- Email: [email protected]
Activation of dihydropyridine sensitive Ca2+ channels in rat hippocampal neurons in culture by parathyroid hormone
Neuroscience Letters 256 (1998) 139–142
Activation of dihydropyridine sensitive Ca2+ channels in rat hippocampal neurons in culture by parathyroid hormone Takae Hirasawa a, Takeshi Nakamura b, Mitsuhiro Morita b, Ikuko Ezawa a, Hiroyoshi Miyakawa b, Yoshihisa Kudo b ,* a Graduate School of Human Life Science, Japan Women’s University, Bunkyo-ku, Mejirodai, Tokyo 112-8681, Japan School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
b
Received 6 August 1998; received in revised form 19 September 1998; accepted 19 September 1998
Abstract We examined the effects of parathyroid hormone (PTH) on rat hippocampal neurons in culture to determine whether it caused a similar intracellular calcium concentration ([Ca2+]i) increase in these cells to that seen with renal epithelial cells and found that PTH induced the effect in about 30% of the neurons. The effects appeared gradually during continuous administration of fulllength PTH1–84 or its active fragment, PTH1–34, but not of an inactive fragment, PTH39–84. However, the active fragment of the PTH-related peptide (PTHrP1–34) had little effect on [Ca2+]i during 60 min of administration. The PTH effect was inhibited by nifedipine, an L-type Ca2+ channel antagonist, and facilitated by S-(-)-BAY K 8644, an L-type Ca2+ channel agonist. Our findings suggest that PTH is one of the causal factors for the age-related increase in the density of voltage gated Ca2+ channels in hippocampal neurons. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Hippocampal neuron; Parathyroid hormone; Parathyroid hormone related peptide; Intracellular Ca2+ concentration; Dihydropyridine sensitive Ca2+ channel; Senile dementia
A dysfunction of Ca2+ homeostasis during aging and its relationship to the incidence of senile dementia have been suggested [6,10,15]. Since a high [Ca2+]i is neurotoxic [4,13], aging appears to be a major risk factor for neurodegenerative conditions [5], and the vulnerability of the aged neurons can be attributed to the elevated [Ca2+]i. An age-related increase in the density of voltage-gated Ca2+ channels (especially L-type) in hippocampal neurons, reported in animal models, has been suggested as a potential contributor to the age-related vulnerability of hippocampal neurons [11,14,16]. However, the possible mechanisms for this increase in Ca2+ channels have not been elucidated. We examined the role of parathyroid hormone (PTH), a major regulator of blood Ca2+ levels, in the regulation of expression of Ca2+ channels in hippocampal neurons. PTH facilitates re-uptake of Ca2+ at the epithelial cells of the distal renal tube, where it has been demonstrated to increase the density of dihydropyridine-sensitive Ca2+ channels by microtubule-dependent exocytosis [2,8]. This PTH-induced * Corresponding author. Tel.: +81 426 767164; fax: +81 426 768841; e-mail: [email protected]
0304-3940/98/$ - see front matter PII S0304- 3940(98) 00782- 4
change in density of L-type Ca2+ channels suggests that a similar mechanism may account for the age-related increase in Ca2+ channels in hippocampal neurons. Primary cultures of hippocampal cells from 18 days fetal rats were prepared using a previously described method [13]. After 7 days [Ca2+]i of cultured cells was measured by fura-2 fluorometry as described elsewhere [17]. Briefly, the dissociated cultured cells were exposed to 7.5 mM fura-2 acetoxymethyl ester (Fura-2/AM) dissolved in a balanced salt solution (BSS; 130 mM NaCl, 5.4 mM KCl, 2.0 mM CaCl2, 5.5 mM glucose, 10 mM HEPES-5 mM NaOH– HCl, pH 7.3) for 60 min at room temperature. The preparation was then transferred to fresh BSS and incubated at 32°C for at least 1 h. The glass coverslip with the fura-2-loaded cells was mounted on the stage of a fluorescence microscope (Olympus IMT-2) and the cells perfused continuously with BSS at 32°C at a rate of 2 ml/min. Fluorescence images of the fura-2 loaded cell, obtained using a 20 × objective lens and a silicon-intensified-target (SIT) video camera (Hamamatsu Photonics, C2400-8), were fed into an image processor (Hamamatsu Photonics Argus 50) for two-dimensional analysis. Drugs and chemicals were obtained from the fol-
