Dopamine stimulates salivary duct cells in the cockroach Periplaneta americana.
This study examines whether the salivary duct cells of the cockroach Periplaneta americana can be stimulated by the neurotransmitters dopamine and serotonin. We have carried out digital Ca2+-imaging experiments using the Ca2+-sensitive dye fura-2 and conventional intracellular recordings from isolated salivary glands. Dopamine evokes a slow, almost tonic, and reversible dose-dependent elevation in [Ca2+]i in the duct cells. Upon stimulation with 10(-)6 mol l-1 dopamine, [Ca2+]i rises from 48+/-4 nmol l-1 to 311+/-43 nmol l-1 (mean +/- s.e.m., N=18) within 200-300 s. The dopamine-induced elevation in [Ca2+]i is absent in Ca2+-free saline and is blocked by 10(-)4 mol l-1 La3+, indicating that dopamine induces an influx of Ca2+ across the basolateral membrane of the duct cells. Stimulation with 10(-)6 mol l-1 dopamine causes the basolateral membrane to depolarize from -67+/-1 to -41+/-2 mV (N=10). This depolarization is also blocked by La3+ and is abolished when Na+ in the bath solution is reduced to 10 mmol l-1. Serotonin affects neither [Ca2+]i nor the basolateral membrane potential of the duct cells. These data indicate that the neurotransmitter dopamine, which has previously been shown to stimulate fluid secretion from the glands, also stimulates the salivary duct cells, suggesting that dopamine controls their most probable function, the modification of primary saliva. (+info)
Increased calcium entry into dystrophin-deficient muscle fibres of MDX and ADR-MDX mice is reduced by ion channel blockers.
1. Single fibres were enzymatically isolated from interosseus muscles of dystrophic MDX mice, myotonic-dystrophic double mutant ADR-MDX mice and C57BL/10 controls. The fibres were kept in cell culture for up to 2 weeks for the study of Ca2+ homeostasis and sarcolemmal Ca2+ permeability. 2. Resting levels of intracellular free Ca2+, determined with the fluorescent Ca2+ indicator fura-2, were slightly higher in MDX (63 +/- 20 nM; means +/- s.d.; n = 454 analysed fibres) and ADR-MDX (65 +/- 12 nM; n = 87) fibres than in controls (51 +/- 20 nM; n = 265). 3. The amplitudes of electrically induced Ca2+ transients did not differ between MDX fibres and controls. Decay time constants of Ca2+ transients ranged between 10 and 55 ms in both genotypes. In 50 % of MDX fibres (n = 68), but in only 20 % of controls (n = 54), the decay time constants were > 35 ms. 4. Bath application of Mn2+ resulted in a progressive quench of fura-2 fluorescence emitted from the fibres. The quench rate was about 2 times higher in MDX fibres (3.98 +/- 1.9 % min-1; n = 275) than in controls (2.03 +/- 1.4 % min-1; n = 204). The quench rate in ADR-MDX fibres (2.49 +/- 1.4 % min-1; n = 87) was closer to that of controls. 5. The Mn2+ influx into MDX fibres was reduced to 10 % by Gd3+, to 19 % by La3+ and to 47 % by Ni2+ (all at 50 microM). Bath application of 50 microM amiloride inhibited the Mn2+ influx to 37 %. 6. We conclude that in isolated, resting MDX muscle fibres the membrane permeability for divalent cations is increased. The presumed additional influx of Ca2+ occurs through ion channels, but is well compensated for by effective cellular Ca2+ transport systems. The milder dystrophic phenotype of ADR-MDX mice is correlated with a smaller increase of their sarcolemmal Ca2+ permeability. (+info)
Thapsigargin inhibits a potassium conductance and stimulates calcium influx in the intact rat lens.
