Search

Purpose: We provided direct evidence for the existence of purinergic innervation in the rat urinary bladder., Materials and Methods: The non-adrenergic non-cholinergic (NANC) innervation was studied in 4-month-old Wistar rats. Electric-field stimulation (EFS) of the detrusor muscle strips in the presence of four autonomic blockers (atropine 10(-6) M, guanethidine 10(-6) M, phentolamine 10(-6) M and propranolol 10(-6) M) showed NANC contractions accounted for about 50% of the maximum contractile response. The adenyl purines released from nerves by EFS were detected by HPLC after conversion to ethenopurines. The amount of total purine released was frequency-dependent and could be totally suppressed by tetradotoxin (10(-6) M). The amount of ATP released was significantly greater than those for ADP, AMP and adenosine (p < 0.05, n = 4). Desensitization induced by alpha, beta-MeATP (10(-6) to 10(-4) M), a P2x receptor agonist, reduced the NANC contraction. In addition, the NANC contraction was also abolished by P2 receptor blocker suramin (10(-4) to 10(-3) M) and P2x receptor blocker PPADS (10(-5) to 10(-4) M.)., Conclusion: The results of the present study give evidence to support purinergic nerve-mediated bladder smooth muscle contractions in the rat. Among the purine nucleotides, ATP is the dominant purinergic neurotransmitter released and P2x receptor activation is responsible for the NANC contractile response.

The functional role of non-adrenergic non-cholinergic (NANC) nerves in the autonomic control of the male mini-pig bladder neck was investigated in the present study. Electrical stimulation of muscle strips from male mini-pig bladder neck showed biphasic response with initial phasic contraction followed by post-contractile relaxation. Electrical stimulation in the presence of four autonomic blockers (atropine 10(-6) M, propanolol 10(-6) M, phentolamine 10(-6) M) showed suppression of 68 +/- 15% of the contractile response (P < 0.05, n = 8) but no significant change in the relaxation response. Alpha-chymotrypsin 2 U/ml, L-NG-monomethyl-L-arginine acetate (a nitric oxide synthetase inhibitor) 10(-4) M, 8-phenylthlophylline (a P1-purinoceptor antagonist) 10(-6) M, and pyridoxal-phosphate-6-azophenyl-2', 4'-disulphoric acid tetrasodium salt (a P2Y-purinoceptor antagonist) 3 x 10(-5) M did not alter the NANC response significantly. On the other hand, reactive blue-2 (a P2Y-purinoceptor antagonist) 3 x 10(-5) significantly reduced the relaxation by 79 +/- 9%. The result suggested that the P2Y-purinoceptor is involved in the electrically induced NANC post-contractile relaxation of the mini-pig bladder neck smooth muscle.

1. Contractile responses of smooth muscle from the Wistar rat urinary bladder were studied with the use of muscarinic agonists and antagonists. 2. McN-A-343 induced only weak contractile responses of the bladder muscle. In contrast, oxotremorine showed higher potency than either acetylcholine or bethanechol in inducing a contractile response (the respective pD2 values were 6.38 +/- 0.25, 4.82 +/- 0.24 and 4.42 +/- 0.14). 3. The M2 antagonists, methoctramine (10(-9) M to 10(-5) M) and gallamine (10(-9) M to 10(-5) M), did not reduce acetylcholine-induced (10(-5) M) contractions of the bladder muscle strip. On the other hand, 4-diphenyl-acetoxy-N-methyl piperidine methiodide (4-DAMP, 10(-10) M to 10(-7) M), an M3 receptor blocker, effectively antagonized the acetylcholine-induced contractions in a concentration-dependent manner. 4-DAMP had a similar pA2 value to those of the non-selective antagonists, atropine and scopolamine (pA2 values were 8.26 +/- 0.05, 8.36 +/- 0.05 and 8.41 +/- 0.11, respectively). Pirenzepine, and M1 blocker, antagonized the contractions at higher concentrations (10(-8) M to 10(-5) M, pA2 = 6.23 +/- 0.04). 4. It is concluded that (1) the dominant muscarinic receptor subtype responsible for smooth muscle contraction in the rat urinary bladder is M3; and (2) the muscarinic agonist oxotremorine was more potent than acetylcholine and bethanechol in inducing a contractile response.

