloro-4-fluorobenzoyl)-4-fluoro-4-[(5-methyl-6-; methylaminopyridin-2-yl)-methylaminomethyl]-piperidine (F 14679), and especially N-(3-chloro-4-fluorobenzoyl)-4-fluoro-4-[(5-methylpyridin-2-yl)-; methylaminomethyl]piperidine (F 13640) as representative ligands of a new chemical class (methylamino-pyridine) that combines both high efficacy and selectivity for 5-HT1A receptors. 5-HT (pEC50 = 6.70 +/- 0.02) induced a pertussis toxin-sensitive, transient high-magnitude Ca2+ response. High-magnitude Ca2+ responses (Emax, percentage versus 5-HT) were also found with F 13640 (107 +/- 4), 5-carboxamidotryptamine (100 +/- 3), and F 14679 (87 +/- 3). In contrast, the prototypical 5-HT1A receptor agonists buspirone, ipsapirone, and 8-(hydroxy-2-(di-n-propylamino)tetralin, and also flesinoxan and eptapirone, were virtually inactive (< or =5). This atypical pattern of 5-HT1A receptor activation contrasts with the broad spectrum of the ligands' partial agonist properties as observed by measuring guanosine 5'-O-(3-[35 S]thio)triphosphate ([35S]GTPgammaS) binding responses with membranes of either CHO-K1 or C6-glial cells stably expressing a human 5-HT1A receptor. Remarkably, differences between ligands that seem small in the [35S]GTPgammaS binding assay translate into huge differences in the magnitude of Ca2+ responses. Therefore, some of these 5-HT1A ligands (i.e., F 13640) may in a selective way induce responses that may be not at all be achieved with other ligands (i.e., buspirone). In conclusion, the pharmacology of 5-HT1A receptor ligands seems to be codetermined by the effector pathway.

Differential Effects of Phosphodiesterase-Sensitive and -Resistant Analogs of cAMP on Initiation of Contraction in Cardiac Ventricular Myocytes

Amplitudes of cardiac contractions initiated by Ca2+-induced Ca2+ release (CICR) are proportional to the magnitude of Ca2+ current (ICa-L). However, large contractions accompanied by little inward current have been reported in some but not all studies in which cells were dialyzed with different analogs of cAMP. This study compares the effects of different phosphodiesterase (PDE)-resistant and PDE-sensitive analogs of cAMP on CICR, and investigates whether cAMP sensitizes CICR so that small currents induce large contractions. Experiments were conducted in voltage-clamped guinea pig ventricular myocytes at 37 degrees C, with different analogs of cAMP added to patch pipette solutions. With PDE sensitive Tris-cAMP, contraction-voltage relations were bell-shaped and proportional to ICa-L. In contrast, dialysis with PDE-resistant dibutyryl-cAMP resulted in sigmoidal contraction-voltage relations and large responses with little inward current. Similarly, in cells loaded with fura-2, large Ca2+ transients were elicited with little inward current in cells dialyzed with PDE-resistant but not PDE-sensitive cAMP. However, large transients were observed with PDE-sensitive cAMP when PDE was inhibited with 3-isobutyl-1-methylxanthine. When the amplitude of ICa-L was varied by partial block with Cd2+, or by partial inactivation, CICR remained proportional to the amplitude of ICa-L. Thus, cAMP altered the relationship between Ca2+ transients and membrane potential but did not sensitize conventional CICR coupled to ICa-L. Our results show that effects of different analogs of cAMP on contraction depend on the PDE resistance of the analog tested. Furthermore, PDE can play a major role in modulating cardiac contraction by altering the relationship between membrane potential and Ca2+ release.

In situ modulation of the human cardiac ryanodine receptor (hRyR2) by FKBP12.6.

