Pituitary adenylate cyclase activating polypeptide and vasoactive intestinal peptide increase cytosolic free calcium concentration in cultured rat hippocampal neurons

Ichiro Tatsuno, Toshihiko Yada, Sandor Vigh, Hiroyoshi Hidaka, Akira Arimura

Research output: Contribution to journalArticle

89 Citations (Scopus)

Abstract

Recently, pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalamus and it was shown to stimulate adenylate cyclase in rat pituitary cells, neurons, and astrocytes. PACAP exhibits a 68% amino acid sequence homology with vasoactive intestinal peptide (VIP); however, it is 1000 times more potent than VIP in stimulating adenylate cyclase. In view of the wide distribution of PACAP and its receptor in the central nervous system, PACAP is likely to act as a neurotransmitter or neuromodulator as well. In the present study, we investigated the effects of PACAP38 on cytosolic-free calcium concentrations ([Ca2+]i) and compared these effects with those of VIP in cultured rat hippocampal neurons. Calcium concentrations, at the single cell level, were measured using fura-2, a calcium sensitive fluorescent dye, and fura-2-loaded neurons were continuously superfused at 37 C and viewed under an inverted microscope. Images of these neurons were recorded at 10-sec intervals by a video camera equipped with an Argus-50/CA system which controls the image acquisition and display. [Ca2+]i was quantitated from the intensities of fluorescence of the cells at two excitation wavelengths of 340 and 380 nm. The ratio of the intensities of emitted fluorescence (340/380 nm) was calibrated to determine [Ca2+]i. PACAP38 (0.1 nM) increased [Ca2+]i in some hippocampal neurons. As the concentration of peptide was increased from 0.1 to 10 nM, the accumulated number of hippocampal neurons responding to PACAP38 progressively increased and reached a plateau at 10 nM. Total neurons (33.0 ± 5.3%, n = 4; 502 neurons) were found to respond to 100 nM PACAP38. The half-maximal concentration (ED50) of PACAP38 was 2.60 ± 0.77 nM. Typically, 60-90 sec after the addition of PACAP38 (10 nM), [Ca2+]i increased from basal levels of 50-100 to 150-300 nM. VIP also increased [Ca2+]i, but required 1 μM or higher concentration for a considerable number of cells to respond. The number of hippocampal neurons responding to VIP at 1 μM was 28.9 ± 9.8% (n = 4; 442 cells) which was comparable to the population of neurons responding to 10 nM PACAP38. The ED50 for VIP was 0.68 ± 0.38 μM which was approximately 260 times higher than the ED50 for PACAP38. Neither 1-10 μM nitrendipine, a L-type voltage-dependent Ca2+ channel blocker, or 1 μM ω-conotoxin GVIA, a N-type voltage-dependent Ca2+ channel blocker, altered the PACAP-induced Ca2+ increment. Removal of Ca2+ from the superfusion media did not influence the PACAP38-induced increase of [Ca2+]i. Furthermore, 1-30 μM forskolin or 1-10 mM (Bu)2CAMP did not increase [Ca2+]i in the same neurons which responded to PACAP38. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide, an inhibitor of protein kinase A, did not affect PACAP38-induced increase in [Ca2+]i. These findings suggest that PACAP increases [Ca2+]i in rat hippocampal neurons by mobilizing Ca2+ from intracellular stores and that this action is not linked to activation of the cAMP-protein kinase A signal transduction system.

Original languageEnglish
Pages (from-to)73-81
Number of pages9
JournalEndocrinology
Volume131
Issue number1
Publication statusPublished - Jul 1992

Fingerprint

Pituitary Adenylate Cyclase-Activating Polypeptide
Vasoactive Intestinal Peptide
Calcium
Neurons
Fura-2
Cyclic AMP-Dependent Protein Kinases
Adenylyl Cyclases
Neurotransmitter Agents
Pituitary Adenylate Cyclase-Activating Polypeptide Receptors
Fluorescence
Nitrendipine
Amino Acid Sequence Homology

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Pituitary adenylate cyclase activating polypeptide and vasoactive intestinal peptide increase cytosolic free calcium concentration in cultured rat hippocampal neurons. / Tatsuno, Ichiro; Yada, Toshihiko; Vigh, Sandor; Hidaka, Hiroyoshi; Arimura, Akira.

In: Endocrinology, Vol. 131, No. 1, 07.1992, p. 73-81.

