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: 50ng/ml, 2.1- and 13.4-fold increases, respectively, P< or =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 (approximately 4-fold increase in WEHI and Ordway lines, P< or =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.

Temporal relationship between renal cyst development, hypertension and cardiac hypertrophy in a new rat model of autosomal recessive polycystic kidney disease.

BACKGROUND/METHODS: We have examined the hypothesis that cyst formation is key in the pathogenesis of cardiovascular disease in a Lewis polycystic kidney (LPK) model of autosomal-recessive polycystic kidney disease (ARPKD), by determining the relationship between cyst development and indices of renal function and cardiovascular disease. RESULTS: In the LPK (n = 35), cysts appear at week 3 (1.1 +/- 0.1 mm) increasing to week 24 (2.8 +/- 2 mm). Immunostaining for nephron-specific segments indicate cysts develop predominantly from the collecting duct. Cyst formation preceded hypertension (160 +/- 22 vs. Lewis control 105 +/- 20 mm Hg systolic blood pressure (BP), n = 12) at week 6, elevated creatinine (109 +/- 63 vs. 59 +/- 6 micromol/l, n = 16) and cardiac mass (0.7 vs. 0.4% bodyweight, n = 15) at week 12, and left ventricular hypertrophy (2,898 +/- 207 vs. 1,808 +/- 192 mum, n = 14) at week 24 (all p < or = 0.05). Plasma-renin activity and angiotensin II were reduced in 10- to 12-week LPK (2.2 +/- 2.9 vs. Lewis 11.9 +/- 4.9 ng/ml/h, and 25.0 +/- 19.1 vs. 94.9 +/- 64.4 pg/ml, respectively, n = 26, p < or = 0.05). Ganglionic blockade (hexamethonium 3.3 mg/kg) significantly reduced mean BP in the LPK (52 vs. Lewis 4%, n = 9, p < or = 0.05). CONCLUSION: Cyst formation is a key event in the genesis of hypertension while the sympathetic nervous system is important in the maintenance of hypertension in this model of ARPKD.

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 < or = 0.05). Growth of MPC cells in Roswell Park Memorial Institute media induced a further significant increase in PNMT gene expression (P < or = 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 [(3)H] noradrenaline uptake activity (threefold > than PC12 American Type Culture Center line, P < or = 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.

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< or =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< or =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< or =0.05), while nNOS was significantly higher (1.76-fold greater, P< or =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.

Functional expression of muscarinic and purinoceptors in the urinary bladder of male and female rats and guinea pigs.

PURPOSE: The objectives of this study were to compare the functional expression of muscarinic and purinergic receptors in the urinary bladder of 2 species, rat and guinea pig under comparable experimental conditions; and to test whether the receptors in males and females differ. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) techniques were used to identify gene expression profiles in bladder smooth muscle (total n=8 rats, 7 guinea pigs) and mechanical responses to nerve stimulation and applied acetylcholine (ACh) in the presence of specific antagonists were used to identify functional receptor sub-types (total n=12 rats, 16 guinea pigs). RESULTS: RT-PCR indicated that M2 and M3 were the predominant muscarinic receptor genes in both the male and female rat and guinea pig bladders. The phasic component of the nerve-induced contraction was greater in guinea pigs vs. rats. The tonic component and the ACh response were inhibited by the M3 receptor antagonist, darifenacin (10(-6) M, P≤0.05), but not by the M2 receptor antagonist, methoctramine (10(-5) M). The antipurinergic drug α, ß-methylene ATP (5 × (-5) M) caused a significant reduction in the amplitude of the phasic response to nerve stimulation in all groups, and this effect was significantly greater in male vs. female rats. mRNA for the purinergic P2X1, P2X2, P2X4, P2X5 and P2X7 receptors was detected in both male and female rats, whereas P2X3 and P2X6 were inconsistently detected in male rats. The P2X1 purinoceptor antagonist pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4', 8'-disulphonate) (PPNDS), only inhibited nerve induced contractions at high concentrations (up to 10(-4) M). CONCLUSIONS: While only minor functional differences were documented in cholinergic and purinergic bladder contractile responses between male and female animals, and between rats and guinea pigs, data such as presented in this study are critical in determining how relative functional contributions may change in the diseased state, providing valuable information towards new treatment options.