Pharmacological dissection of receptor-associated and voltage-sensitive ionic channels involved in catecholamine release

The experiments were designed to quantify pharmacologically the degree of participation of channels associated with the nicotinic cholinoceptor compared with voltage-sensitive channels during the evoked release of [³H]noradrenaline from prelabelled 3–7-day old cultured bovine adrenal chromaffin cells. To achieve this purpose we studied (a) the release of [³H]noradrenaline evoked by secretagogues known to trigger the secretory response through activation of receptor-associated channels (acetylcholine, nicotine), voltage-sensitive Na⁺ (veratridine) and Ca²⁺ (high [K⁺]) channels or direct, channel-independent promotion of Ca²⁺ entry (ionomycin); and (b) the selective blockade of some of those responses using ionic manipulations (Na⁺ deprivation, high Mg²⁺) or drugs known to block the activity of receptoroperated channels (imipramine, cocaine), voltage-dependent Na⁺ (tetrodotoxin) or Ca²⁺ (nitrendipine) channels.

Inhibition by nitrendipine, a potent Ca²⁺ antagonist, of the secretory responses to both nicotine and high [K⁺] indicates a preferential Ca²⁺ entry through voltage-sensitive channels during the secretory process. Blockade by cocaine and imipramine of the release of [³H]noradrenaline evoked by acetylcholine and nicotine, without alteration of the responses to high [K⁺], veratridine or ionomycin, speaks in favor of a selective inactivation of the nicotinic receptor-associated channel. Since Na⁺ deprivation abolished [³H]noradrenaline release produced by nicotine, it seems that Na⁺ entry through the receptor-linked ionophore might be a primary event in the initiation of the secretory process; the fact that tetrodotoxin did not affect the release favors this view.

However, veratridine induced a tetrodotoxin-sensitive secretory response, suggesting the presence of voltage-sensitive Na⁺ channels which might physiologically be used to propagate action potentials through gap junctions between adjacent chromaffin cells, only in the intact gland. Propagated action potentials will activate voltage-sensitive Ca²⁺ channels, producing a massive Ca²⁺ entry and an explosive ejection of catecholamines simultaneously from many cells; during stressful situations this could constitute an efficient mechanism for the amplification of the secretory signal initiated by depolarization of chromaffin cells at restricted subsynaptic areas by acetylcholine liberated at splanchnic nerve-chromaffin cell junctions.     

Aging does not alter cytosolic calcium levels of cortical synaptosomes in Fischer 344 rats

Several lines of experimental evidence support an association between altered Ca²⁺ regulation and aging. It has been supposed that free cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) may decrease or increase in aged animals. In this study, both resting and KCl-stimulated [Ca²⁺]_i were measured in purified cortical synaptosomes from young (3 mo.), middle-aged (12 mo.), and old (24 mo.) Fischer 344 rats. Two additional groups of rats were included, one middle-aged and one old which were trained on a treadmill for 6 months prior to experimentation. The [Ca²⁺]_i was determined using the fluorescent Ca²⁺ chelator fura-2. Net KCl-dependent changes (ΔK) in [Ca²⁺]_i were determined by the difference between stimulatory (100 μM Ca²⁺/60 mM KCl) and resting (100 μM Ca²⁺/5 mM KCl buffer) conditions among the 3 age groups. Significant increases in [Ca²⁺]_i were observed in each age group upon depolarization with 60 mM KCl. However, there were no significant age-dependent differences in either resting [Ca²⁺]_i or KCl-stimulated [Ca²⁺]_i.     

