Adverse effects of an active fragment of parathyroid hormone on rat hippocampal organotypic cultures

Abstract

Adverse effects of an active fragment of parathyroid hormone (PTH(1 - 34)), a blood Ca(2+) level-regulating hormone, were examined using rat hippocampal slices in organotypic culture. Exposure of cultured slice preparations to 0.1 microM PTH(1 - 34) for 60 min resulted in a gradual increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)); this effect was most obvious in the apical dendritic region of CA1 subfield. When PTH(1 - 34) at a lower concentration (1 nM) was added to the culture medium and its toxic effects examined using a propidium iodide intercalation method, significant toxicity was seen 3 days after exposure and increased with time. Cells in the CA1 region seemed more vulnerable to the hormone than cells in other regions. At 1 week of exposure, the toxic effects were dose-dependent over the range of 0.1 pM to 0.1 microM, the minimum effective dose being 10 pM. The adverse effects were not induced either by the inactive fragment, PTH(39 - 84), or by an active fragment of PTH-related peptide (PTHrP(1 - 34)), an intrinsic ligand of the brain PTH receptor. The PTH(1 - 34)-induced adverse effects were significantly inhibited by co-administration of 10 microM nifedipine, an L-type Ca(2+) channel blocker, but not by co-administration of blockers of the other types of Ca(2+) channel. The present study demonstrates that sustained high levels of PTH in the brain might cause degeneration of specific brain regions due to Ca(2+) overloading via activation of dihydropyridine-sensitive Ca(2+) channels, and suggests that PTH may be a risk factor for senile dementia. British Journal of Pharmacology (2000) 129, 21 - 28

Figure 1

Effects of PTH_1–34 on [Ca²⁺]_i in hippocampal organotypic slice cultures. (a) Pseudocolor ratio images (F340/F380) of hippocampal organotypic slices loaded with fura-2 as Ca²⁺ indicator during exposure to PTH_1–34 (0.1 μM), using a low magnification objective lens (4×). The numbers indicate the time in min after exposure to PTH_1–34. The colored scale bar shows the F340/F380 ratio. (b) Time-course of the increase in [Ca²⁺]_i in the CA1, CA3 and dentate gyrus regions indicated as squares in the control image in (a) observed in PTH_1–34 treated (filled squares) and untreated control preparations (filled circles).

Figure 2

Time-course of the development of the toxic effects of PTH_1–34 in hippocampal organotypic slice cultures, detected by PI intercalation. (a) Representative fluorescence images of PI-stained organotypic slice cultures exposed to PTH_1–34 (1 nM) and PI (2 μM) for the number of days indicated on each panel. PI fluorescence (emission >580 nm) was measured using a SIT camera following excitation with 520–540 nm light. Organotypic slice culture preparations were prepared separately for each day tested. (b) The PI fluorescence intensity of the whole preparation was measured in arbitrary units, using an image analyser (Argus 50) from digitized images; the mean and standard error for the intensity are indicated (n=6). The open columns indicate the averaged fluorescence intensity of day-matched control preparations and the filled columns indicate the intensity after 1–5 days exposure to PTH_1–34. A statistically significant difference was seen from 3 days of exposure to PTH_1–34 (^**P<0.01 and ^***P<0.001 as evaluated according to Tukey's multiple comparison method with control).

Figure 3

Histological observation on the organotypic slice culture exposed to PTH_1–34. Neuronal cells in the organotypic slice culture exposed to PTH_1–34 (1 nM) was visualized by Nissl staining. (a) The control preparation cultured for 14 days. (b) The preparation cultured for 7 days and then exposed to PTH_1–34 (1 nM) for another 7 days. Note the part shown by arrow heads. Neuronal cells located in the CA1 subfield were lost in the PTH treated prepration.

Figure 4

Dose-response relationship of the adverse effects of PTH_1–34 on organotypic hippocampal slice culture preparations as detected by PI intercalation. (a) Representative PI fluorescence images in organotypic slice cultures exposed for 5 days to PTH_1–34 (0.1 pM, 10 pM, 10 nM and 0.1 μM), together with PI (2 μM) were obtained as in Figure 2. (b) The intensities of the PI fluorescence in the CA1, CA3 and DG region was measured separately, as in Figure 2, to show the regional specificity of the adverse effects of PTH_1–34; the mean and standard error for the intensity are indicated (n=6). ^*P<0.05 and ^**P<0.01 as evaluated according to Tukey's multiple comparison method with control.

Figure 5

Effects of PTH_1–34 and related peptides on organotypic slice cultures. (a) Representative fluorescence images of control samples and preparations exposed for 5 days to 1 nM PTH_1–34, PTH_39–84 or PTHrP_1–34 plus PI (2 μM) as an indicator of degenerating cells. The images were obtained as described in Figure 2. (b) Averaged fluorescence intensity for six preparations treated with each peptide. A statistically significant difference was seen between the results for PTH_1–34 and the control (^***P<0.001, Student's t-test), but no significant difference was seen using PTH_39–84, an inactive fragment of PTH, or PTHrP_1–34, an active fragment of PTHrP in the central nervous system.

Figure 6

Detection of mRNA coding for PTH/PTHrP receptor and PTH₂ receptor in rat whole brain minus cerebellum and hippocampal organotypic slice culture using PCR method. As describe in Methods, specific mRNA primers for PTH/PTHrP receptor and PTH₂ receptor were used for whole brain minus cerebellum (1) and hippocampal organotypic slice culture (2). Expected size of mRNA for PTH/PTHrP receptor (251 bp) and for PTH₂ receptor (294 bp) detected by each primers in both preparations. Size markers were shown on both sides.