1998 Elsevier Science Ireland Ltd. All rights reserved
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lowing sources: PTH1–84, PTH1–34 and PTH39–84 were from Peptide Institute (Osaka, Japan), fura-2/AM was from Doijindo Chemical Laboratory (Kumamoto, Japan) and nifedipine and S-(-)-Bay K8644 were from Sigma. To examine whether PTH had the same effects on the calcium level in hippocampal cells as on that in renal epithelial cells, full-size human PTH (PTH1–84), its active fragment (PTH1–34), an inactive fragment (PTH39–84) and the active fragment of the PTH-related peptide (PTHrP1–34) were applied continuously to cultured hippocampal cells
for 60 min (2 ml/min). Significant effects were seen using PTH1–84 at concentrations greater than 10−8 M. As shown in Fig. 1a,b, 10−7 M PTH1–84 induced a slow, but clear, increase in [Ca2+]i in about 30% of the cells. The effects were seen more than 20 min after PTH1–84 was applied. The [Ca2+]i, calculated using a conventional calibration curve, was greater than 500 nM at the end of administration. A similar gradual increase in [Ca2+]i was seen using PTH1–34, the active fragment of PTH (Fig. 1b). The time course of the [Ca2+]i increase was similar to that seen in renal epithelial
Fig. 1. Effect of PTH1–84 and related peptides on [Ca2+]i in cultured hippocampal cells. (a) Effect of PTH1–84 (10−7 M) on [Ca2+]i; image analysis showing an increase in [Ca2+]i after 30 and 60 min. (b) The time-course of the increase in [Ca2+]i induced by PTH1–84 (10−7 M) (filled circles), PTH1–34 (10−7 M) (open circles), PTH39–84 (10−7 M) (filled squares), PTHrP1–34 (open triangles) and balanced salt solution (control) (open squares). Inset indicates the effect of PTHrP at faster time resolution. The results are the mean ± SE (n = 20). *P , 0.05; **P , 0.01 and ***P , 0.001 (Student’s t-test between control). Fig. 2. Identification of PTH-sensitive cells using immuno-cytochemistry and Ca2+ imaging. Cells loaded with fura-2 and exposed to PTH1–34 were subjected to Ca2+ imaging and then fixed and immuno-cytochemically labeled with antibody specific for GFAP, a glial cell marker, and MAP2, a neuronal cell marker. (a) Fluorescence of MAP2-positive cells (fluorescein labeling). (b) Location of PTH-sensitive cells detected by Ca2+ imaging. PTH-sensitive GFAP-positive cells are indicated by vertical pink arrow heads, PTH-sensitive MAP2-positive cells by horizontal
T. Hirasawa et al. / Neuroscience Letters 256 (1998) 139–142
cells [2]. The fragment PTH39–84, known to be inactive as a hormone in Ca2+ regulation, had no apparent effect at a concentration of 10−7 M. These results indicated that the PTH effect may be induced through specific receptors. In contrast, 10−7 M PTHrP1–34, a peptide found abundantly in the brain and an intrinsic agonist of brain PTH receptors, induced only a fast transient increase in the [Ca2+]i of some cells (about 30%), as indicated in the inset of Fig. 1, which was measured faster time resolution, but failed to produce the same gradual increase in [Ca2+]i even after prolonged administration (Fig. 1b). As shown in Fig. 1a, only one-third of the cells responded to PTH. Since the primary cultures employed in the present study contained both neuronal and glial cells, either might have been sensitive to PTH. An immuno-cytochemical method combined with Ca2+ fluorometery was used to determine whether the PTH-sensitive cells were neuronal or glial cells [3]. As shown in Fig. 2, major population of PTHsensitive cells could be identified as neurons which were
Fig. 3. The effects of a dihydropyridine-sensitive Ca2+ channel blocker and activator on the PTH1–34 induced increase in [Ca2+]i. The slow gradual increase in [Ca2+]i induced by PTH1–34 (10−7 M) (a) was blocked by nifedipine (b). Nifedipine (10−5 M) was administered for 5 min before and during the continuous administration of PTH1–34. Bay K8644 (10−7 M), an L-type Ca2+ channel agonist, induced a rapid increase in [Ca2+]i when administered after 20 min pretreatment with PTH1–34 (10−7 M) (c). Each point is the mean ± SE (n = 7).
