Histamine-induced calcium entry in rat cerebellar astrocytes: evidence for capacitative and non-capacitative mechanisms

Abstract

We have investigated the effects of histamine on the intracellular calcium concentration ([Ca2+]i) of cultured rat cerebellar astrocytes using fura-2-based Ca2+ imaging microscopy. Most of the cells responded to the application of histamine with an increase in [Ca2+]i which was antagonized by the H1 receptor blocker mepyramine. When histamine was applied for several minutes, the majority of the cells displayed a biphasic Ca2+ response consisting of an initial transient peak and a sustained component. In contrast to the initial transient [Ca2+]i response, the sustained, receptor-activated increase in [Ca2+]i was rapidly abolished by chelation of extracellular Ca2+ or addition of Ni2+, Mn2+, Co2+ and Zn2+, but was unaffected by nifedipine, an antagonist of L-type voltage-activated Ca2+ channels. These data indicate that the sustained increase in [Ca2+]i was dependent on Ca2+ influx. When intracellular Ca2+ stores were emptied by prolonged application of histamine in Ca2+-free conditions, Ca2+ re-addition after removal of the agonist did not lead to an 'overshoot' of [Ca2+]i indicative of store-operated Ca2+ influx. However, Ca2+ stores were refilled despite the absence of any substantial change in the fura-2 signal. Depletion of intracellular Ca2+ stores using cyclopiazonic acid in Ca2+-free saline and subsequent re-addition of Ca2+ to the saline resulted in an increase in [Ca2+]i that was significantly enhanced in the presence of histamine. The results suggest that besides capacitative mechanisms, a non-capacitative, voltage-independent pathway is involved in histamine-induced Ca2+ entry into cultured rat cerebellar astrocytes.

Figure 4. [Ca²⁺]_i increases induced by Ca²⁺ removal/Ca²⁺ re-addition protocols are unaffected by prior application of histamine

A, cytosolic Ca²⁺ changes upon removal (0 mm Ca²⁺, 0.5 mm EGTA) and re-addition (2 mm Ca²⁺) of external Ca²⁺ with (dotted trace) or without (continuous trace) prior application of histamine (HA, 100 μm). Each trace is the mean of 19 experiments. The experimental protocol is illustrated by representative traces in the insets above (left, with histamine; right, without histamine). Histamine was applied either for 2 min or for 6 min. B, Ca²⁺ transients monitored as fura-2 ratio induced by histamine in the absence (left) and in the presence (right) of external Ca²⁺, showing that the Ca²⁺ stores refilled after re-addition of external Ca²⁺.

Figure 5. Manganese reversibly inhibits Ca²⁺ entry

Ca²⁺ responses monitored as fura-2 ratio. The sustained component evoked by histamine and by re-addition of external Ca²⁺ in the presence of cyclopiazonic acid (CPA) was reversibly reduced by 1 mm Mn²⁺.

Figure 6. Capacitative and non-capacitative Ca²⁺ entry in store-depleted cells (1)

Capacitative Ca²⁺ entry following repeated removal and re-addition of external Ca²⁺ to cells treated with CPA (10 μm). A, two consecutive responses to Ca²⁺ re-addition after treatment with CPA in a Ca²⁺-free solution. B, similar protocol to that in A, but with the first Ca²⁺ step in the presence of histamine. C, statistical analysis of all experiments. The amplitudes of the cytosolic [Ca²⁺] increases upon the second Ca²⁺ re-addition were normalized to the second [Ca²⁺]_i increase, which was taken as 100 %. When compared to control (1st entry), histamine produced a significant (* P < 0.01) enhancement of the amplitude of the first [Ca²⁺]_i increase.

Figure 7. Capacitative and non-capacitative Ca²⁺ entry in store-depleted cells (2)

Capacitative Ca²⁺ entry following repeated removal and re-addition of external Ca²⁺ to cells treated with CPA (10 μm). A, two consecutive responses to Ca²⁺ re-addition after treatment with CPA in a Ca²⁺-free solution. B, similar protocol to that in A, but with the second Ca²⁺ step in the presence of histamine. C, statistical analysis of all experiments. The amplitudes of the cytosolic [Ca²⁺] increases upon the second Ca²⁺ re-addition were normalized to the first [Ca²⁺]_i increase of the protocol which was taken as 100 %. When compared to control (2nd entry), histamine produced a significant (*P < 0.001) enhancement of the amplitude of the second [Ca²⁺]_i increase.

Figure 1. Histamine-induced [Ca²⁺]_i increases in cultured astrocytes

A, response patterns obtained with varying durations of agonist application: transient response to short application of 100 μm histamine (HA, 5 s, left panel); biphasic (middle panel) or oscillatory responses (right panel) upon longer applications of histamine. The panels show data from three different cells. B, concentration dependence of the histamine-induced [Ca²⁺]_i increases. Because response amplitudes decline upon repetitive stimulation with the same agonist concentration, the dose-response relation was determined using the following protocol. Two applications of a single test concentration of histamine (between 0.3 and 1000 μm) were separated by an application of 100 μm histamine. The duration of each application was 5 s, the interval between successive histamine applications 3 min. Peak amplitudes of the responses to the test concentrations were normalized to the response to 100 μm histamine and averaged. A fit to the dose-response curve yielded an EC₅₀ value of 6.4 ± 0.8 μm and a maximally effective histamine concentration of 100 μm. Error bars indicate s.e.m.; the number of experiments (n) is indicated above the error bars.

Figure 2. The sustained component of the [Ca²⁺]_i response to prolonged application of histamine is due to Ca²⁺ entry

A, effect of removal and re-addition of extracellular Ca²⁺ during the sustained phase of the histamine-induced [Ca²⁺]_i response. B, response to a prolonged application of histamine in Ca²⁺-free extracellular solution. Re-addition of Ca²⁺ in the continuous presence of the agonist induced a sustained increase in [Ca²⁺]_i. C, reversible inhibition of the plateau phase of the histamine-induced [Ca²⁺]_i increase by Ni²⁺ (1 mm). D, effect of nifedipine (10 μm), an inhibitor of L-type voltage-activated Ca²⁺ channels, on the histamine-induced Ca²⁺ plateau.

Figure 3. Pharmacological characterization of the sustained component of the histamine-induced [Ca²⁺]_i response

A, inhibition of the plateau component of the histamine-induced [Ca²⁺]_i increase by the H₁ receptor antagonist mepyramine (2 μm). B, abolition of the plateau component of the histamine-induced [Ca²⁺]_i increase by the phospholipase C inhibitor U73122 (5 μm).