The ryanodine receptor complex (RyR), a large oligomeric assembly that functions as a Ca(2+)-release channel in the sarcoplasmic reticulum (SR)/endoplasmic reticulum (ER), comprises four RyR subunits and four FK506-binding proteins (FKBP). The precise mode of interaction and modulation of the cardiac RyR (RyR2) channel by FKBP12/FKBP12.6 remains to be fully defined. We have generated a series of Chinese-hamster ovary (CHO) cell lines stably expressing discrete levels of recombinant human RyR2 (hRyR2) (CHO(hRyR2)). Confocal microscopy of CHO(hRyR2) cells co-expressing either FKBP12 or FKBP12.6 demonstrated that FKBP12.6 was sequestered from the cytoplasm to ER membranes as the cellular levels of hRyR2 increased. There was negligible hRyR2-induced subcellular redistribution of FKBP12. The magnitude of Ca(2+) release in CHO(hRyR2) cells in response to stimulation by 4-chloro- m -cresol was in direct proportion to the expression levels of hRyR2. However, in CHO(hRyR2) cells co-expressing FKBP12.6, Ca(2+) release triggered by the addition of 4-chloro- m -cresol was markedly decreased. In contrast, co-expression of FKBP12 did not affect agonist-induced Ca(2+) release in CHO(hRyR2) cells. Resting cytoplasmic [Ca(2+)] in CHO(hRyR2) remained unaltered after co-expression of FKBP12 or FKBP12.6, but estimation of the ER Ca(2+) load status showed that co-expression of FKBP12.6, but not FKBP12, promoted superfilling of the ER Ca(2+) store which could not be released by RyR2 after agonist activation. The effects of FKBP12.6 on hRyR2-mediated intracellular Ca(2+) handling could be antagonized using rapamycin (5 microM). These results suggest that FKBP12.6 associates with hRyR2 in situ to modulate precisely the functionality of hRyR2 Ca(2+)-release channel.

Alterations in Ca2+ cycling by lysoplasmenylcholine in adult rabbit ventricular myocytes.

We previously reported that lysoplasmenylcholine (LPlasC) altered the action potential (AP) and induced afterdepolarizations in rabbit ventricular myocytes. In this study, we investigated how LPlasC alters excitation-contraction coupling using edge-motion detection, fura-PE3 fluorescent indicator, and perforated and whole cell patch-clamp techniques. LPlasC increased contraction, myofilament Ca(2+) sensitivity, systolic and diastolic free Ca(2+) levels, and the magnitude of Ca(2+) transients concomitant with increases in the maximum rates of shortening and relaxation of contraction and the rising and declining phases of Ca(2+) transients. In some cells, LPlasC induced arrhythmias in a pattern consistent with early and delayed aftercontractions. LPlasC also augmented the caffeine-induced Ca(2+) transient with a reduction in the decay rate. Furthermore, LPlasC enhanced L-type Ca(2+) channel current (I(Ca,L)) and outward currents. LPlasC-induced alterations in contraction and I(Ca,L) were paralleled by its effect on the AP. Thus these results suggest that LPlasC elicits distinct, potent positive inotropic, lusitropic, and arrhythmogenic effects, resulting from increases in Ca(2+) influx, Ca(2+) sensitivity, sarcoplasmic reticular (SR) Ca(2+) release and uptake, SR Ca(2+) content, and probably reduction in sarcolemmal Na(+)/Ca(2+) exchange.

Regulation of Ca2+ channel and phosphatase activities by polyamines in intestinal and vascular smooth muscle--implications for cellular growth and contractility.

Polyamines added extracellularly to intestinal and vascular smooth muscle cells cause relaxation through inhibition of Ca2+ channel activity. Intracellularly applied polyamines also affect Ca2+ channel properties. Polyamines do not readily pass over the plasma membrane because of their positive charges but in permeabilized smooth muscle preparations they have free access to the cytoplasm. In this system they increase sensitivity of the contractile machinery to Ca2+ through inhibition of myosin phosphatase activity. The magnitude of Ca2+ channel and phosphatase inhibition depends on the number of positive charges on the polyamine molecule. Polyamines have an obligatory, but yet undefined, role in regulation of cell growth and proliferation. Several groups of protein kinases, such as tyrosine and mitogen activated protein (MAP)-kinases transmit the growth signal from the plasma membrane to the cell nucleus where mitosis and protein synthesis are initiated. The data reviewed here show that polyamines may affect such signal transmission via inhibition of phosphatase activity.

Bivalent ligands with rigid double-stranded DNA spacers reveal structural constraints on signaling by Fc epsilon RI.