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MAP2 (microtubules associated protein)-positive cells, though some glial cells which were glial fibrillatory acidic protein (GFAP)-positive cells responded a little to PTH1–34. Nifedipine, a dihydropyridine Ca2+ channel blocker, and S-(-)-Bay K8644, a dihydropyridine Ca2+ channel agonist, were used to examine whether the PTH-induced increase in the [Ca2+]i resulted from mechanisms similar to those reported in distal renal tube epithelial cells. As shown in Fig. 3a, slow and gradual increase in [Ca2+]i was observed in the one third of the cells during exposure to the PTH1–34 as already shown in Fig. 1. We confirmed the reproducibility of the effects of PTH1–34 in more than five separate experiments. In Fig. 3 seven neurons showed representative effects to PTH1–34 in a single experiment were selected and displayed as a representative effects of PTH1–34. The increase in [Ca2+]i seen after administration of PTH1–34 was completely inhibited by nifedipine (10−5 M) when applied for five minutes before, and during, PTH1–34 application (Fig. 3b). This effect was also reproducible and we could not find any PTH positive neurons in the images obtained after the treatment with nifedipine. The effect of S-(-)-Bay K8644, a dihydropyridine agonist, in a concentration of 10−7 M, which itself had no significant effects on resting [Ca2+]i, was examined on seven cells which showed minor response to PTH1–34 within 20 min of administration. The agonist caused a rapid increase in [Ca2+]i in those cells as shown in Fig. 3c. The facilitatory effects of S-(-)-Bay K8644 on the neurons were also reproducible, but the effect were rather difficult to discriminate in neurons sensitive to PTH1–34. These results indicated that the density and/or sensitivity of L-type Ca2+ channels increased in a certain population of the cultured hippocampal neurons during PTH exposure and that the [Ca2+]i increased due to the spontaneous activation of these channels. The present results indicated that the effect of PTH on hippocampal neurons were apparently similar to that on renal distal tubule epithelial cells [2,8]. Since PTH39–84, an inactive fragment, had no effect, the effect of active forms of PTH probably results from the activation of specific receptors. Recent molecular biological studies have shown that the same PTH receptor found in peripheral tissues (the PTH1 receptor) [1] and a new type of PTH receptor (the PTH2 receptor) are expressed in the central nervous system [19]. Thus, the effects of PTH on the [Ca2+]i of hippocampal cells might result from the activation of certain of these receptors. Although the physiological roles in the brain of PTH receptors and PTHrP, their intrinsic ligand, have not yet been elucidated, the same receptors would also be stimulated by PTH and its active fragment. However, in the present study, a large difference was seen between the effects of PTH and PTHrP. Since the PTH1 receptor for PTHrP is reported to be the same as that for peripheral PTH, other types of PTH receptors might mediate the slow onset gradual increase in [Ca2+]i seen in hippocampal neurons. One possible candidate is the PTH2 receptor, which is also found in the central nervous system [19]. However, we cannot exclude the possibility of the existence
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of other, as yet undiscovered, PTH receptor subtypes that might be responsible for these effects of PTH on hippocampal neurons. Further studies examining this question are now under investigation. The PTH-induced increase in [Ca2+]i was found to be mediated by activation of L-type Ca2+ channels, which became expressed on the cell membranes during prolonged exposure to PTH. Although more than 20 min was required for PTH to cause a marked elevation of [Ca2+]i, this time period seems too short for the results to be ascribed to the induction of L-type channels on the plasma membranes via mRNA translation. However, the analogous effect of PTH on renal epithelial cells suggests that the channels