GABA induces Ca2+ transients in astrocytes

By using the Ca(2+)-sensitive indictor Fura-2/AM, the cytosolic Ca2+ levels [Ca2+]i were measured in type 1 astrocytes in rat cortical astroglial primary cultures, after stimulation with GABA, muscimol (GABAA agonist), or baclofen (GABAB agonist). We report the first evidence that stimulation of both GABAA and GABAB receptors evokes Ca2+ transients in type I astrocytes. Two types of Ca2+ responses were seen: the single-phase curve, which was the most common, and the biphasic, which consisted of an initial rise that persisted at the maximal or submaximal level. Both types of Ca2+ responses appeared with some latency. The responses were obtained in astrocytes grown for 12-16 days in culture and the response frequencies for all three agonists were 18% of the total number of examined cells. However, when the astrocytes were grown in a mixed astroglial/neuronal culture the response frequencies for all three agonists increased to 35% of the total number of examined cells. In some cells, the responses after GABA stimulation were blocked to baseline levels after exposure to bicuculline (GABAA antagonist). In other cells, bicuculline only slightly reduced the GABA-evoked responses, and the addition of phaclofen (GABAB antagonist) did not potentiate this partial inhibition. However, the muscimol-evoked rises in [Ca2+]i were completely inhibited after exposure to bicuculline, while the responses after baclofen could only be partly blocked by phaclofen. GABA evoked rises in [Ca2+]i which alternatively were inhibited (mostly) or persisted in Ca(2+)-free buffer. The rises in [Ca2+]i persisted, but were reduced, in Ca(2+)-free buffer after stimulation with muscimol, but were inhibited after baclofen stimulation. The GABA uptake blockers guvacine, 4,5,6,7-tetrahydroisoxazolo(4,5-c)pyridin-3-ol and nipecotic acid were also able to reduce the GABA-evoked rises in [Ca2+]i. However, the L-type Ca2+ channel antagonist nifedipine failed to influence on the GABA-evoked Ca2+ transients. The results suggest that type 1 astrocytes in primary culture express GABA receptors which can elevate [Ca2+]i directly or indirectly via Ca2+ channels and/or via release from internal Ca2+ stores. The results also suggest that GABA can have intracellular Ca(2+)-mobilizing sites since the GABA-evoked responses were reduced after incubation with GABA uptake blockers.     

Stimulation by DIF causes an increase of intracellular Ca2+ in Dictyostelium discoideum

Using fluorescence-activated cell sorting (FACS), we have studied the effect of the differentiation-inducing factor (DIF) on cellular Ca2+ in Dictyostelium discoideum. We have shown previously that freshly starved or postaggregation amoebae are heterogenous with respect to the amounts of cellular Ca2+ that they contain; the L or "low Ca2+" class exhibits a prespore tendency and the H or "high Ca2+" class exhibits a prestalk tendency. Upon adding DIF, within 2 min there is an approximately twofold increase in the relative fraction of amoebae falling in the H class. A major part of the increase is caused by Ca2+ influx from the extracellular medium. Therefore a rise in the level of cellular Ca2+ is an early step in the signal transduction pathway following stimulation by DIF. Also, in parallel with the cellular heterogeneity in respect of Ca2+ content, there is a heterogeneity in the response to DIF, which appears to be restricted to L cells.     

Regulation of the TRP Ca2+ channel by INAD in Drosophila photoreceptors

Drosophila vision involves a G protein-coupled phospholipase C-mediated signaling pathway that leads to membrane depolarization through activation of Na+ and Ca2+ channels. InaD mutant flies have a M442K point mutation and display a slow recovery of the Ca2+ dependent current. We report that anti-INAD antibodies coimmunoprecipitate TRP, identified by its electrophoretic mobility, cross reactivity with anti-TRP antibody, and absence in a null allele trp mutant. This interaction is abolished by the InaD point mutation in vitro and in vivo. Interaction was localized to the 19 amino acid C-terminus of TRP by overlay assays, and to the PDZ domain of INAD, encompassing the point mutation. Given the impaired electrophysiology of the InaD mutant, this novel interaction suggests that INAD functions as a regulatory subunit of the TRP Ca2+ channel.     