Degranulation of mast cells and basophils during the allergic response is initiated by Ag-induced cross-linking of cell surface IgE-Fc epsilon RI receptor complexes. To investigate how separation distances between cross-linked receptors affect the competency of signal transduction, we synthesized and characterized bivalent dinitrophenyl (DNP)-modified dsDNA oligomers with rigid spacing lengths of approximately 40-100 A. All of these bivalent ligands effectively bind and cross-link anti-DNP IgE with similar affinities in the nanomolar range. The 13-mer (dsDNA length of 44 A), 15-mer (51 A), and flexible 30-mer ligands stimulate similar amounts of cellular degranulation, about one-third of that with multivalent Ag, whereas the 20-mer (68 A) ligand is less effective and the rigid 30-mer (102 A) ligand is ineffective. Surprisingly, all stimulate tyrosine phosphorylation of Fc epsilon RI beta, Syk, and linker for activation of T cells to similar extents as multivalent Ag at optimal ligand concentrations. The magnitudes of Ca(2+) responses stimulated by these bivalent DNP-dsDNA ligands are small, implicating activation of Ca(2+) mobilization by stimulated tyrosine phosphorylation as a limiting process. The results indicate that structural constraints on cross-linked IgE-Fc epsilon RI complexes imposed by these rigid DNP-dsDNA ligands prevent robust activation of signaling immediately downstream of early tyrosine phosphorylation events. To account for these results, we propose that activation of a key downstream target is limited by the spacing between cross-linked, phosphorylated receptors and their associated components.

Dark-stimulated calcium ion fluxes in the chloroplast stroma and cytosol.

Using transgenic Nicotiana plumbaginifolia seedlings in which the calcium reporter aequorin is targeted to the chloroplast stroma, we found that darkness stimulates a considerable flux of Ca(2+) into the stroma. This Ca(2+) flux did not occur immediately after the light-to-dark transition but began approximately 5 min after lights off and increased to a peak at approximately 20 to 30 min after the onset of darkness. Imaging of aequorin emission confirmed that the dark-stimulated luminescence emanated from chloroplast-containing tissues of the seedling. The magnitude of the Ca(2+) flux was proportional to the duration of light exposure (24 to 120 h) before lights off; the longer the duration of light exposure, the larger the dark-stimulated Ca(2+) flux. On the other hand, the magnitude of the dark-stimulated Ca(2+) flux did not appear to vary as a function of circadian time. When seedlings were maintained on a 24-h light/dark cycle, there was a stromal Ca(2+) burst after lights off every day. Moreover, the waveform of the Ca(2+) spike was different during long-day versus short-day light/dark cycles. The dark-stimulated Ca(2+) flux into the chloroplastidic stroma appeared to affect transient changes in cytosolic Ca(2+) levels. DCMU, an inhibitor of photosynthetic electron transport, caused a significant increase in stromal Ca(2+) levels in the light but did not affect the magnitude of the dark-stimulated Ca(2+) flux. This robust Ca(2+) flux likely plays regulatory roles in the sensing of both light/dark transitions and photoperiod.

A single-channel method for evaluation of very magnitudes of Ca2+ ion fluxes through epsilon4/zeta1 N-methyl-D-aspartate receptor channels in bilayer lipid membranes.

A single-channel method for evaluating agonist selectivity in terms of the very number of Ca2+ ions passed through the epsilon4/zeta1 N-methyl-D-aspartate (NMDA) receptor ion channel in bilayer lipid membranes (BLMs) is described. The number of Ca2+ passed through the single-channel was obtained from single-channel recordings in a medium where the primary permeant ion is Ca2+. The recombinant epsilon4/zeta1 NMDA channel was partially purified from Chinese hamster ovary cells expressing the channel and incorporated in BLMs formed by the tip-dip method. It was found that the epsilon4/zeta1 channel in BLMs is permeable to Ca2+ and Na+, but the number of Ca2+ passed through the channel is much fewer than that of Na+. The integrated Ca2+ currents induced by three typical agonists NMDA, L-glutamate and L-CCG-IV were obtained at concentration of 50 microM, where the integrated currents for all the agonists reached their saturated values. The integrated Ca2+ currents obtained are (3.1+/-0.21) x 10(-13) C/s for NMDA, (4.6+/-0.31) x 10(-13) C/s for L-glutamate and (5.7+/-0.25) x 10(-13) C/s for L-CCG-IV, respectively, suggesting that the three kinds of agonists have different efficacies to induce permeation of Ca2+. The range of the agonist selectivity thus obtained is much narrower than that of binding affinities for the NMDA receptors from rat brain. The present method is able to detect Ca2+ permeation with a detection limit of approximately 10(5) Ca2+ ions/s.

