Histamine elevates free intracellular calcium in mouse retinal dopaminergic cells via H1-receptors

Abstract

Purpose: Previously, retinopetal axons containing histamine and dopaminergic neurons expressing histamine H(1)-receptor had been localized in mouse retinas using anatomic techniques. The goal of these experiments was to demonstrate that these receptors are functional.

Methods: Dopaminergic cells were acutely isolated from retinas of transgenic mice expressing red fluorescent protein under control of the tyrosine hydroxylase promoter and loaded with the calcium indicator Fura-2.

Results: Under control conditions, there were spontaneous oscillations in the levels of free intracellular calcium in dopaminergic cells. These oscillations were abolished in nominally calcium-free extracellular medium and in 1 μM tetrodotoxin, findings suggesting that the oscillations were mediated by calcium entry across the plasma membrane in response to sodium-dependent action potentials. Histamine increased the mean free intracellular calcium in the dopaminergic cells by increasing the frequency and/or amplitude of the calcium oscillations. The effects of histamine were dose-dependent and reached maximum at 5 μM. With this dose, there was a 65% increase in the mean free intracellular calcium concentration. The histamine H(1)-receptor antagonist, pyrilamine, blocked the effects of 5 μM histamine when applied at 50 μM. The selective histamine H(1)-receptor agonists, 2-(3-trifluoromethylphenyl) histamine and methylhistaprodifen significantly increased mean free intracellular calcium when applied at 5 μM.

Conclusions: Histamine released from retinopetal axons in the mouse retina can elevate intracellular calcium levels in the perikarya of dopaminergic cells via the activation of histamine H(1)-receptors.

Figure 1.

Morphology of dopaminergic neurons. (A) A whole mount image of a transgenic mouse retina in which dopaminergic cells express a red fluorescent protein. Three large dopaminergic cells are visible. A smaller red cell of a type that was not examined in this study is also observed (arrowhead). (B) Isolated dopaminergic cells (red) were fixed for 10 minutes in 4% paraformaldehyde and coverslipped in mounting medium containing the nuclear dye 4,6-diamino-2-phenylindole dihydrochloride (DAPI; blue). A stack of nine 0.5-μm optical sections was collected with a confocal scanning microscope. (C) An acutely dissociated dopaminergic cell used in an experiment. Dotted box: region of the field from which the emitted Fura-2 fluorescence signal was collected. Scale bar: (A) 20 μm; (B, C) 10 μm.

Figure 2.

Effects of external Ca²⁺ concentration on dopaminergic cells. (A) Oscillations of [Ca²⁺]_i levels were observed in normal (2 mM) external Ca²⁺. (B) Oscillations were greatly decreased in 0.5 mM Ca²⁺ and (C) were absent in nominally 0 Ca²⁺. (D) A statistically significant difference in the [Ca²⁺]_i levels was observed between the normal Ca²⁺ (n = 7) and nominally 0 Ca²⁺ (n = 6) groups (P = 0.0183). Data are the mean ± SD.

Figure 3.

Superfusion of TTX in normal external Ca²⁺ blocked the [Ca²⁺]_i oscillations. (A) Horizontal bar indicates when 1 μM TTX was present. (B) The decrease in [Ca²⁺]_i was statistically significant (n = 6; P = 0.0050). Data are the mean ± SD.

Figure 4.

Superfusion of histamine in normal external Ca²⁺ increased mean [Ca²⁺]_i. (A) Horizontal bar indicates the presence of 5 μM histamine. (B) An increase in the concentration of histamine is associated in an increase in the mean [Ca²⁺]_i. Curve is fitted from 0.5 to 5 μM histamine and is given by Δ[Ca²⁺]_i = Δ[Ca²⁺]_i,max/[1 + (K_[1/2]/histamine)ⁿ], where Δ[Ca²⁺]_i,max = 61 ± 12 nM, K_[1/2] = 1.9 ± 0.5 μM, and n = 2.1 ± 0.8. Error bars, ±SEM.

Figure 5.

Superfusion of 5 μM histamine (dark bar) in the presence of 50 μM H₁-receptor antagonist pyrilamine (light bar) in normal external Ca²⁺ had no effect on mean [Ca²⁺]_i (P = 0.9753).

Figure 6.

Superfusion of H₁-receptor agonists in normal external Ca²⁺ increased mean [Ca²⁺]_i. (A) TFMH 5 μM; (B) MH. Horizontal bars indicate when the drugs were present.