or their regulatory factors may already be translated and located on intracellular vesicles [2] and that the activation of PTH receptors may facilitate their delivery or incorporation into the plasma membrane. Very similar mechanisms have been demonstrated for neuronal cell receptors during plastic change in synaptic transmission [9,12]. The present results demonstrate that PTH, via overloading of the [Ca2+]i, may exert toxic effects in the hippocampal neurons. Regulation of blood Ca2+ levels is achieved by a well-designed Ca2+ homeostatic mechanism. However, chronic and severe Ca2+ loss from the blood cannot be compensated by those processes and results in the activation of osteoclast cells, which then absorb osteocytes and thus release Ca2+ into the blood. However, in osteoporosis patients, even if the PTH level is increased, the density of osteocytes available to supply Ca2+ may also be decreased and inadequate for the provision of sufficient Ca2+. In such patients, blood PTH levels may remain high for a considerable period [7], and PTH may become a risk factor for the degradation of central nervous system cells. The effective dose required to cause the increase in [Ca2+]i in hippocampal neurons was higher than that found in such patients. However, if the administration period was prolonged, similar effects might be produced by much lower concentrations of PTH. Our preliminary studies on cultured hippocampal cells show that overnight exposure to PTH1–34 induces significant cell death at a concentration of less than 10−9 M (data not shown). Sustained high plasma PTH levels may result in PTH entering the brain through the blood–brain barrier, which has been demonstrated to become leaky during senescence [18]. PTH that has entered the brain may affect brain PTH receptors, causing activation of the L-type Ca2+ channel and resulting in an increase in [Ca2+]i. This process should be examined as a possible causal factor of senile dementia.
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This study was supported by a grant from the Organization of Pharmaceutical Safety and Research (OPSR) Japan. We thank Dr. Takuo Fujita (Calcium Research Institute) for his valuable suggestions. [1] Abou Samura, A.B., Juppner, H., Force, T., Freeman, M.W., Kong, X.F., Schipani, E., Urena, P., Richards, J., Bonventre,
[19]
J.V. and Potts, J.T., Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol triphosphates and increases intracellular free calcium, Proc. Natl. Acad. Sci. USA, 89 (1992) 2732–2736. Bacskai, B.J. and Friedman, P.A., Activation of latent Ca2+ channels in renal epithelial cells by parathyroid hormone, Nature, 347 (1990) 388–391. Barry de, J., Ogura, A. and Kudo, Y., Ca2+ mobilization in cultured rat cerebellar cells: astrocytes are activated by t-ACPD, Eur. J. Neurosci., 3 (1991) 1146–1154. Choi, D.W., Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage, Trends Neurosci., 7 (1988) 369–372. Disterhorft, J.F., Moyer, J.R. Jr. and Thompson, L.T., The calcium rationale in aging and Alzheimer’s disease. Evidence from an animal model of normal aging, Ann. N.Y. Acad. Sci., 747 (1994) 382–406. Fujita, T., Biological effects of aging on bone and the central nervous system, Exp. Gerontol., 25 (1990) 317–321. Fujita, T., Ohue, T., Fujii, Y., Miyauchi, A. and Takagi, Y., Effect of calcium supplementation on bone density and parathyroid function in elderly subjects, Miner Electrolyte Metab., 21 (1995) 229–231. Gesek, F.A. and Friedman, P.A., On the mechanism of parathyroid hormone stimulation of calcium uptake by mouse distal convoluted tubule cells, J. Clin. Invest., 90 (1992) 749–758. Isaac, J.T.R., Nicoll, R.A. and Malenka, R.C., Evidence for silent synapses: implications for the expression of LTP, Neuron, 15 (1995) 425–434. Kipen, E., Helme, R.D., Wark, J.D. and Flicker, L., Bone density, vitamin D nutrition, and parathyroid hormone levels in women with dementia, J. Am. Geriatr. Soc., 43 (1995) 1088– 1091. Landfield, P.W., Aging-related increase in hippocampal calcium channels, Life Sci., 59 (1996) 399–404. Liao, D., Hessler, N.A. and Malinow, R., Activation of postsynaptically silent synapses during pairing-induced LTP in CA1 region of hippocampal slice, Nature, 375 (1995) 400–404. Ogura, A., Miyamoto, M. and Kudo, Y., Neuronal death in vitro: parallelism between survivability of hippocampal neurons and sustained elevation of cytosolic Ca2+ after exposure to glutamate receptor agonists, Exp. Brain Res., 73 (1988) 447–458. Porter, N.M., Thibault, O., Thibault, V., Chen, K.