The role of Ca2+ in the generation of spontaneous astrocytic Ca2+ oscillations

Astrocytes in the rat thalamus display spontaneous [Ca²⁺]_i oscillations that are due to intracellular release, but are not dependent on neuronal activity. In this study we have investigated the mechanisms involved in these spontaneous [Ca²⁺]_i oscillations using slices loaded with Fluo-4 AM (5 μM) and confocal microscopy. Bafilomycin A1 incubation had no effect on the number of spontaneous [Ca²⁺]_i oscillations indicating that they were not dependent on vesicular neurotransmitter release. Oscillations were also unaffected by ryanodine. Phospholipase C (PLC) inhibition decreased the number of astrocytes responding to metabotropic glutamate receptor (mGluR) activation but did not reduce the number of spontaneously active astrocytes, indicating that [Ca²⁺]_i increases are not due to membrane-coupled PLC activation. Spontaneous [Ca²⁺]_i increases were abolished by an IP3 receptor antagonist, whilst the protein kinase C (PKC) inhibitor chelerythrine chloride prolonged their duration, indicating a role for PKC and inositol 1,4,5,-triphosphate receptor activation. BayK8644 increased the number of astrocytes exhibiting [Ca²⁺]_i oscillations, and prolonged the responses to mGluR activation, indicating a possible effect on store-operated Ca²⁺ entry. Increasing [Ca²⁺]_o increased the number of spontaneously active astrocytes and the number of transients exhibited by each astrocyte. Inhibition of the endoplasmic reticulum Ca²⁺ ATPase by cyclopiazonic acid also induced [Ca²⁺]_i transients in astrocytes indicating a role for cytoplasmic Ca²⁺ in the induction of spontaneous oscillations. Incubation with 20 μM Fluo-4 reduced the number of astrocytes exhibiting spontaneous increases.

This study indicates that Ca²⁺ has a role in triggering Ca²⁺ release from an inositol 1,4,5,-triphosphate sensitive store in astrocytes during the generation of spontaneous [Ca²⁺]_i oscillations.     

Spatial and dynamic changes in intracellular Ca measured by confocal laser-scanning microscopy in bullfrog sympathetic ganglion cells

Confocal laser scanning microscopy (CLSM) was used to record spatial and dynamic changes in the intracellular Ca²⁺ ([Ca²⁺]_i) of bullfrog sympathetic ganglion cells in excised tissue or in culture. A CLSM utilizing Ar ion laser (488 nm) and recording fluo-3 fluorescence yielded the sliced image of ganglion cells, while conventional epifluorescence microscopy provided the cell image of a convex structure. A high K⁺ (50 mM) solution, caffeine (3–10 mM) and electrical stimulation (10–20 Hz, 0.5–10 s) caused a homogeneous increase in fluo-3 fluorescence with or without regional differences, possibly due to intracellular organelles and other constituents. Scanning a single horizontal line across the cytoplasm with He-Cd laser (325 nm) and recording indo-1 fluorescence demonstrated that the rate of rise in [Ca²⁺]_i following action potentials depends on the distance from the cell membrane and on the cytoplasmic constituents, showing an inward spread of ‘Ca²⁺-wave’ at variable speeds of 17–219 μm/s. These results suggest that heterogeneity of the cytoplasmic structures and constituents affects dynamic and spatial changes of [Ca²⁺]_i in response to stimuli in neurones. Such heterogenic changes in [Ca²⁺]_i would better be studied by CLMS.     

Regulation of intracellular Ca concentration by interleukin-1β in rat cortical synaptosomes: an age-related study

The pro-inflammatory cytokine interleukin-1β (IL-1β) is released by cells during injury and stress, and increased neuronal expression of IL-1β is a feature of age-related neurodegeneration. We have recently reported that IL-1β has a biphasic effect on the K⁺-induced rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cortical synaptosomes, exerting an inhibitory effect on the K⁺-induced rise in [Ca²⁺]_i at lower (3.5 ng/mL) concentrations and a stimulatory effect on the K⁺-induced rise in [Ca²⁺]_i at higher (100 ng/mL) concentrations. In the present study, we observed that the K⁺-induced rise in [Ca²⁺]_i was inhibited to a similar extent by the lower concentration of IL-1β in cortical synaptosomes prepared from young (3-month-old), middle-aged (12-month-old) and aged (24-month-old) rats. In contrast, cortical synaptosomes prepared from the aged rats exhibited an increased susceptibility to the higher concentration of IL-1β, resulting in a marked elevation in [Ca²⁺]_i. We propose that the age-related increase in neuronal concentration of IL-1β promotes a dramatic elevation in [Ca²⁺]_i following membrane depolarization, thereby altering Ca²⁺ homeostasis and exacerbating neuronal vulnerability to excitotoxicity.     

