Single-cell Calcium Imaging Research Articles

Role of Na +/H + exchangers, excitatory amino acid receptors and voltage-operated Ca 2+ channels in human immunodeficiency virus type 1 gp120-mediated increases in intracellular Ca 2+ in human neurons and astrocytes

Human immunodeficiency virus type 1 (HIV-1) dementia is the commonest form of dementia in North American people less than 60 years of age. HIV-1 envelope glycoprotein gp120 has been implicated in the neurotoxicity observed in, and the pathogenesis of, HIV-1 dementia. Recombinant gp120 (gp120) was pressure-applied on to cultured human fetal neurons and astrocytes and, by using single-cell calcium imaging, we determined the mechanisms responsible for gp120-induced increases in the levels of intracellular calcium ([Ca 2+] i). Significant dose-related increases in [Ca 2+] i were observed in neurons and astrocytes. In neurons, 5 pM gp120 increased [Ca 2+] i by 290±13 nM and increases of 2210±211 nM were found at 209 nM, the highest concentration of gp120 tested. The apparent ec 50 value for gp120 of 223±40 pM in neurons was not significantly different from that in astrocytes. Immunoelution of gp120 with polyclonal anti-gp120 and Ca 2+-free conditions blocked increases in [Ca 2+] i by gp120. Increases in [Ca 2+] i were significantly ( P<0.005) attenuated by the Na +/H + exchange blocker 5-( N-methyl- N-isobutyl)-amiloride in neurons and astrocytes. The L-type calcium channel blockers nimodipine, diltiazem and CdCl 2+NiCl 2 significantly ( P<0.005) reduced increases in [Ca 2+] i in neurons, but not astrocytes. Increases in [Ca 2+] i by gp120 were not significantly affected by blockers of N-, P- and Q-type calcium channels. The N-methyl- d-aspartate receptor antagonists (±)-2-amino-5-phosphonopentanoic acid (AP5), memantine and dizocilpine significantly ( P<0.01) lowered gp120-induced increases in [Ca 2+] i in neurons. AP5 and memantine, but not dizocilpine, significantly ( P<0.01) reduced increases in [Ca 2+] i by gp120 in astrocytes. Gp120 appears to activate astrocyte Na +/H + exchangers to release glutamate and potassium and, subsequent to this, increases in [Ca 2+] i in neurons and astrocytes result from activation of excitatory amino acid receptors on astrocytes and neurons, and voltage-operated calcium channels on neurons. Drugs that block gp120-induced changes in [Ca 2+] i in neurons and astrocytes may help in the treatment of HIV-1 dementia.

Ca2+-Induced Increases in Steady-State Concentrations of Intracellular Calcium Are Not Required for Inhibition of Parathyroid Hormone Secretion

It has been well established that increases in extracellular calcium concentration ([Ca2+]) inhibit parathyroid hormone (PTH) secretion. The effects of [Ca2+] are mediated through a G-protein-coupled receptor that has been cloned and characterized. Additionally, it has been demonstrated in parathyroid cells that an increase in [Ca2+] results in an increase in steady-state levels of intracellular calcium ([Ca2+]i). At present, it has not been fully resolved whether changes in [Ca2+]i are related to changes in PTH secretion. In the current study, the effect of increased [Ca2+] on PTH secretion and the connection regarding changes in concentrations of intracellular calcium [Ca2+]i have been examined in primary cultures of bovine parathyroid cells. PTH secretion was measured by radioimmunoassay and intracellular calcium was determined by single cell calcium imaging. Bovine parathyroid cells pre-incubated with either 0.5 or 1 mM calcium responded to rapid increases in [Ca2+] (≥0.5 mM) with an immediate and sustained increase in steady-state levels of [Ca2+]i that persisted for time intervals greater than 15 minutes. Although the magnitude of the sustained increase in [Ca2+]i varied among individual cells (∼40% to >300%), the overall pattern and course of time were similar in all cells examined (n = 142). In all trials, [Ca2+]i immediately returned to baseline levels following the addition of the calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Additional control studies, however, suggest that sustained increases in [Ca2+]i do not correlate with regulation of parathyroid hormone secretion. Sustained elevations of [Ca2+]i were not observed when [Ca2+] was gradually increased by the addition of 0.1 mM increments at 1 minute intervals. Furthermore, the effect on inhibition of PTH secretion was the same regardless of whether [Ca2+] was increased by gradual or rapid addition.

Receptor-activated Ca influx in human airway smooth muscle: use of Ca imaging and perforated patch-clamp techniques.

Previous studies have demonstrated a dihydropyridine-insensitive, receptor-activated calcium influx pathway in cultured human airway smooth muscle (ASM) cells. To further define the biophysical characteristics of this pathway, the relationship between membrane potential and cytosolic free calcium ([Ca2+]i) was studied with the combined methods of the patch-clamp technique and single cell calcium imaging. The nystatin perforated-patch method was used to maintain normal intracellular calcium buffering and receptor-activated signal transduction processes in voltage-clamped cells. Single voltage-clamped human ASM cells responded to exposure to histamine (200 microM) with an initial transient rise in [Ca2+]i followed by a secondary sustained elevation that was dependent on extracellular calcium. Before agonist activation, step changes in holding potential produced only slight changes in [Ca2+]i, whereas, after activation, cells developed a sustained rise in [Ca2+]i that showed a large variation as a function of membrane potential. Depolarization from -80 to 0 mV caused a fall in the steady-state [Ca2+]i to basal levels or slightly below. Repolarization to -80 mV caused the redevelopment of the sustained phase of the calcium response. When calcium was removed from the extracellular fluid by the addition of a stoichiometric excess of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), the voltage dependence of the sustained phase was abolished. In a series of experiments, agonist addition evoked a 54-fold increase in the voltage dependence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation of Retinal Mice Neurospheres by Trophic and Contact Factors: Functional Identification Using Single Cell Calcium Imaging

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