-C. and Landfield, P.W., Calcium channel density and hippocampal cell death with age in long-term culture, J. Neurosci., 17 (1997) 5629–5639. Satrustegui, J., Villalba, M., Pereira, R., Bogonez, E. and Martinez-Serrano, A., Cytosolic and mitochondrial calcium in synaptosomes during aging, Life Sci., 59 (1996) 429–434. Thibault, O. and Landfield, P.W., Increase in single L-type calcium channels in hippocampal neurons during aging, Science, 272 (1996) 1017–1020. Uchino, S., Kudo, Y., Watanabe, W., Nakajima-Iijima, S. and Mishina, M., Inducible expression of N-methyl-D-aspartate (NMDA) receptor channels from cloned cDNA in CHO cells, Mol. Brain Res., 44 (1997) 1–11. Ueno, M., Akiguchi, I., Hosokawa, M., Shinnou, M., Sakamoto, H., Takemura, M. and Higuchi, K., Age-related changes in the brain transfer of blood-borne horseradish peroxidase in the hippocampus of senescence-accelerated mouse, Acta Neuropathol., 93 (1997) 233–240. Usdin, T.B., Gruber, C. and Bonner, T.I., Identification and functional expression of a receptor selectively recognizing parathyroid hormone, the PTH2 receptor, J. Biol. Chem., 270 (1995) 15455–15458.
Activation of dihydropyridine sensitive Ca2+ channels in rat hippocampal neurons in culture by parathyroid hormone Takae Hirasawa a, Takeshi Nakamura b, Mitsuhiro Morita b, Ikuko Ezawa a, Hiroyoshi Miyakawa b, Yoshihisa Kudo b ,* a Graduate School of Human Life Science, Japan Women’s University, Bunkyo-ku, Mejirodai, Tokyo 112-8681, Japan School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
b
Received 6 August 1998; received in revised form 19 September 1998; accepted 19 September 1998
Abstract We examined the effects of parathyroid hormone (PTH) on rat hippocampal neurons in culture to determine whether it caused a similar intracellular calcium concentration ([Ca2+]i) increase in these cells to that seen with renal epithelial cells and found that PTH induced the effect in about 30% of the neurons. The effects appeared gradually during continuous administration of fulllength PTH1–84 or its active fragment, PTH1–34, but not of an inactive fragment, PTH39–84. However, the active fragment of the PTH-related peptide (PTHrP1–34) had little effect on [Ca2+]i during 60 min of administration. The PTH effect was inhibited by nifedipine, an L-type Ca2+ channel antagonist, and facilitated by S-(-)-BAY K 8644, an L-type Ca2+ channel agonist. Our findings suggest that PTH is one of the causal factors for the age-related increase in the density of voltage gated Ca2+ channels in hippocampal neurons. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Hippocampal neuron; Parathyroid hormone; Parathyroid hormone related peptide; Intracellular Ca2+ concentration; Dihydropyridine sensitive Ca2+ channel; Senile dementia
A dysfunction of Ca2+ homeostasis during aging and its relationship to the incidence of senile dementia have been suggested [6,10,15]. Since a high [Ca2+]i is neurotoxic [4,13], aging appears to be a major risk factor for neurodegenerative conditions [5], and the vulnerability of the aged neurons can be attributed to the elevated [Ca2+]i. An age-related increase in the density of voltage-gated Ca2+ channels (especially L-type) in hippocampal neurons, reported in animal models, has been suggested as a potential contributor to the age-related vulnerability of hippocampal neurons [11,14,16]. However, the possible mechanisms for this increase in Ca2+ channels have not been elucidated. We examined the role of parathyroid hormone (PTH), a major regulator of blood Ca2+ levels, in the regulation of expression of Ca2+ channels in hippocampal neurons. PTH facilitates re-uptake of Ca2+ at the epithelial cells of the distal renal tube, where it has been demonstrated to increase the density of dihydropyridine-sensitive Ca2+ channels by microtubule-dependent exocytosis [2,8]. This PTH-induced * Corresponding author. Tel.: +81 426 767164; fax: +81 426 768841; e-mail: [email protected]