Optical identification of calcium-dependent action potentials transiently expressed in the embryonic rat brainstem

Using multiple-site optical recording of transmembrane potential changes, we have found a new type of calcium-dependent action potential expressed transiently in the embryonic rat dorsal motor nucleus of the vagus nerve. Slice preparations with vagus nerve fibers attached were dissected from 12- to 16-day-old embryonic (E12–E16) rat brainstems, and they were stained with a voltage-sensitive merocyanine–rhodanine dye (NK2761). Electrical activities in response to vagal stimuli were optically recorded simultaneously from many sites using 1020- or 128-element photodiode array measuring systems. In brainstem preparations, two types of action potential-related optical signals were identified. One was detected from the dorsolateral region, and was related to sensory nerve activity (Type I). The other was detected from the dorsomedial region, and corresponded to the action potential in the dorsal motor nucleus of the vagus nerve (Type II). We found a difference in the ionic basis of the Type I vs Type II signals. The Type I signal was not altered in Ca²⁺-free bathing solution and was eliminated by tetrodotoxin, suggesting that the sensory nerve activity is mediated by Na⁺ currents. The Type II signal at early developmental stages (E12–E13, and some preparations in E14) was also independent of Ca²⁺. However, the Type II signal in later developmental stages (E15–E16, and some preparations in E14) did depend upon Ca²⁺: it was eliminated in Ca²⁺-free Ringer's solution, blocked by Cd²⁺, Ni²⁺ or Mn²⁺, and elicited in Sr²⁺-containing Ringer's solution, where CaCl₂ was replaced with SrCl₂. These results suggest that the cation which dominates the motoneuron action potential changes from Na⁺ to Ca²⁺ during development, and this change occurs around E14. With pharmacological analysis using Ca²⁺ channel blockers, we show that the Ca²⁺ channel mediating the motoneuron action potential is distinct from T-, L-, N-, P- or Q-type channels.

Because the vagal action potential in adult mammals is mainly mediated by Na⁺, we suggest that a Ca²⁺ action potential mediated by a new type of Ca²⁺ channel is expressed transiently in the rat dorsal motor nucleus of the vagus nerve at particular stages of development.     

Vip Modulates Intracellular Calcium Oscillations in Human Lymphoblasts

Vasoactive intestinal polypeptide (VIP) has been shown to stimulate adenylate cyclase in a human lymphoblast cell line (MOLT 4). In the present study, we monitored fluorescence in cell suspensions and in single fura-2 loaded MOLT 4 lymphoblasts to determine if VIP modulates intracellular calcium concentrations ([Ca²⁺]_i), and if this modulation is mediated by adenylate cyclase. The distribution of [Ca²⁺]_i in resting and stimulated cells was non-homogeneous, with gradients of high [Ca²⁺]_i present in the subplasmalemmal space. In a subset of cells (10-30% of all cells studied), [Ca²⁺]_i showed La³⁺-sensitive, temporal changes in the form of [Ca²⁺]_i oscillations with a baseline [Ca²⁺]_i value of 115±10 nM, an oscillation amplitude of 150±18 nM and a mean period of 9.2±2s. The remaining non-oscillating cells showed a constant [Ca²⁺]_i level of 75±5 nM (n=65 cells from 4 experiments). In the subset of cells with spontaneous [Ca²⁺]_i oscillations, VIP dose-dependendy (10^-12 to 10^-8M) increased the amplitude of oscillations but did not stimulate their frequency. The stimulatory effect of VIP was correlated with baseline [Ca²⁺]_i in these cells, was attenuated in the presence of La³⁺ (25 μM), but was unaffected by cell depolarization (126 mM KC1). Dibutyryl cyclic AMP (10^-4 to 10^-3 M) and forskolin (10^-4M) had no effect on [Ca²⁺]_i oscillations, or on [Ca²⁺]_i in cells without oscillations. In cell suspensions, baseline [Ca²⁺]_i was found to be 55. 1±11.2 nM (meanS.E.M., n=11); VIP, cyclic AMP analogues or forskolin had no significant effect on [Ca²⁺]_i. These findings suggest that: a) VIP modulates the amplitude of [Ca²⁺]_i oscillations generated by a cytosolic [Ca²⁺] oscillator in a subset of cells at a concentration of 10^-12M, a thousand-fold below the K_D for the VIP receptor; b) baseline [Ca²⁺] values may be related to both the ability of cells to generate spontaneous [Ca²⁺] oscillations and of oscillating cells to respond to VIP; c) due to the small number of responding cells, VIP-induced [Ca²⁺]_i changes are not detectable when studied in cell suspensions.     