1. An increase in lens cell calcium has long been associated with cortical cataract. Recently, it has been shown that thapsigargin induces a rise in lens cell calcium by release from endoplasmic reticulum stores. The effects of this rise on the optical and membrane characteristics of the lens were studied in the isolated rat lens. 2. The electrical characteristics of the isolated, perifused rat lens were measured using a two-internal microelectrode technique that permits measurement of plasma membrane conductance (Gm), membrane potential (Vm) and junctional conductance in the intact lens. 3. Thapsigargin (1 microM) induced a rapid overall depolarization of Vm that was accompanied by first a decrease and then an increase in Gm. 4. Replacing external Na+ with tetraethylammonium (TEA) abolished the decrease in Gm. However, a transient increase phase was still observed. 5. The changes in conductance were further characterized by measuring 22Na+ and 45Ca2+ influxes into the isolated lens. Thapsigargin (1 microM) induced a transient increase in 45Ca2+, but did not affect Na+ influx. 6. The Ca2+ channel blocker La3+ (10 microM) totally inhibited the thapsigargin-induced Ca2+ influx. It also blocked the increase in Gm observed in control and in Na+-free-TEA medium. In the absence of external calcium, thapsigargin induced a small depolarization in Vm. 7. These data indicate that thapsigargin induces both a decrease in K+ conductance and an increase in Ca2+ conductance. These probably result from release of stored Ca2+ and subsequent activation of store-operated Ca2+ channels (capacitative Ca2+ entry). 8. Thapsigargin application over the time course of these experiments (24 h) had no effect on junctional conductance or on the transparency of the lens. (+info)
Sequential activation of different Ca2+ entry pathways upon cholinergic stimulation in mouse pancreatic acinar cells.
1. We have studied capacitative calcium entry (CCE) under different experimental conditions in fura-2-loaded mouse pancreatic acinar cells by digital microscopic fluorimetry. CCE was investigated during [Ca2+]i decay after cell stimulation with a supramaximal concentration of ACh (10 microM) or during Ca2+ readmission in Ca2+-depleted cells (pretreated with thapsigargin or ACh). 2. La3+ and Zn2+ (100 microM) inhibited CCE during Ca2+ readmission but had negligible effects during ACh decay. In contrast flufenamic acid (100 microM), an inhibitor of non-selective cation channels, genistein (10 microM), a broad-range tyrosine kinase inhibitor, and piceatannol (10 microM), an inhibitor specific for non-receptor Syk tyrosine kinase, inhibited CCE during ACh decay but not during Ca2+ reintroduction. 3. Simultaneous detection of Mn2+ entry and [Ca2+]i measurement showed that, in the presence of extracellular calcium, application of 100 microM Mn2+ during ACh decay resulted in manganese influx without alteration of calcium influx, whilst when applied during Ca2+ readmission, Mn2+ entry was significantly smaller and induced a clear inhibition of CCE. 4. Application of the specific protein kinase C inhibitor GF109293X (3 microM) reduced CCE in Ca2+-depleted cells, whereas the activator phorbol 12-myristate, 13-acetate (3 microM) increased Ca2+ entry. 5. Based on these results we propose that cholinergic stimulation of mouse pancreatic acinar cells induces Ca2+ influx with an initial phase operated by a non-specific cation channel, sensitive to flufenamic acid and tyrosine kinase inhibitors but insensitive to lanthanum and divalent cations, followed by a moderately Ca2+-selective conductance inhibited by lanthanum and divalent cations. (+info)
Calcium block of Na+ channels and its effect on closing rate.
Calcium ion transiently blocks Na+ channels, and it shortens the time course for closing of their activation gates. We examined the relation between block and closing kinetics by using the Na+ channels natively expressed in GH3 cells, a clonal line of rat pituitary cells. To simplify analysis, inactivation of the Na+ channels was destroyed by including papain in the internal medium. All divalent cations tested, and trivalent La3+, blocked a progressively larger fraction of the channels as their concentration increased, and they accelerated the closing of the Na+ channel activation gate. For calcium, the most extensively studied cation, there is an approximately linear relation between the fraction of the channels that are calcium-blocked and the closing rate. Extrapolation of the data to very low calcium suggests that closing rate is near zero when there is no block. Analysis shows that, almost with certainty, the channels can close when occupied by calcium. The analysis further suggests that the channels close preferentially or exclusively from the calcium-blocked state. (+info)
Isolation and characterization of a Ca2+ -binding polysaccharide associated with coccoliths of Emiliania huxleyi (Lohmann) Kamptner.
