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45P University of Oxford
BPS 75th Anniversary Meeting December 2006

 

Muscarinic receptor function in the streptozotocin-diabetic rat urinary bladder

Bahareh Vahabi, Neil McKay, Kim Lawson and Donna Sellers. Biomedical Research Centre, Sheffield Hallam University, Sheffield, S1 1WB

 

Diabetic patients have an increased incidence of bladder dysfunction, including bladder overactivity ( Salinas et al., 1999 ). The pathophysiological mechanisms underlying this remain unclear. In normal bladder smooth muscle contraction is mediated by the M3 muscarinic receptor subtype. However, it has been suggested that in pathological conditions the larger population of M2 receptors may be involved (Braverman & Ruggieri, 2003). The aim of the present study was to characterise the muscarinic receptor subtype mediating contraction of bladder smooth muscle in the streptozotocin-induced diabetic rat.

Male Wistar rats (200-250g) were administered streptozotocin (STZ, 65mgkg-1, i.p.). Strips of detrusor smooth muscle were isolated from bladder of rats 1, 4, 8 and 12 weeks after STZ or from weight matched controls. Tissues were mounted in tissue baths containing Krebs-bicarbonate solution (in mM, 118.3 NaCl, 11.7 D-glucose, 24.9 NaHCO3, 4.7 KCl, 1.15 MgSO4, 1.15 KH2PO4, 1.9 CaCl2) with 5µM indomethacin at 37°C and gassed with 95% O2/5% CO2. After equilibration (2g tension for 60 minutes) cumulative concentration contractile response curves (CRCs) to carbachol (0.01-100µM) were determined in the presence and absence of muscarinic antagonists (4-DAMP (3-30nM), pirenzepine (3-30µM) and methoctramine (3-30µM)). Isometric tension was measured via UF1 force transducers connected to a Powerlab using Chart software. Individual EC50 values (molar concentration producing half maximal response) were determined via non-linear regression of the CRCs using Prism software (GraphPad, San Diego). Dissociation constants (pKB) were calculated and, from Schild plots, pA2 values with slopes were determined. Data is presented as means ± SEM.

Pirenzepine and methoctramine, which have high affinity at M1 and M2 muscarinic receptors respectively, both showed low affinity in the control, 1, 4, 8 and 12 week diabetic rat bladder detrusor. However, the M3 selective antagonist 4-DAMP had a high affinity in rat detrusor from all groups. Schild plots had slopes close to unity indicating action at a single receptor. (students t test)

 

 

4-DAMP

Methoctramine

Pirenzepine

 

pKB

pA2

Slope

pKB

pA2

Slope

pKB

pA2

Slope

Control

 

9.15±0.07

(n=23)

9.0

1.17±0.15

5.77±0.05

(n=7)

5.9

0.83±0.05

6.88±0.04

(n=23)

6.9

0.98±0.01

1 week

diabetic

8.99±0.06

(n=24)

8.8

1.19±0.05

5.78±0.06

(n=8)

5.9

0.83±0.10

6.82±0.05

(n=24)

7.0

0.91±0.01

4 week

diabetic

9.05±0.04

(n=24)

8.9

1.12±0.04

5.50±0.07

(n=24)

5.7

0.87±0.10

6.79±0.06

(n=24)

6.8

1.01±0.03

8 week

diabetic

9.18±0.12

(n=20)

8.9

1.21±0.04

5.37±0.10

(n=20)

5.4

0.83±0.05

6.79±0.09

(n=20)

6.8

0.96±0.20

12 week diabetic

9.05±0.05

(n=20)

8.9

1.18±0.01

6.27±0.39

(n=20)

5.6

0.93±0.08

7.02±0.04

(n=20)

7.0

1.00±0.02

 

These results suggest that direct contractile responses of diabetic rat detrusor muscle to muscarinic receptor stimulation in vitro are mediated solely via the M3-muscarinic receptor subtype with little contribution from the M2 receptor population.

 

Braverman A.S & Ruggieri M.R (2003) Am J Physiol Regul Integr Comp Physiol 285:701-708
Salinas Casado et al. (1999) Arch Esp Urol 52 (2): 149-156