Muscarinic modulation of acetylcholine release from slices of guinea pig nucleus basalis magnocellularis

Spontaneous and electrically evoked endogenous acetylcholine release and [³H]-choline efflux from slices of guinea pig nucleus basalis magnocellularis (nbM) were studied. Tetrodotoxin reduced the spontaneous endogenous release by 55%, while the Ca²⁺-free medium reduced it by about 30%. Evoked [³H]-choline efflux was Na⁺ and Ca²⁺ dependent and frequency related. Physostigmine, 30 μM, nearly halved the stimulation-evoked efflux; atropine, 0.15 μM, not only antagonized, but even reversed this effect into facilitation. Pirenzepine, 1 μM, and AFDX 116, 1 μM, were less effective than atropine, and reversed the inhibitory effect of physostigmine only when applied together. 4-DAMP, 0.01 μM, was ineffective. These findings indicate that acetylcholine release in guinea pig nbM slices is inhibited by the cooperation of muscarinic autoreceptors, possibly belonging to the M₁ and M₂ subclasses.     

Mechanism of histamine-induced calcium efflux from cultured bovine adrenal chromaffin cells: possible involvement of an Na/Ca exchange mechanism

The effect of stimulation of the histamine receptor on Ca²⁺ mobilization in cultured bovine adrenal chromaffin cells was examined. Histamine (10⁻⁵ M) increased the intracellular free Ca²⁺ ([Ca²⁺]_i) to a peak in the presence or absence of extracellular Ca²⁺, followed by decrease with time. Histamine (10⁻⁸–10⁻⁵ M) also stimulated ⁴⁵Ca²⁺ efflux from cultured bovine adrenal chromaffin cells in a concentration dependent manner. Its stimulatory effect on ⁴⁵Ca²⁺ efflux was inhibited by the specific histamine H₁ receptor antagonist mepyramine. The increase in histamine-stimulated ⁴⁵Ca²⁺ efflux was inhibited by deprivation of extracellular Na⁺ and by the Na⁺/Ca²⁺ exchange inhibitor amiloride. In addition, histamine stimulated ²²Na⁺ influx into the cells, and this action was inhibited by amiloride. These results suggest that stimulation of the histamine H₁ receptor regulates Na⁺/Ca²⁺ exchange in cultured bovine adrenal chromaffin cells.     

Disruption of ionic and cell volume homeostasis in cerebral ischemia: The perfect storm

The mechanisms of brain tissue damage in stroke are strongly linked to the phenomenon of excitotoxicity, which is defined as damage or death of neural cells due to excessive activation of receptors for the excitatory neurotransmitters glutamate and aspartate. Under physiological conditions, ionotropic glutamate receptors mediate the processes of excitatory neurotransmission and synaptic plasticity. In ischemia, sustained pathological release of glutamate from neurons and glial cells causes prolonged activation of these receptors, resulting in massive depolarization and cytoplasmic Ca²⁺ overload. High cytoplasmic levels of Ca²⁺ activate many degradative processes that, depending on the metabolic status, cause immediate or delayed death of neural cells. This traditional view has been expanded by a number of observations that implicate Cl⁻ channels and several types of non-channel transporter proteins, such as the Na⁺,K⁺,2Cl⁻ cotransporter, Na⁺/H⁺ exchanger, and Na⁺/Ca²⁺ exchanger, in the development of glutamate toxicity. Some of these ion transporters increase tissue damage by promoting pathological cell swelling and necrotic cell death, while others contribute to a long-term accumulation of cytoplasmic Ca²⁺. This brief review is aimed at illustrating how the dysregulation of various ion transport processes combine in a ‘perfect storm’ that disrupts neural ionic homeostasis and culminates in the irreversible damage and death of neural cells. The clinical relevance of individual transporters as targets for therapeutic intervention in stroke is also briefly discussed.     