Voltage- and calcium-dependent inactivation of calcium channels in Lymnaea neurons.

Ca(2+) channel inactivation in the neurons of the freshwater snail, Lymnaea stagnalis, was studied using patch-clamp techniques. In the presence of a high concentration of intracellular Ca(2+) buffer (5 mM EGTA), the inactivation of these Ca(2+) channels is entirely voltage dependent; it is not influenced by the identity of the permeant divalent ions or the amount of extracellular Ca(2+) influx, or reduced by higher levels of intracellular Ca(2+) buffering. Inactivation measured under these conditions, despite being independent of Ca(2+) influx, has a bell-shaped voltage dependence, which has often been considered a hallmark of Ca(2+)-dependent inactivation. Ca(2+)-dependent inactivation does occur in Lymnaea neurons, when the concentration of the intracellular Ca(2+) buffer is lowered to 0.1 mM EGTA. However, the magnitude of Ca(2+)-dependent inactivation does not increase linearly with Ca(2+) influx, but saturates for relatively small amounts of Ca(2+) influx. Recovery from inactivation at negative potentials is biexponential and has the same time constants in the presence of different intracellular concentrations of EGTA. However, the amplitude of the slow component is selectively enhanced by a decrease in intracellular EGTA, thus slowing the overall rate of recovery. The ability of 5 mM EGTA to completely suppress Ca(2+)-dependent inactivation suggests that the Ca(2+) binding site is at some distance from the channel protein itself. No evidence was found of a role for serine/threonine phosphorylation in Ca(2+) channel inactivation. Cytochalasin B, a microfilament disrupter, was found to greatly enhance the amount of Ca(2+) channel inactivation, but the involvement of actin filaments in this effect of cytochalasin B on Ca(2+) channel inactivation could not be verified using other pharmacological compounds. Thus, the mechanism of Ca(2+)-dependent inactivation in these neurons remains unknown, but appears to differ from those proposed for mammalian L-type Ca(2+) channels.

Sub‐chronic effect of neem based pesticide (Vepacide) on acetylcholinesterase and ATPases in rat

Acetylcholinesterases (AChE), Na+‐K+, Mg2+ and Ca2+‐ATPases were monitored in rat brain when treated orally with 80, 160 and 320 mg/kg of Vepacide, an active ingredient from neem seed oil, daily for 90 days. Brain AChE, Na+‐K+ and Ca2+‐ATPases were inhibited whereas Mg2+‐ATPase levels were enhanced in both the sexes after 45 and 90 days of treatment. The relative sensitivities of these ATPases to Vepacide indicated that Ca2+‐ATPase being more sensitive than Na+‐K+‐ATPase in both the sexes. The magnitude of Ca2+‐ATPase inhibited by this compound was higher than that of brain AChE. It appears to be sexual dimorphism in the alterations of brain AChE, Na+‐K+ and Mg2+‐ATPases by Vepacide with females being significant when compared with males. After 28 days of post treatment the alterations observed were approached to those of controls both in male and female rats showing reversal of the toxicity. These results indicated that the ATPases were potently inhibited by Vepacide and seemed to be its precise target among the enzyme studied. This can be used as biochemical marker of exposure to this neem derived product.

ATP-sensitive K+ channel openers prevent Ca2+ overload in rat cardiac mitochondria.