0304-3940/98/$ - see front matter PII S0304- 3940(98) 00782- 4
change in density of L-type Ca2+ channels suggests that a similar mechanism may account for the age-related increase in Ca2+ channels in hippocampal neurons. Primary cultures of hippocampal cells from 18 days fetal rats were prepared using a previously described method [13]. After 7 days [Ca2+]i of cultured cells was measured by fura-2 fluorometry as described elsewhere [17]. Briefly, the dissociated cultured cells were exposed to 7.5 mM fura-2 acetoxymethyl ester (Fura-2/AM) dissolved in a balanced salt solution (BSS; 130 mM NaCl, 5.4 mM KCl, 2.0 mM CaCl2, 5.5 mM glucose, 10 mM HEPES-5 mM NaOH– HCl, pH 7.3) for 60 min at room temperature. The preparation was then transferred to fresh BSS and incubated at 32°C for at least 1 h. The glass coverslip with the fura-2-loaded cells was mounted on the stage of a fluorescence microscope (Olympus IMT-2) and the cells perfused continuously with BSS at 32°C at a rate of 2 ml/min. Fluorescence images of the fura-2 loaded cell, obtained using a 20 × objective lens and a silicon-intensified-target (SIT) video camera (Hamamatsu Photonics, C2400-8), were fed into an image processor (Hamamatsu Photonics Argus 50) for two-dimensional analysis. Drugs and chemicals were obtained from the fol-
1998 Elsevier Science Ireland Ltd. All rights reserved
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lowing sources: PTH1–84, PTH1–34 and PTH39–84 were from Peptide Institute (Osaka, Japan), fura-2/AM was from Doijindo Chemical Laboratory (Kumamoto, Japan) and nifedipine and S-(-)-Bay K8644 were from Sigma. To examine whether PTH had the same effects on the calcium level in hippocampal cells as on that in renal epithelial cells, full-size human PTH (PTH1–84), its active fragment (PTH1–34), an inactive fragment (PTH39–84) and the active fragment of the PTH-related peptide (PTHrP1–34) were applied continuously to cultured hippocampal cells
for 60 min (2 ml/min). Significant effects were seen using PTH1–84 at concentrations greater than 10−8 M. As shown in Fig. 1a,b, 10−7 M PTH1–84 induced a slow, but clear, increase in [Ca2+]i in about 30% of the cells. The effects were seen more than 20 min after PTH1–84 was applied. The [Ca2+]i, calculated using a conventional calibration curve, was greater than 500 nM at the end of administration. A similar gradual increase in [Ca2+]i was seen using PTH1–34, the active fragment of PTH (Fig. 1b). The time course of the [Ca2+]i increase was similar to that seen in renal epithelial
Fig. 1. Effect of PTH1–84 and related peptides on [Ca2+]i in cultured hippocampal cells. (a) Effect of PTH1–84 (10−7 M) on [Ca2+]i; image analysis showing an increase in [Ca2+]i after 30 and 60 min. (b) The time-course of the increase in [Ca2+]i induced by PTH1–84 (10−7 M) (filled circles), PTH1–34 (10−7 M) (open circles), PTH39–84 (10−7 M) (filled squares), PTHrP1–34 (open triangles) and balanced salt solution (control) (open squares). Inset indicates the effect of PTHrP at faster time resolution. The results are the mean ± SE (n = 20). *P , 0.05; **P , 0.01 and ***P , 0.001 (Student’s t-test between control). Fig. 2. Identification of PTH-sensitive cells using immuno-cytochemistry and Ca2+ imaging. Cells loaded with fura-2 and exposed to PTH1–34 were subjected to Ca2+ imaging and then fixed and immuno-cytochemically labeled with antibody specific for GFAP, a glial cell marker, and MAP2, a neuronal cell marker. (a) Fluorescence of MAP2-positive cells (fluorescein labeling). (b) Location of PTH-sensitive cells detected by Ca2+ imaging. PTH-sensitive GFAP-positive cells are indicated by vertical pink arrow heads, PTH-sensitive MAP2-positive cells by horizontal