C-occolithophoridae, a group of mostly unicellular algae, possess a cell wall containing calcified plates, called coccoliths. The coccoliths from the species Emilania huxleyi (Lohmann) Kamptner contain a water-soluble acid polysaccharide. In this paper we describe the isolation and some characteristic properties of the polysaccharide, in particular its Ca2+ -binding capacity. A large-scale cultivation of the Coccolithophoridae was worked out and a new procedure for isolating coccoliths was developed. The polysaccharide obtained from the coccoliths contained two types of monobasic acid groups in a total amount of 1.8 mumol/mg polysaccharide. One type consisted of weakly acid groups which were identified as uronic acids. The nature of the stronger acid groups remains to be established. The ratio between the respective groups was 1:0.8. Studies with 45Ca2+ demonstrated that the isolated polysaccharide is capable of binding Ca2+. Equilibrium dialysis revealed that the maximum amount of Ca2+ which can be bound in 0.92 +/- 0.05 mumol/mg polysaccharide. Flow-rate dialysis experiments strongly suggested the presence of two classes of Ca2+ -binding sites differing in affinity for Ca2+. High-affinity sites (dissociation constant Kd for Ca2+ :2.2 +/- 1.0 X 10(-5) M) were found to be present in amounts (0.38 +/- 0.04 mumol/mg polysaccharide) approximately equivalent to the strongly acid monovalent groups mentioned above (0.8 mumol/mg polysaccharide). Low-affinity sites (Kd for Ca2+: -11 +/- 39 X 10(-5) M) were estimated at 0.74 +/- 0.11 mumol/mg polysaccharide. Although this figure could be determined less accurately, it is suggested that the uronic acids (1.0 mumol/mg polysaccharide) are identical to the low-affinity sites. Preferential binding of Ca2+ occurred in a 100-fold excess of Na+ and Mg2+ as was shown by gel filtration. A 100-fold excess of Sr2+ inhibited Ca2+ binding to a great extent while no Ca2+ was bound in the presence of an equimolar amount of La3+. The dissociation constants of the high-affinity sites for Na+, Mg2+, Sr2+ and La3+ (in the presence of Ca2+) were determined with the flow-rate dialysis technique. They confirm the order of binding preference found with gel filtration. A polysaccharide with similar properties could be isolated from subfossil coccoliths of E. hyxleyi (about 1000 years old). The possible role of the polysaccharide as a heterogeneous matrix in coccolith formation is discussed. (+info)
Evidence for calcium inactivation during hormone release in the rat neurohypophysis.
1. A study has been made of the relationship between 45Ca uptake into and hormone release from isolated rat neurohypophyses incubated in vitro. 2. Hormone secretion is triggered by high-K (56 mM) but long exposure to the stimulus does not generate a maintained release of hormone. 3. When hormone release began to wane, addition of Ba of La increased hormone output which suggests that the decline in output did not result from depletion of the neurosecretory granules at the nerve terminals. 4. 45Ca uptake is enhanced in the presence of high-K concentration, but the initial high rate declines during long exposure to the potassium stimulus with a time constant similar to that of the decline in hormone release. 5. After a period of incubation in a K-rich, calcium-free medium, addition of calcium to the medium induced hormone release. The magnitude of this release was dependent on the time of exposure to excess potassium. 6. After inactivation of secretion, mobilization of internal calcium by means of a calcium ionophore increased hormone release. (+info)
Manganese-dependent propagated action potentials and their depression by electrical stimulation in guinea-pig myocardium perfused by sodium-free media.
1. Propagated action potentials were recorded in right ventricular papillary muscles from guinea-pig heart while exposed to Na-free, Ca-free and Mg-free solutions containing Mn. 2. When Na was totally replaced by 95 mM-Mn the overshoot was about 45 mV while the resting potential was about -90mV. 3. The overshoot of action potentials was increased by about 20-30 mV per tenfold increase of Mn concentration over the range of 2-50 mM. 4. Similar increases of overshoots with increasing of Mn concentration also occurred in the presence of 0-6 mM-Ca. Increasing of Ca from 5 to 20 mM had little influence on the overshoot but shortened the duration of the Mn-dependent action potential in the presence of 5 mM-MN. 5 Mn-dependent action potentials were not depressed by 3 X 10(5) M tetrodotoxin but by La. 6. These results suggest that Mn passes through the slow inward current channel to generate the action potential seen under the Na-free condition. 7. The overshoot and duration of the Mn-dependent action potential decreased with stimulation. At stimulus frequencies (Hz) of 0-5, 0-2, 0-1, 0-017 and 0-0033 the overshoot of action potential in 5 mM-Mn Tyrode decreased by 0-5-1 mV per an action potential. This depression of the action potential is explained by assuming intracellular accumulation of Mn. (+info)