Requirement of neural activity for the maintenance of dopaminergic neurons in rat midbrain slice cultures

Chronic treatment of organotypic midbrain slice cultures with L-type Ca²⁺ channel blocker nicardipine (3–10 μM) or verapamil (10 μM) for 18 days resulted in a drastic decrease in the number of dopaminergic neurons. A voltage-dependent Na⁺ channel blocker tetrodotoxin (1 μM) was also effective in decreasing the number of dopaminergic neurons. Concurrent application of forskolin (20 μM) or dibutyryl cyclic AMP (1 mM) counteracted the effects of nicardipine and tetrodotoxin. These results suggest that spontaneous neuronal activity within midbrain slice cultures, causing Ca²⁺ influx through L-type Ca²⁺ channels that maintains intracellular cyclic AMP levels, is required for the maintenance of dopaminergic neurons.     

妊高征肾病患者血清CysC水平与红细胞膜ATP酶活性的关系

目的：探讨了妊高征肾病患者血清胱抑素蛋白酶抑制素C（CysC）水平与红细胞膜Na^＋、K^＋-ATP酶和Ca^2＋、Mg^2＋-ATP酶活性改变参与妊高征肾病发生的可能机制。方法：应用乳胶免疫增强比浊法和Reinil制膜法测定了32例妊高征肾病患者血清CysC水平与红细胞膜Na^＋、K^＋-ATP酶和Ca^2＋、Mg^2＋-ATP酶含量,并与35名正常孕妇组作比较。结果：妊高征肾病患者血清CysC水平非常显著地高于正常孕妇组（P〈0．01）,而红细胞膜Na^＋、K^＋-ATP酶和Ca^2＋、Mg2^2＋-ATP酶含量显著地低于正常孕妇组（P〈0．01）。结论：妊高征肾病的发生发展与血清CysC水平和红细胞膜Na^＋、K^＋-ATP酶和Ca^2＋、Mg^2＋-ATP酶活性有密切的关系。     

Modulation of the dihydropyridine-insensitive Ca influx by 8-bromo-guanosine-3′:5′-monophosphate, cyclic (8-Br-cGMP) in bovine adrenal chromaffin cells

Pretreatment of chromaffin cells with the permeable analogue of cGMP, 8-Br-cGMP (100 μM), leads to a reduction (35%) of depolarization-evoked intracellular calcium concentration ([Ca²⁺]_i) increases. There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca²⁺ influx pathways. Combined incubations with nifedipine 10 μM and 8-Br-cGMP reduced KCl-evoked intracellular Ca²⁺ concentration to a greater extent that each compound separately. Moreover, 8-Br-cGMP failed to affect the [Ca²⁺]_i transient induced by the L-type Ca²⁺ channel agonist Bay K 8644 (1μM) under conditions of low depolarization. Neomycin (0.2 mM) and θ-Aga Toxin-IVA (AgTx) (1μM) inhibited the calcium transient to a similar extent, and this inhibition was not enhanced by the presence of 8-Br-cGMP. It is concluded that 8-Br-cGMP modulated the dihydropyridine-insensitive Ca²⁺ influx pathway in the chromaffin cell.     

Effects of free Ca on the [Ca + Mg-Dependent adenosinetriphosphatase (ATPase) of Alzheimer and normal fibroblasts

Differences between Alzheimer and control fibroblasts [Ca²⁺ + Mg²⁺]-dependent ATPase activity at free Ca²⁺ concentration considerably higher than physiologic concentrations were observed. At 50 μM free Ca²⁺, Alzheimer and control fibroblast homogenates exhibited maximum velocity values ranging from 8 to 25 nmoles phosphate released/min/mg protein. Higher free Ca²⁺ (350 μM) inhibited control fibroblast ATPase activity approximately 77%; whereas, Alzheimer fibroblasts retained greater than 75% starting activity. Although the pathophysiological significance of these findings is at present unclear, these data suggest the Ca²⁺ pump of Alzheimer fibroblasts behaves differently in the presence of high free Ca²⁺. Such behavior may be of potential diagnostic value.     