Ca2+ limits the development of the light response in Drosophila photoreceptors

The development of the light response was followed in Drosophila photoreceptors at 25 degrees C. In whole-cell recordings from dissociated ommatidia, responses to light were first detected at 82 h post-puparium formation; over the next 8 h sensitivity to light increased exponentially by 5 or 6 orders of magnitude. The end of this phase coincided with the maturation of the rhabdomere as measured by whole-cell capacitance. There was a modest 5-10fold further increase in sensitivity over the final 10 h of pupal development (90-100 h). During a narrow developmental time window (82-87 h) no responses could be detected using non-invasive recording techniques (electroretinogram or suction electrode), and responses to light could only be elicited in whole-cell recordings when micromolar concentrations of Ca2+ are included in the pipette. It seems unlikely that cytosolic Ca2+ per se is the limiting factor, and we suggest instead that the failure to respond to light is due to the lack of Ca2+ in the InsP3-sensitive intracellular stores and that the presence of Ca2+ in these stores is an absolute requirement for phototransduction in Drosophila.     

Cyclic ADP-ribose induces Ca2+ release from caffeine-insensitive Ca2+ pools in canine salivary gland cells

Cyclic ADP-ribose (cADPR), a novel putative messenger of the ryanodine receptor, was examined regarding its ability to mobilize Ca2+ from intracellular Ca2+ stores in isolated cells of parotid and submandibular glands of the dog. cADPR induced a rapid and transient Ca2+ release in the digitonin-permeabilized cells of salivary glands. cADPR-induced Ca2+ release was inhibited by ryanodine receptor antagonists ruthenium red, ryanodine, benzocaine, and imperatoxin inhibitor but not by the inositol 1,4,5-trisphosphate (IP3)-receptor antagonist heparin. Thapsigargin, at a concentration of 3 to 30 microM, inhibited IP3-induced Ca2+ release, while higher concentrations were required to inhibit cADPR-induced Ca2+ release. Cross-potentiation was observed between cADPR and ryanodine or SrCl2, suggesting that cADPR sensitizes the Ca2+-induced Ca2+ release mechanism. Cyclic AMP plays a stimulatory role on cADPR- and IP3-induced Ca2+ release in digitonin-permeabilized cells. Calmodulin also potentiated cADPR-induced Ca2+ release, but inhibited IP3-induced Ca2+ release. Acetylcholine and ryanodine caused the rise in intracellular free Ca2+ concentration ([Ca2+]i) in intact submandibular and parotid cells. Caffeine did not produce any increase in Ca2+ release or [Ca2+]i rise in any preparation. ADP-ribosyl cyclase activity was found in the centrifuged particulate fractions of the salivary glands. These results suggest that cADPR serves as an endogenous modulator of Ca2+ release from Ca2+ pools through a caffeine-insensitive ryanodine receptor channel, which are different from IP3-sensitive pools in canine salivary gland cells. This system is positively regulated by cyclic AMP and calmodulin.     

Intracellular Ca2+ and adaptation of voltage responses to light in Hermissenda photoreceptors

Fluorescent imaging of Ca2+ and intracellular recordings were used to assess Ca2+ increases and voltage responses during light presentations in Hermissenda B photoreceptors. Ca2+ levels increased and were sustained during a relatively long exposure to light. Repeated presentations of a brief light induced an elevation of intracellular Ca2+ that persisted throughout short interlight intervals, but which dissipated during long interlight intervals. In all instances, the magnitude of the intracellular Ca2+ signal was inversely related to the amplitude of the light-induced generator potential. Blocking of voltage-dependent Ca2+ channels did not significantly affect the magnitude of the Ca2+ signal, suggesting that the intracellular Ca2+ response arises primarily from release from intracellular stores. These results indicate that Ca2+ plays an important role in the modulation of the voltage responses to light, acting to suppress the response during repetitive or prolonged stimulation.     