Research output: Contribution to journalArticle

@article{37f93d1a58774dfaa1a2aba533375b4b,
title = "Pituitary adenylate cyclase activating polypeptide and vasoactive intestinal peptide increase cytosolic free calcium concentration in cultured rat hippocampal neurons",
abstract = "Recently, pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalamus and it was shown to stimulate adenylate cyclase in rat pituitary cells, neurons, and astrocytes. PACAP exhibits a 68{\%} amino acid sequence homology with vasoactive intestinal peptide (VIP); however, it is 1000 times more potent than VIP in stimulating adenylate cyclase. In view of the wide distribution of PACAP and its receptor in the central nervous system, PACAP is likely to act as a neurotransmitter or neuromodulator as well. In the present study, we investigated the effects of PACAP38 on cytosolic-free calcium concentrations ([Ca2+]i) and compared these effects with those of VIP in cultured rat hippocampal neurons. Calcium concentrations, at the single cell level, were measured using fura-2, a calcium sensitive fluorescent dye, and fura-2-loaded neurons were continuously superfused at 37 C and viewed under an inverted microscope. Images of these neurons were recorded at 10-sec intervals by a video camera equipped with an Argus-50/CA system which controls the image acquisition and display. [Ca2+]i was quantitated from the intensities of fluorescence of the cells at two excitation wavelengths of 340 and 380 nm. The ratio of the intensities of emitted fluorescence (340/380 nm) was calibrated to determine [Ca2+]i. PACAP38 (0.1 nM) increased [Ca2+]i in some hippocampal neurons. As the concentration of peptide was increased from 0.1 to 10 nM, the accumulated number of hippocampal neurons responding to PACAP38 progressively increased and reached a plateau at 10 nM. Total neurons (33.0 ± 5.3{\%}, n = 4; 502 neurons) were found to respond to 100 nM PACAP38. The half-maximal concentration (ED50) of PACAP38 was 2.60 ± 0.77 nM. Typically, 60-90 sec after the addition of PACAP38 (10 nM), [Ca2+]i increased from basal levels of 50-100 to 150-300 nM. VIP also increased [Ca2+]i, but required 1 μM or higher concentration for a considerable number of cells to respond. The number of hippocampal neurons responding to VIP at 1 μM was 28.9 ± 9.8{\%} (n = 4; 442 cells) which was comparable to the population of neurons responding to 10 nM PACAP38. The ED50 for VIP was 0.68 ± 0.38 μM which was approximately 260 times higher than the ED50 for PACAP38. Neither 1-10 μM nitrendipine, a L-type voltage-dependent Ca2+ channel blocker, or 1 μM ω-conotoxin GVIA, a N-type voltage-dependent Ca2+ channel blocker, altered the PACAP-induced Ca2+ increment. Removal of Ca2+ from the superfusion media did not influence the PACAP38-induced increase of [Ca2+]i. Furthermore, 1-30 μM forskolin or 1-10 mM (Bu)2CAMP did not increase [Ca2+]i in the same neurons which responded to PACAP38. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide, an inhibitor of protein kinase A, did not affect PACAP38-induced increase in [Ca2+]i. These findings suggest that PACAP increases [Ca2+]i in rat hippocampal neurons by mobilizing Ca2+ from intracellular stores and that this action is not linked to activation of the cAMP-protein kinase A signal transduction system.",
author = "Ichiro Tatsuno and Toshihiko Yada and Sandor Vigh and Hiroyoshi Hidaka and Akira Arimura",
year = "1992",
month = "7",
language = "English",
volume = "131",
pages = "73--81",
journal = "Endocrinology",
issn = "0013-7227",
publisher = "The Endocrine Society",
number = "1",

}

TY - JOUR

T1 - Pituitary adenylate cyclase activating polypeptide and vasoactive intestinal peptide increase cytosolic free calcium concentration in cultured rat hippocampal neurons