Modulation of Ca channels by activation of adenosine A1 receptors in rat striatal glutamatergic nerve terminals

We determined that activation of adenosine A₁ receptors in striatal synaptosomes with 100 nM N⁶-cyclopentyladenosine (CPA) inhibited both the release of endogenous glutamate and the increase of intracellular free Ca²⁺ concentration ([Ca²⁺]_i), due to 4-aminopyridine (4-AP) stimulation, by 28 and 19%, respectively. Furthermore, CPA enhanced the inhibition of endogenous glutamate release due to ω-conotoxin GVIA (ω-Cgtx GVIA), ω-Cgtx MVIIC or ω-Cgtx GVIA plus ω-Cgtx MVIIC. Similar effects were observed in the [Ca²⁺]_i signal. The inhibitory effects of CPA and ω-Cgtx GVIA were additive, but the effects of CPA and ω-Cgtx MVIIC were only partially additive. These results suggest that P/Q-type Ca²⁺ channels and other type(s) of Ca²⁺ channel(s), coupled to glutamate release, are inhibited subsequently to activation of adenosine A₁ receptors.     

Effectiveness in learning complex problem solving and salivary ion indices of psychological stress and activation

Following Hinton et al. (1992, Biol. Psychol. 33, 63–71) and Richter et al. (1995, Biol. Psychol. 39, 131–142) ionic concentration of [K⁺] in unstimulated saliva was predicted to rise with perceived challenge, while lowered [Na⁺] was expected when experiencing psychological stress (PS). Subjects had to learn an engaging complex problem-solving ‘game', via positive and negative feed-back on three ‘games' lasting 2.5–3.0 h overall. Comparisons were made between three groups: (1) high success; (2) partial success (‘strugglers'); and (3) total failure to learn. Saliva was sampled after resting and after each of three ‘games'. Successful learners had a significant rise in [K⁺] on the first ‘game' followed by a significant fall, consistent with task-challenge reaction followed by fast autonomic adaptation with successful learning. The ‘strugglers' [Na⁺] fell significantly over the ‘games', indicating mineralocorticoid-induced PS response of Na⁺ reabsorption. The ‘total failure' subjects had generally significantly higher [K⁺] than the successful ones, showing raised tonic sympathetic relative to parasympathetic activity — this outcome being interpreted from interference theories. The ‘failures' also had significantly higher tonic [Na⁺] on ‘games' — indicating low PS as predicted from McGrath's (1976) theory.     

The aminoglycoside G418 suppresses muscarinic receptor-activated calcium release in stably transfected murine N1E-115 neuroblastoma cells

The aminoglycoside G418 inhibited the release of calcium (Ca²⁺) from internal stores coupled to muscarinic receptors in murine N1E-115 neuroblastoma cells carrying the aminoglycoside resistance gene neomycin phosphotransferase (NPT). No significant effect was observed on responses coupled to histamine or bradykinin receptors. Cells were transfected using the eukaryotic expression vector pHβAPr-1-neo and selected using G418. Two groups were differentiated either in the continued presence of G418 or in the absence of G418. Carbachol (1 mM), histamine (200 μM) and bradykinin (100 nM) were administered to cells for thirty seconds and changes in [Ca²⁺]_i were measured with fluorescence video microscopy of single cells loaded with the Ca²⁺ indicator fura-2. The effects of G418 on carbachol evoked Ca²⁺ release included a 73% reduction in the number of cells responding, a two fold increase in the time to reach half-maximal response, a 35% reduction of the peak [Ca²⁺]_i in response to agonist and an elevation of resting [Ca²⁺]_i from 99± 14 nM (mean ± S.E.M.) to 155 ± 27 nM. Acute application (20 min) of G418 to transfected cells differentiated without G418 also reduced the percentage of cells responding to carbachol. This effect was less pronounced in non-transfected parent cells. Thus, the mechanism might involve a metabolite of G418 produced in cells expressing NPT. These results indicate that G418 attenuates Ca²⁺ release coupled to muscarinic receptors.     