The thapsigargin-evoked increase in [Ca2+]i involves an InsP3-dependent Ca2+ release process in pancreatic acinar cells

In pancreatic acinar cells, as in many other cell types, the tumour promoter thapsigargin (TG) evokes a significant increase of intracellular free Ca2+ ([Ca2+]i). The increases of [Ca2+]i evoked by TG was associated with significant changes of plasma membrane Ca2+ permeability, with [Ca2+]i values following changes in extracellular [Ca2+]. Plasma membrane Ca2+ extrusion is activated rapidly as a consequence of the rise in [Ca2+]i evoked by TG and the rate of extrusion is linearly dependent on [Ca2+]i up to 1 microM Ca2+. In contrast, the activation of the Ca2+ entry pathway is delayed and the apparent rate of Ca2+ entry is independent of [Ca2+]i. In the presence of 20 mM caffeine, which reduces the resting levels of inositol trisphosphate (InsP3), the increase of [Ca2+]i evoked by TG was significantly reduced. The reduction was manifest both as a decrease of the amplitude of the [Ca2+]i peak (30% reduction) and, more importantly, as a reduction of the apparent maximal rate of [Ca2+]i increase (from 12.3 +/- 1.0 to 6.1 +/- 0.6 nM Ca2+/s). The inhibition evoked by caffeine was reversible and the removal of caffeine in the continuous presence of TG evoked a further increase of [Ca2+]i. The amplitude of the [Ca2+]i increase upon caffeine removal was reduced as a function of the time of TG exposure. Addition of TG in the presence of 1 mM La3+, which is known to inhibit the plasma membrane Ca(2+)-activated adenosine triphosphatase, induced a much higher peak of [Ca2+]i. This increase was associated with an augmentation of the apparent rate of [Ca2+]i increase (from 12.3 +/- 1.2 to 16.1 +/- 1.9 nM Ca2+/s).(ABSTRACT TRUNCATED AT 250 WORDS)     

Light-induced cGMP-phosphodiesterase activity in intact rat retinal rod cells as revealed by rapid-freezing enzyme cytochemistry

Cyclic guanosine monophosphate (cGMP)-phosphodiesterase (PDE) in the outer segment of vertebrate retinal rod cells is one of key enzymes mediating phototransduction. We report here on light-induced PDE activity in intact rat retinal rod cells processed by rapid-freezing enzyme cytochemistry, a new morphological technique that is a combination of rapid-freezing, freeze-substitution fixation, and subsequent enzyme cytochemistry. This technique quickly immobilizes and preserves both enzyme activity and the cell ultrastructure in a state approximating living conditions; consequently, it has proved useful for cytochemical detection of light-induced PDE activity. Using this technique we observed that the catalytic site of PDE molecules in rapid-frozen outer segments was predominantly located in the extradiscal spaces; PDE activity was significantly greater in light than in darkness; and illumination elicited marked increases in PDE activity in dark-adapted cells. Light-induced PDE activity was first cytochemically detected after 3 s of illumination and reached a peak within 5 s, at which time it was in virtually the same as that seen in fully light adapted cells. In addition, cytochemical guanosine triphosphatase (GTPase) activity in dark-adapted cells, as well as corresponding PDE activity, increased in a time-dependent manner with illumination duration; this acceleration in GTPase activity closely paralleled the PDE activity. Thus, our results suggest, in part, the existence of reciprocal regulation of PDE and activated transducin alpha subunit, thereby modulating light adaptation in rod cells.     

Salicylic acid induces a cytosolic Ca2+ elevation in yeast

Cytosolic free calcium ion concentration ([Ca2+]cyt) after a salicylic acid (SA)-stimulus was monitored in cells of the yeast Saccharomyces cerevisiae expressing apoaequorin, which constitutes a Ca(2+)-sensitive luminescent protein, aequorin, when combined with coelenterazine. SA induced a transient [Ca2+]cyt elevation that was dependent on the concentration of SA and pH of the SA solution. The SA-induced [Ca2+]cyt elevation was not reduced in Ca(2+)-deficient medium, suggesting that Ca2+ was mobilized from an intracellular Ca2+ store(s). Benzoic acid, butyric acid and sorbic acid did not induced a [Ca2+]cyt elevation.     

