Comparative studies of PC12 and mouse pheochromocytoma-derived rodent cell lines as models for the study of neuroendocrine systems

We have compared PC12 cell lines derived from different laboratories and the newly developed mouse pheochromocytoma (MPC) cell line. Morphologically, there were distinct differences in size, shape, adherence, and clumping behavior, which varied in response to different culture media, growth substrates, and nerve growth factor. Quantitative messenger ribonucleic acid (mRNA) analysis showed significant variability in the expression of the catecholaminergic biosynthetic enzymes tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT), the noradrenaline transporter (NAT), and neuron-specific enolase (NSE) between all lines examined. Of most significance were the increased levels of PNMT mRNA in the MPC cells, which were to 15-fold greater than in the PC12 cell lines grown under the same conditions in Dulbecco modified Eagle medium (P ≤ 0.05). Growth of MPC cells in Roswell Park Memorial Institute media induced a further significant increase in PNMT gene expression (P ≤ 0.05). Immunohistochemistry for TH, PNMT, and NAT was generally consistent with mRNA analysis, with the MPC cells demonstrating strong immunoreactivity for PNMT. The MPC cells showed the highest levels of desipramine-sensitive [3H] noradrenaline uptake activity (threefold > than PC12 American Type Culture Center line, P ≤ 0.05), despite relatively low levels of NAT mRNA. These results indicate that PC12 cell lines should be carefully chosen for optimal utility in the study of chromaffin cell or sympathetic neuron biology and that cell features will be influenced by type of media and substrate chosen. Furthermore, they confirm that the new MPC cell line is likely a useful model for the study of adrenergic mechanisms or studies involving NAT

Lack of functional expression of NMDA receptors in PC12 cells

PC12 cells are an established model for studying the role of N-methyl-d-aspartate (NMDA) receptors in excitotoxicity and function as multimeric assemblies of NR1 with at least one NR2(A-D) subunit. We examined NR1 splice variant and NR2 subunit expression in four PC12 cell-lines (ATCC, WEHI, Ordway and Flinders), correlated mRNA expression with protein expression, and used patch-clamp recordings to test functionality. PCR indicated strong expression of the NR1 splice variants NR1-2a and NR1-4a in all cell-lines, with the remainder weakly detected or absent. Real-time PCR showed variable levels of NR1 mRNA expression (all splice variants) between cell-lines and a significant increase in response to nerve growth factor in the WEHI and Ordway lines (NGF: 50 ng/ml, 2.1- and 13.4-fold increases, respectively, P ≤ 0.05). mRNA for NR2A or NR2B was not detected in any PC12 cell-line. NR2C mRNA expression varied between lines and increased after NGF treatment (∼4-fold increase in WEHI and Ordway lines, P ≤ 0.05). In the Ordway line, NR2D mRNA was seen only after NGF treatment. Immunohistochemistry confirmed protein expression for NR1, NR2C and NR2D, and while fluorescence intensity changes in response to NGF paralleled mRNA responses, the degree of increase was of reduced magnitude. Whole-cell patch-clamping of NGF treated cells failed to detect functional NMDA receptors in any of the cell-lines. Our study demonstrates that in contrast to neurons from the CNS, PC12 cells do not express a normal complement of NMDA receptor-subunits, and this may be one factor limiting functional responses to NMDA/glutamate and consequently the use of PC12 cells as a neuronal model

Unique levels of expression of N-methyl-d-aspartate receptor subunits and neuronal nitric oxide synthase in the rostral ventrolateral medulla of the spontaneously hypertensive rat

The rostral ventrolateral medulla (RVLM) is the major brainstem region contributing to sympathetic control of blood pressure. We have compared the expression of N-methyl-D-aspartate (NMDA) receptor subunits (NR1, NR2A-D), NR1 splice variants (NR1-1a/1b, -2a/2b, -3a/3b, -4a/4b), and the neuronal and inducible isoforms of NO synthase (nNOS and iNOS) in the RVLM of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR), based on the hypothesis that altered NMDA receptor make-up or altered expression of endogenous NO may be associated with the increase in sympathetic output described from this site in hypertension. Total RNA was extracted and reverse transcribed from the RVLM of mature male WKY and SHR (16-23 weeks). Conventional polymerase chain reaction (PCR) indicated that only the NR1 splice variants NR1-2a, NR1-2b, NR1-4a and NR1-4b were expressed in the RVLM of either species. Quantitative real-time PCR indicated that for both strains of rat, mRNA for the NR1 subunit (all splice variants) was the most abundant (16.5-fold greater, P≤0.05, relative to the NR2A subunit). Amongst the NR2A-D subunits, NR2C was the most abundant (7- and 1.7-fold greater relative to the NR2A subunit, P≤0.05, WKY and SHR, respectively). Relative to WKY, mRNA levels for the NR2C and NR2D subunits in the SHR RVLM were significantly lower (0.3- and 0.25-fold less, P≤0.05), while nNOS was significantly higher (1.76-fold greater, P≤0.05). This was confirmed immunohistochemically for nNOS expression. These results demonstrate differential expression levels of NMDA receptor subunits and NOS isoforms in the RVLM region of SHR when compared to WKY rats

"THE COMING OF AGE OF ISLAND STUDIES"

This paper presents insights into the emerging academic field of 'island studies', defined as the interdisciplinary study of islands on their own terms. This exposé is undertaken in two ways: conceptually, by means of a critical and judicious review of the literature across a number of disciplines; and analytically in relation to what is probably the most popular scholarly piece of non-fiction based on an island society written to date -"Coming of Age in Samoa" by Margaret Mead. Copyright (c) 2004 by the Royal Dutch Geographical Society KNAG.