AU - Tatsuno, Ichiro

AU - Yada, Toshihiko

AU - Vigh, Sandor

AU - Hidaka, Hiroyoshi

AU - Arimura, Akira

PY - 1992/7

Y1 - 1992/7

N2 - Recently, pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalamus and it was shown to stimulate adenylate cyclase in rat pituitary cells, neurons, and astrocytes. PACAP exhibits a 68% amino acid sequence homology with vasoactive intestinal peptide (VIP); however, it is 1000 times more potent than VIP in stimulating adenylate cyclase. In view of the wide distribution of PACAP and its receptor in the central nervous system, PACAP is likely to act as a neurotransmitter or neuromodulator as well. In the present study, we investigated the effects of PACAP38 on cytosolic-free calcium concentrations ([Ca2+]i) and compared these effects with those of VIP in cultured rat hippocampal neurons. Calcium concentrations, at the single cell level, were measured using fura-2, a calcium sensitive fluorescent dye, and fura-2-loaded neurons were continuously superfused at 37 C and viewed under an inverted microscope. Images of these neurons were recorded at 10-sec intervals by a video camera equipped with an Argus-50/CA system which controls the image acquisition and display. [Ca2+]i was quantitated from the intensities of fluorescence of the cells at two excitation wavelengths of 340 and 380 nm. The ratio of the intensities of emitted fluorescence (340/380 nm) was calibrated to determine [Ca2+]i. PACAP38 (0.1 nM) increased [Ca2+]i in some hippocampal neurons. As the concentration of peptide was increased from 0.1 to 10 nM, the accumulated number of hippocampal neurons responding to PACAP38 progressively increased and reached a plateau at 10 nM. Total neurons (33.0 ± 5.3%, n = 4; 502 neurons) were found to respond to 100 nM PACAP38. The half-maximal concentration (ED50) of PACAP38 was 2.60 ± 0.77 nM. Typically, 60-90 sec after the addition of PACAP38 (10 nM), [Ca2+]i increased from basal levels of 50-100 to 150-300 nM. VIP also increased [Ca2+]i, but required 1 μM or higher concentration for a considerable number of cells to respond. The number of hippocampal neurons responding to VIP at 1 μM was 28.9 ± 9.8% (n = 4; 442 cells) which was comparable to the population of neurons responding to 10 nM PACAP38. The ED50 for VIP was 0.68 ± 0.38 μM which was approximately 260 times higher than the ED50 for PACAP38. Neither 1-10 μM nitrendipine, a L-type voltage-dependent Ca2+ channel blocker, or 1 μM ω-conotoxin GVIA, a N-type voltage-dependent Ca2+ channel blocker, altered the PACAP-induced Ca2+ increment. Removal of Ca2+ from the superfusion media did not influence the PACAP38-induced increase of [Ca2+]i. Furthermore, 1-30 μM forskolin or 1-10 mM (Bu)2CAMP did not increase [Ca2+]i in the same neurons which responded to PACAP38. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide, an inhibitor of protein kinase A, did not affect PACAP38-induced increase in [Ca2+]i. These findings suggest that PACAP increases [Ca2+]i in rat hippocampal neurons by mobilizing Ca2+ from intracellular stores and that this action is not linked to activation of the cAMP-protein kinase A signal transduction system.

AB - Recently, pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalamus and it was shown to stimulate adenylate cyclase in rat pituitary cells, neurons, and astrocytes. PACAP exhibits a 68% amino acid sequence homology with vasoactive intestinal peptide (VIP); however, it is 1000 times more potent than VIP in stimulating adenylate cyclase. In view of the wide distribution of PACAP and its receptor in the central nervous system, PACAP is likely to act as a neurotransmitter or neuromodulator as well. In the present study, we investigated the effects of PACAP38 on cytosolic-free calcium concentrations ([Ca2+]i) and compared these effects with those of VIP in cultured rat hippocampal neurons. Calcium concentrations, at the single cell level, were measured using fura-2, a calcium sensitive fluorescent dye, and fura-2-loaded neurons were continuously superfused at 37 C and viewed under an inverted microscope. Images of these neurons were recorded at 10-sec intervals by a video camera equipped with an Argus-50/CA system which controls the image acquisition and display. [Ca2+]i was quantitated from the intensities of fluorescence of the cells at two excitation wavelengths of 340 and 380 nm. The ratio of the intensities of emitted fluorescence (340/380 nm) was calibrated to determine [Ca2+]i. PACAP38 (0.1 nM) increased [Ca2+]i in some hippocampal neurons. As the concentration of peptide was increased from 0.1 to 10 nM, the accumulated number of hippocampal neurons responding to PACAP38 progressively increased and reached a plateau at 10 nM. Total neurons (33.0 ± 5.3%, n = 4; 502 neurons) were found to respond to 100 nM PACAP38. The half-maximal concentration (ED50) of PACAP38 was 2.60 ± 0.77 nM. Typically, 60-90 sec after the addition of PACAP38 (10 nM), [Ca2+]i increased from basal levels of 50-100 to 150-300 nM. VIP also increased [Ca2+]i, but required 1 μM or higher concentration for a considerable number of cells to respond. The number of hippocampal neurons responding to VIP at 1 μM was 28.9 ± 9.8% (n = 4; 442 cells) which was comparable to the population of neurons responding to 10 nM PACAP38. The ED50 for VIP was 0.68 ± 0.38 μM which was approximately 260 times higher than the ED50 for PACAP38. Neither 1-10 μM nitrendipine, a L-type voltage-dependent Ca2+ channel blocker, or 1 μM ω-conotoxin GVIA, a N-type voltage-dependent Ca2+ channel blocker, altered the PACAP-induced Ca2+ increment. Removal of Ca2+ from the superfusion media did not influence the PACAP38-induced increase of [Ca2+]i. Furthermore, 1-30 μM forskolin or 1-10 mM (Bu)2CAMP did not increase [Ca2+]i in the same neurons which responded to PACAP38. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide, an inhibitor of protein kinase A, did not affect PACAP38-induced increase in [Ca2+]i. These findings suggest that PACAP increases [Ca2+]i in rat hippocampal neurons by mobilizing Ca2+ from intracellular stores and that this action is not linked to activation of the cAMP-protein kinase A signal transduction system.

UR - http://www.scopus.com/inward/record.url?scp=0026634305&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026634305&partnerID=8YFLogxK

M3 - Article

VL - 131

SP - 73

EP - 81

JO - Endocrinology

JF - Endocrinology

SN - 0013-7227

IS - 1

ER -