Limbic system-associated membrane protein (LAMP) induces neurite outgrowth and intracellular Ca2+ increase in primary fetal neurons

The ability of cell adhesion molecules (CAMs) to transduce cell surface signals into intracellular responses is critical for developing neurons, particularly during axonal pathfinding and targeting. It has been suggested that different CAMs can promote neuronal outgrowth via activation of common neuronal CAM-specific second-messenger pathways, although the elements involved in this cascade could differ. Limbic system-associated membrane protein (LAMP), a member of the Ig superfamily, is a molecule that promotes cell adhesion and neurite outgrowth from specific populations of fetal neurons. In the present study, we show that LAMP can induce several types of calcium (Ca2+) signals. Neurite outgrowth is promoted if fetal hippocampal neurons are grown on lamp-transfected CHO cells. This LAMP-induced outgrowth of neurons is mediated in part through activation of L-type Ca channels. Application of soluble LAMP to cultures of fetal hippocampal neurons caused a sustained (up to 60 min) elevation of intracellular Ca2+ as measured by fluo-3 fluorescence on a confocal microscope. The number of responding hippocampal neurons was initially low, but increased with age in culture and the [Ca2+]i elevation was only partially decreased by an L-type Ca(2+)-channel blocker. In contrast, at all times in culture, only a small fraction of neurons from visual cortex responded to LAMP application and only with transient elevation of cytosolic Ca2+ (< 15 min). Based on these observations, LAMP appears to function primarily through homophilic interactions and acts in part by modulating intracellular Ca2+ levels during neurite outgrowth by increasing the Ca2+ influx through L-type calcium channels, but has additional effects on intracellular Ca2+ signaling at later developmental stages.     

Effects of removing extracellular Ca2+ on excitation and adaptation in Limulus ventral photoreceptors

In Limulus ventral photoreceptors, removing extracellular calcium (Ca2+o) increases the median latency of light-evoked discrete waves. Removal greatly lengthens the time-to-peak of responses in the dark-adapted cell, but not in the light-adapted cell. Removal does not block light-adaptation or the light-induced rise in intracellular calcium (Ca2+i). These results are interpreted in terms of the hypothesis that both sensitivity and the kinetics of excitation are dependent on Ca2+i, and that Ca2+i is dependent on Ca2+o in the dark-adapted cell, but in the light is dependent largely on Ca2+ released from intracellular compartments.     

ATP, thapsigargin and cAMP increase Ca2+ in rat hepatocytes by activating three different Ca2+ influx pathways

OBJECTIVE: To evaluate the risk and efficacy of pulmonary lobectomy in dogs with pneumonia. DESIGN: Retrospective study. ANIMALS: 59 dogs with pneumonia. PROCEDURE: Review of medical records and telephone conversations. RESULTS: 54.2% of dogs had resolution of pneumonia after lobectomy, 20.3% died in the perioperative period, and 25.4% survived the perioperative period but pneumonia did not resolve. Pneumonia was caused by bacteria (25 dogs), fungi (12), foreign bodies (8), parasites (1), viruses (1), and allergies (1). In 11 dogs, the etiologic agent was not isolated. Bacterial or fungal pneumonias were significantly less likely to resolve compared with foreign body pneumonia or when an etiologic agent was not isolated. Perioperative mortality rate increased significantly with an increase in number of pulmonary lobes removed. Complications during surgery significantly increased perioperative mortality rate. Surgical era (1972 to 1983 vs 1984 to 1994) was a significant predictor of mortality, with the odds of dying in the perioperative period being 11 times greater between 1972 to 1983. The odds of failure to resolve pneumonia was 3 times greater during 1972 to 1983. CLINICAL IMPLICATIONS: Number of pulmonary lobes removed and complications during surgery significantly affect perioperative mortality rate. Identification of etiologic agents may help in predicting dogs likely to resolve pneumonia after surgery.     

Cellular Ca2+ ATPase activity in diabetes mellitus

Basal and maximal Ca2+ ATPase activity was studied in erythrocytes of 29 healthy controls, 15 patients with insulin-dependent diabetes mellitus (IDDM) and 22 patients with non-insulin-dependent diabetes mellitus (NIDDM). Basal and maximal Ca2+ ATPase activity was significantly decreased in insulin-dependent diabetes mellitus (8.4 +/- 0.5 and 22.5 +/- 1.1 pmol/10(6) RBC/min) and non-insulin-dependent diabetes mellitus (7.3 +/- 1.0 and 18.6 +/- 1.8 pmol/10(6) RBC/min) compared to healthy controls (9.3 +/- 1.0 and 24.6 +/- 1.1 pmol/10(6) RBC/min). Maximal Ca2+ ATPase activity showed a significant correlation to systolic blood pressure in both insulin-dependent diabetes mellitus and non-insulin-dependent diabetes mellitus. There was no significant correlation of maximal Ca2+ ATPase activity to fasting serum glucose concentration and to HbA1 levels. Maximal Ca2+ ATPase activity was significantly correlated to creatinine clearance in non-insulin-dependent diabetes mellitus, but not in insulin-dependent diabetes mellitus. It is concluded that a decreased cellular Ca2+ ATPase activity may predispose to the development of hypertension in diabetes mellitus.     

