A comparative study of NMDA receptor expression and modulation in the RVLM and CVLM of normotensive and spontaneously hypertensive rats

1 page(s

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, β-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.15 page(s

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

Age dependent response to anti-hypertensive therapy in a new model of rat polycystic kidney disease

2 page(s

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 μmol/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 μm, n = 14) at week 24 (all p ≤ 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 ≤ 0.05). Ganglionic blockade (hexamethonium 3.3 mg/kg) significantly reduced mean BP in the LPK (52 vs. Lewis 4%, n = 9, p ≤ 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