1. Mitochondrial dysfunction, secondary to excessive accumulation of Ca2+, has been implicated in cardiac injury. We here examined the action of potassium channel openers on mitochondrial Ca2+ homeostasis, as these cardioprotective ion channel modulators have recently been shown to target a mitochondrial ATP-sensitive K+ channel. 2. In isolated cardiac mitochondria, diazoxide and pinacidil decreased the rate and magnitude of Ca2+ uptake into the mitochondrial matrix with an IC50 of 65 and 128 microM, respectively. At all stages of Ca2+ uptake, the potassium channel openers depolarized the mitochondrial membrane thereby reducing Ca2+ influx through the potential-dependent mitochondrial uniporter. 3. Diazoxide and pinacidil, in a concentration-dependent manner, also activated release of Ca2+ from mitochondria. This was prevented by cyclosporin A, an inhibitor of Ca2+ release through the mitochondrial permeability transition pore. 4. Replacement of extramitochondrial K+ with mannitol abolished the effects of diazoxide and pinacidil on mitochondrial Ca2+, while the K+ ionophore valinomycin mimicked the effects of the potassium channel openers. 5. ATP and ADP, which block K+ flux through mitochondrial ATP-sensitive K+ channels, inhibited the effects of potassium channel openers, without preventing the action of valinomycin. 6. In intact cardiomyocytes, diazoxide also induced mitochondrial depolarization and decreased mitochondrial Ca2+ content. These effects were inhibited by the mitochondrial ATP-sensitive K+ channel blocker 5-hydroxydecanoic acid. 7. Thus, potassium channel openers prevent mitochondrial Ca2+ overload by reducing the driving force for Ca2+ uptake and by activating cyclosporin-sensitive Ca2+ release. In this regard, modulators of an ATP-sensitive mitochondrial K+ conductance may contribute to the maintenance of mitochondrial Ca2+ homeostasis.

Effects of pH changes on calcium-mediated potentials in rat hippocampal neurons in vitro

The effects of changes in extra- and intracellular pH (pH o and pH i, respectively) on potentials mediated by the influx of Ca 2+ ions were investigated in intracellular “current-clamp” recordings from CA1 pyramidal neurons in rat hippocampal slices. In neurons which exhibited a “regular-spiking” discharge in response to depolarizing current injection at pH 7.3, perfusion with pH 7.7 medium led to the development of burst firing. Conversely, neurons which were “burst-firing” at pH 7.3 became regular spiking upon exposure to pH 6.9 medium. In addition, the rebound depolarization following a current-evoked hyperpolarization to >−60 mV, which in part reflects activation of a low-voltage-activated Ca 2+ conductance, was reduced at pH o 6.9 and enhanced at pH o 7.7. Neither the burst firing pattern of discharge nor the augmented rebound depolarization observed during perfusion with pH 7.7 medium was due to the reduction in [Cl −] o consequent upon the increase in [HCO − 3] o at a constant P CO2. The magnitudes of the fast afterhyperpolarization which follows a single depolarizing current-evoked action potential and the slow afterhyperpolarization which follows a train of action potentials were attenuated and enhanced, respectively, during perfusion with pH 6.9 and pH 7.7 media, compared with responses obtained at pH 7.3. Reducing pH i at a constant pH o (by exposure to pH 7.3 HCO 3 −/CO 2-free medium buffered with 30 mM HEPES) also attenuated fast and slow afterhyperpolarizations. In tetrodotoxin- and tetraethylammonium-poisoned slices, perfusion with pH 6.9 and pH 7.7 media reduced and increased, respectively, the magnitude of current-evoked Ca 2+-dependent depolarizing potentials and their associated slow afterhyperpolarizations, compared to responses obtained at pH 7.3. In contrast, reducing pH i at a constant pH o elicited only a small reduction in the magnitude of Ca 2+ spikes but markedly attenuated the subsequent slow afterhyperpolarization. The results suggest that, in rat CA1 hippocampal pyramidal neurons, Ca 2+-dependent depolarizing potentials mediated by the influx of Ca 2+ ions through voltage-activated Ca 2+ channels are sensitive to changes in pH o. These effects of changes in pH o are not dependent upon changes in pH i consequent upon the changes in pH o. Changes in pH o also affect the magnitudes of fast and slow afterhyperpolarizations mediated by Ca 2+-dependent K + conductances. In these cases, however, the effects of changes in pH o are mimicked by changes in pH i at a constant pH o, suggesting in turn that the effects of changes in pH o on fast and slow afterhyperpolarizations may be mediated both by changes in Ca 2+ influx (reflecting mainly changes in pH o) and by direct effects of changes in pH i (consequent upon changes in pH o) on Ca 2+-dependent K + conductances.