P2 receptors in satellite glial cells in trigeminal ganglia of mice

There is strong evidence for the presence of nucleotide (P2) receptors in sensory neurons, which might play a role in the transmission of pain signals. In contrast, virtually nothing is known about P2 receptors in satellite glial cells (SGCs), which are the main glial cells in sensory ganglia. We investigated the possibility that P2 receptors exist in SGCs in murine trigeminal ganglia, using Ca²⁺ imaging, patch-clamp recordings, and immunohistochemistry. We found that ATP caused an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in SGCs. As adenosine had no effect on [Ca²⁺]_i, and the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid largely blocked the response to ATP we conclude that P1 receptors did not contribute to the responses. We obtained the following evidence that the responses to ATP were mediated by metabotropic P2Y receptors: (i) persistence of the responses in Ca²⁺-free solution, (ii) inhibition of the response by cyclopiazonic acid, (iii) [Ca²⁺]_i increases in response to the P2Y agonists uridine triphosphate, adenosine thiodiphosphate, and 2-methylthio ADP, and (iv) failure of the P2X agonist ,β-methylene ATP to elicit a response. Agonists of P2Y₁ receptors and uridine triphosphate, an agonist at P2Y₂ and P2Y₄ receptors, induced [Ca²⁺]_i increases suggesting that at least these P2Y receptor subtypes are present on SGCs. Using an antibody against the P2Y₄ receptor, we found immunopositive SGCs. Patch-clamp recordings of SGCs did not reveal any inward current due to ATP. Therefore, there was no evidence for the activation of ionotropic P2X receptors under the present conditions.

The results indicate the presence of functional nucleotide (P2Y) receptors in SGCs.     

A proton pumping pyrophosphatase in acidocalcisomes of Herpetomonas sp

Acidocalcisomes are acidic calcium storage organelles found in several microorganisms. They are characterized by their acidic nature, high electron density, high content of polyphosphates and several cations. Electron microscopy contrast tuned images of Herpetomonas sp. showed the presence of several electron dense organelles ranging from 100 to 300 nm in size. In addition, X-ray element mapping associated with energy-filtering transmission electron microscopy showed that most of the cations, namely Na, Mg, P, K, Fe and Zn, are located in their matrix. Using acridine orange as an indicator dye, a pyrophosphate-driven H⁺ uptake was measured in cells permeabilized by digitonin. This uptake has an optimal pH of 6.5–6.7 and was inhibited by sodium fluoride (NaF) and imidodiphosphate (IDP), two H⁺-pyrophosphatase inhibitors. H⁺ uptake was not promoted by ATP. Addition of 50 μM Ca²⁺ induced the release of H⁺, suggesting the presence of a Ca²⁺/H⁺ countertransport system in the membranes of the acidic compartments. Na⁺ was unable to release protons from the organelles. The pyrophosphate-dependent H⁺ uptake was dependent of ion K⁺ and inhibited by Na⁺ Herpetomonas sp. immunolabeled with monoclonal antibodies raised against a Trypanosoma cruzi V-H⁺-pyrophosphatase shows intense fluorescence in cytoplasmatic organelles of size and distribution similar to the electron-dense vacuoles.

Together, these results suggest that the electron dense organelles found in Herpetomonas sp. are homologous to the acidocalcisomes described in other trypanosomatids. They possess a vacuolar H⁺-pyrophosphatase and a Ca²⁺/H⁺ antiport. However, in contrast to the other trypanosomatids so far studied, we were not able to measure any ATP promoted H⁺ transport in the acidocalcisomes of this parasite.     

Role of intracellular calcium in fatigue in single skeletal muscle fibers isolated from the rat

The aim of the present study was to demonstrate the role of intracellular calcium ([Ca²⁺]_i) in the performance of fatigued muscle fibers isolated from the skeletal muscle of the rat. We measured developed tension of a single myocyte during short tetanic electrical stimulation of various intensities along with [Ca²⁺]_i dynamics by fura-2. The performance of individual muscle fiber was assessed by developed tension during 100 Hz tetanic stimulation (‘100 Hz force’). We regarded the muscle fiber fatigued when, after repeated tetanic stimulations, the developed tension declined to 50% of the initial level. When fatigue was induced by maximal stimulation (100 Hz tetani), the 100 Hz force measured immediately following completion of fatigue was considerably decreased (48% of control). This change in the muscle performance was associated with significant increase in the resting [Ca²⁺]_i (280% of control) and decrease in Ca²⁺ transient (54% of control). The 50% relaxation time after cessation of tetanic stimulation (RT₅₀) was also prolonged. In contrast, when fatigue was induced by low frequency electrical stimulation (30 Hz tetani), neither the 100 Hz force, RT₅₀, nor Ca²⁺ transient in fatigue were significantly different from the controls, while the resting [Ca²⁺]_i increased only slightly. These findings suggest a tight relationship between [Ca²⁺]_i and the performance of fatigued single isolated skeletal muscles. Also, the results show that performance of the fatigued muscle fiber may in part depend on the protocol used to produce muscle fatigue.