T. Hirasawa et al. / Neuroscience Letters 256 (1998) 139–142
cells [2]. The fragment PTH39–84, known to be inactive as a hormone in Ca2+ regulation, had no apparent effect at a concentration of 10−7 M. These results indicated that the PTH effect may be induced through specific receptors. In contrast, 10−7 M PTHrP1–34, a peptide found abundantly in the brain and an intrinsic agonist of brain PTH receptors, induced only a fast transient increase in the [Ca2+]i of some cells (about 30%), as indicated in the inset of Fig. 1, which was measured faster time resolution, but failed to produce the same gradual increase in [Ca2+]i even after prolonged administration (Fig. 1b). As shown in Fig. 1a, only one-third of the cells responded to PTH. Since the primary cultures employed in the present study contained both neuronal and glial cells, either might have been sensitive to PTH. An immuno-cytochemical method combined with Ca2+ fluorometery was used to determine whether the PTH-sensitive cells were neuronal or glial cells [3]. As shown in Fig. 2, major population of PTHsensitive cells could be identified as neurons which were
Fig. 3. The effects of a dihydropyridine-sensitive Ca2+ channel blocker and activator on the PTH1–34 induced increase in [Ca2+]i. The slow gradual increase in [Ca2+]i induced by PTH1–34 (10−7 M) (a) was blocked by nifedipine (b). Nifedipine (10−5 M) was administered for 5 min before and during the continuous administration of PTH1–34. Bay K8644 (10−7 M), an L-type Ca2+ channel agonist, induced a rapid increase in [Ca2+]i when administered after 20 min pretreatment with PTH1–34 (10−7 M) (c). Each point is the mean ± SE (n = 7).
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MAP2 (microtubules associated protein)-positive cells, though some glial cells which were glial fibrillatory acidic protein (GFAP)-positive cells responded a little to PTH1–34. Nifedipine, a dihydropyridine Ca2+ channel blocker, and S-(-)-Bay K8644, a dihydropyridine Ca2+ channel agonist, were used to examine whether the PTH-induced increase in the [Ca2+]i resulted from mechanisms similar to those reported in distal renal tube epithelial cells. As shown in Fig. 3a, slow and gradual increase in [Ca2+]i was observed in the one third of the cells during exposure to the PTH1–34 as already shown in Fig. 1. We confirmed the reproducibility of the effects of PTH1–34 in more than five separate experiments. In Fig. 3 seven neurons showed representative effects to PTH1–34 in a single experiment were selected and displayed as a representative effects of PTH1–34. The increase in [Ca2+]i seen after administration of PTH1–34 was completely inhibited by nifedipine (10−5 M) when applied for five minutes before, and during, PTH1–34 application (Fig. 3b). This effect was also reproducible and we could not find any PTH positive neurons in the images obtained after the treatment with nifedipine. The effect of S-(-)-Bay K8644, a dihydropyridine agonist, in a concentration of 10−7 M, which itself had no significant effects on resting [Ca2+]i, was examined on seven cells which showed minor response to PTH1–34 within 20 min of administration. The agonist caused a rapid increase in [Ca2+]i in those cells as shown in Fig. 3c. The facilitatory effects of S-(-)-Bay K8644 on the neurons were also reproducible, but the effect were rather difficult to discriminate in neurons sensitive to PTH1–34. These results indicated that the density and/or sensitivity of L-type Ca2+ channels increased in a certain population of the cultured hippocampal neurons during PTH exposure and that the [Ca2+]i increased due to the spontaneous activation of these channels. The present results indicated that the effect of PTH on hippocampal neurons were apparently similar to that on renal distal tubule epithelial cells [2,8]. Since PTH39–84, an inactive fragment, had no effect, the effect of active forms of PTH probably results from the activation of specific receptors. Recent molecular biological studies have shown that the same PTH receptor found in peripheral