Cellular and molecular regulation of serotonin N-acetyltransferase activity in chicken retinal photoreceptors

Serotonin N-acetyltransferase (AA-NAT; arylalkylamine N-acetyltransferase; EC 2.3.1.87) is the penultimate enzyme in melatonin synthesis and large changes in the activity of this enzyme appear to regulate the rhythm in melatonin synthesis. Recent advances have made it possible to study the mRNA encoding chicken AA-NAT, which has only been detected in the retina and pineal gland. Within the retina, AA-NAT mRNA is expressed primarily in photoreceptors. The levels of chicken retinal AA-NAT mRNA and activity exhibit 24-hour rhythms with peaks at night. These rhythms appear to reflect circadian clock control of AA-NAT mRNA abundance and independent effects of light and darkness on both mRNA levels and enzyme activity. The effects of darkness and light may occur through alterations in cAMP-dependent protein phosphorylation, which increases AA-NAT activity in photoreceptor cell cultures. The cAMP-dependent increase of AA-NAT enzyme activity reflects, at least in part, increased mRNA levels and inhibition of enzyme inactivation by a posttranslational mechanism. This review discusses a hypothetical model for the cellular and molecular regulation of AA-NAT activity by circadian oscillators and light in chicken retinal photoreceptor cells.     

PLA2 activity regulates Ca2+ storage-dependent cellular proliferation

The objective of this study is to determine the role of arachidonic acid (AA) in cell proliferation by inhibiting AA synthetic enzyme phospholipase A2 (PLA2) and to determine its involvement in the role of the second messenger intracellular calcium (Ca2+). Methods used to determine the effects on proliferation of cell cultures of primary meningioma and astrocytoma U373-MG included treatment with micromolar concentrations of PLA2 inhibitors 4-bromophenacylbromide and quinacrine. Effects of these drugs on proliferation were further investigated by the application of concentrations that inhibit growth by 50% while antagonizing these agents with AA replacement. Free cytosolic Ca2+ was measured with the use of fluorescent dye Fura-2 during PLA2 agonist/antagonist studies. These Ca2+ measurements were performed in the absence of extracellular Ca2+ to identify the contribution of intracellular Ca2+ sources. PLA2 inhibition resulted in decreased growth of cultured astrocytoma and meningioma cells in a dose-dependent manner in the micromolar range. This inhibitory effect was antagonized by the addition of AA. PLA2 inhibition caused an elevation of basal-cytosolic-free [Ca2+] while depleting internal Ca2+ stores. These Ca2+ changes were also antagonized by the addition of AA. In conclusion, these results demonstrate that AA, a PLA2 enzyme product, is involved in regulating the growth rate of these cell types. The PLA2 pathway also regulates the maintenance of the internal Ca2+ stores. Ca2+ is known to be a growth-related intracellular second messenger. These results suggest that the growth regulatory functions of AA are mediated by Ca2+-dependent mechanisms.     

Ca2+ ATPase activity in essential and renal hypertension

In 15 patients with essential hypertension, 16 patients with renal hypertension and in 12 healthy subjects Ca2+ ATPase activity was determined in red blood cells both in the basal state and after maximal stimulation with calmodulin. Normal subjects showed a basal and maximal activity of 7.1 +/- 3.6 and 16.0 +/- 2.3 pmol phosphate/min.10(6) RBC, respectively. Renal hypertensives had a similar basal Ca2+ ATPase activity (5.4 +/- 4.1 pmol phosphate/min.10(6) RBC) and a lowered maximal Ca2+ ATPase activity (9.8 +/- 5.4 pmol phosphate/min.10(6) RBC, p < 0.05). In essential hypertensives basal and maximal Ca2+ ATPase activity was 9.0 +/- 5.3 and 35.4 +/- 14.4 pmol phosphate/min.10(6) RBC, respectively, the latter being significantly increased (p < 0.01). This finding, which is in contrast to earlier results indicating a lowered Ca2+ ATPase activity in essential hypertension, may be explained as a consequence of an increased Ca2+ influx in essential hypertension. A lowered Ca2+ ATPase activity does not seem to be involved in the pathogenesis of essential hypertension.     