tissues (the PTH1 receptor) [1] and a new type of PTH receptor (the PTH2 receptor) are expressed in the central nervous system [19]. Thus, the effects of PTH on the [Ca2+]i of hippocampal cells might result from the activation of certain of these receptors. Although the physiological roles in the brain of PTH receptors and PTHrP, their intrinsic ligand, have not yet been elucidated, the same receptors would also be stimulated by PTH and its active fragment. However, in the present study, a large difference was seen between the effects of PTH and PTHrP. Since the PTH1 receptor for PTHrP is reported to be the same as that for peripheral PTH, other types of PTH receptors might mediate the slow onset gradual increase in [Ca2+]i seen in hippocampal neurons. One possible candidate is the PTH2 receptor, which is also found in the central nervous system [19]. However, we cannot exclude the possibility of the existence
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of other, as yet undiscovered, PTH receptor subtypes that might be responsible for these effects of PTH on hippocampal neurons. Further studies examining this question are now under investigation. The PTH-induced increase in [Ca2+]i was found to be mediated by activation of L-type Ca2+ channels, which became expressed on the cell membranes during prolonged exposure to PTH. Although more than 20 min was required for PTH to cause a marked elevation of [Ca2+]i, this time period seems too short for the results to be ascribed to the induction of L-type channels on the plasma membranes via mRNA translation. However, the analogous effect of PTH on renal epithelial cells suggests that the channels or their regulatory factors may already be translated and located on intracellular vesicles [2] and that the activation of PTH receptors may facilitate their delivery or incorporation into the plasma membrane. Very similar mechanisms have been demonstrated for neuronal cell receptors during plastic change in synaptic transmission [9,12]. The present results demonstrate that PTH, via overloading of the [Ca2+]i, may exert toxic effects in the hippocampal neurons. Regulation of blood Ca2+ levels is achieved by a well-designed Ca2+ homeostatic mechanism. However, chronic and severe Ca2+ loss from the blood cannot be compensated by those processes and results in the activation of osteoclast cells, which then absorb osteocytes and thus release Ca2+ into the blood. However, in osteoporosis patients, even if the PTH level is increased, the density of osteocytes available to supply Ca2+ may also be decreased and inadequate for the provision of sufficient Ca2+. In such patients, blood PTH levels may remain high for a considerable period [7], and PTH may become a risk factor for the degradation of central nervous system cells. The effective dose required to cause the increase in [Ca2+]i in hippocampal neurons was higher than that found in such patients. However, if the administration period was prolonged, similar effects might be produced by much lower concentrations of PTH. Our preliminary studies on cultured hippocampal cells show that overnight exposure to PTH1–34 induces significant cell death at a concentration of less than 10−9 M (data not shown). Sustained high plasma PTH levels may result in PTH entering the brain through the blood–brain barrier, which has been demonstrated to become leaky during senescence [18]. PTH that has entered the brain may affect brain PTH receptors, causing activation of the L-type Ca2+ channel and resulting in an increase in [Ca2+]i. This process should be examined as a possible causal factor of senile dementia.
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This study was supported by a grant from the Organization of Pharmaceutical Safety and Research (OPSR) Japan. We thank Dr. Takuo Fujita (Calcium Research Institute) for his valuable suggestions. [1] Abou Samura, A.B., Juppner, H., Force, T., Freeman, M.W., Kong, X.F., Schipani, E., Urena, P., Richards, J., Bonventre,
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