Increase of empty pool-activated Ca2+ influx using an intracellular Ca2+ chelating agent

We demonstrate here that stimulated 45Ca2+ influx in A7r5 vascular smooth muscle cells induced either by receptor activation with [Arg]8 vasopressin or by the SR-Ca(2+)-ATPase inhibitor thapsigargin was increased more than threefold if cells were preloaded with the intracellular calcium chelator BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). The increased influx is probably due to an attenuation of negative feedback of Ca2+ on its own entry accompanied by increased Ca2+ storage capacity of BAPTA-loaded cells leading to diminished cellular Ca2+ release. We propose that BAPTA preloading could be a useful approach to investigate receptor-induced Ca2+ influx.     

Region-specific activity of the plasma membrane Ca2+ pump and delayed activation of Ca2+ entry characterize the polarized, agonist-evoked Ca2+ signals in exocrine cells

The initial release of Ca2+ from the intracellular Ca2+ stores is followed by a second phase during which the agonist-dependent Ca2+ response becomes sensitive to the extracellular Ca2+, indicating the involvement of the plasma membrane (PM) Ca2+ transport systems. The time course of activation of these transport systems, which consist of both Ca2+ extrusion and Ca2+ entry pathways, is not well established. To investigate the participation of these processes during the agonist-evoked Ca2+ response, isolated pancreatic acinar cells were exposed to maximal concentrations of an inositol 1,4,5-trisphosphate-mobilizing agonist (acetylcholine, 10 microM) in different experimental conditions. Following the increase of [Ca2+]i, there was an almost immediate activation of the PM Ca2+ extrusion system, and maximal activity was reached within less than 2s. The rate of Ca2+ extrusion was dependent on the level of [Ca2+]i, with a steep activation at values just above the resting [Ca2+]i and reached a plateau value at 700 nM Ca2+. In contrast, the PM Ca2+ entry pathway was activated with a much slower time course. There was also a delay of 3-4 s between the maximal effective depletion of the intracellular Ca2+ stores and the activation of this entry pathway. By use of digital imaging data, the PM Ca2+ transport systems were also analyzed independently in two regions of the cells, the lumenal and the basal poles. With respect to the activation of the Ca2+ entry pathways, no significant difference existed between these two regions. In contrast, the PM Ca2+ pump displayed a different pattern of activity in these regions. In the basal pole, the pump activity was more sensitive to changes of [Ca2+]i and had a higher maximal activity. Also, in the lumenal pole, the pump became saturated at values of [Ca2+]i around 700 nM, whereas at the basal pole [Ca2+]i had a biphasic effect on the pump activity, and higher [Ca2+]i inhibited the pump. It is argued that these differences in sensitivity to the levels of [Ca2+]i and the different relationship between [Ca2+]i and the rate of extrusion at the two functional poles of the pancreatic acinar cells indicate that the plasma membrane Ca2+ ATPase might play an important role in the polarization of the Ca2+ response.     

Substance P induces brief, localized increase in [Ca2+]i in dorsal horn neurons

We determined the spatial and temporal dynamics of the increase in intracellular Ca2+ levels [Ca2+]i produced by substance P (SP) in dorsal horn neurons. A microinjection technique was used to apply minute amounts of SP to small areas of cultured neurons loaded with the Ca2+ indicator fura-2. Five successive applications of SP to the soma produced short-lasting (< 50 s) increases in [Ca2+]i that became gradually smaller, indicating receptor desensitization. Focal application of SP to a distal locus in a neurite produced a brief (12 s) increase in [Ca2+]i that travelled down the dendrite but did not spread into cell soma. Prolonged application of SP to these neurons caused the appearance of varicosities in their dendrites.