Synaptosomes are nerve-end particles (NEP) isolated by using the technique of differential centrifugation. The synaptosome offers a good model for biochemical and pharmacological studies of the nerve endings. No report has been made on synaptosome isolation from the urinary bladder. The purpose of our work was to develop the use of synaptosome in the research of neurophysiology and neuropharmacology of the urinary bladder. Synaptosome-rich fraction was prepared from tissue homogenate of male Wistar rat urinary bladder by differential centrifugation (1000, 17,000 and 100,000 g) with discontinuous sucrose gradient. Electron microscopy showed synaptosomes as thin-walled bags containing a large number of synaptic vesicles. Two types of synaptosomes were easily discerned: those containing small agranular vesicles, and those containing dense-cored vesicles. The acetylcholine, norepinephrine, epinephrine and dopamine contents in the preparation were measured by the method of high-performance liquid chromatography. The respective concentrations were 300.4 +/- 30.1, 962.8 +/- 58.5, 617.3 +/- 59.8 and 1354.8 +/- 144.2 pmol/mg synaptosomal protein. In conclusion, it has been demonstrated that synaptosome-rich fractions can be prepared from the rat urinary bladder. Thus it is possible to apply this methodology for the investigation of the neurobiology of urinary bladders.

Three-month-old male Wistar rats were rendered diabetic with a single intravenous injection of streptozotocin (60 mg/kg body weight). Two weeks after induction of diabetes, synaptosome-rich fractions were prepared from urinary bladder tissue homogenate of the diabetic rats and control rats by differential centrifugation (1000 x g, 17,000 x g and 100,000 x g) with discontinuous sucrose gradient. Synaptosomal acetylcholine, norepinephrine, epinephrine and dopamine were measured by the method of high-performance liquid chromatography. The respective neurotransmitter concentrations for the diabetic rats were 1537.8 +/- 65.3, 4757.7 +/- 361.9, 3720.7 +/- 276.1, and 2447.8 +/- 196.8 pmol/mg synaptosomal protein, respectively; those for the control rats were 338.1 +/- 25.0, 1009.0 +/- 54.6, 645.3 +/- 52.2, and 1426.1 +/- 123.9 pmol/mg protein, respectively. Thus, the synaptosomal concentrations for all the measured neurotransmitters were significantly higher in the diabetic rats (P < 0.05 for each comparison). In conclusion, it has been demonstrated that the vesicle-bound acetylcholine and catecholamines in the synaptosome-rich fraction of the urinary bladder were significantly increased in 2-week diabetic rats. This finding would suggest impaired neurotransmitter release from both the bladder sympathetic and parasympathetic efferent nerve endings in early streptozotocin-induced diabetes.

Synaptosome-rich fractions were prepared from tissue homogenate of the urinary bladder of the spontaneously hypertensive rat and normotensive Wistar-Kyoto rat by differential centrifugation (1000 x g, 17 000 x g and 100 000 x g) with discontinuous sucrose gradient. Synaptosomal acetylcholine, norepinephrine, epinephrine and dopamine were measured by the method of high-performance liquid chromatography. The respective neurotransmitter concentrations for the normotensive rats were 300.4 +/- 30.1, 962.8 +/- 58.5, 617.3 +/- 59.8, and 1354.8 +/- 144.2 pmol/mg synaptosomal protein. For the hypertensive rats, the acetylcholine concentration (203.8 +/- 23.0 pmol/mg protein) was significantly lower (P < 0.05), while the norepinephrine, epinephrine and dopamine concentrations (1459.0 +/- 180.3, 971.3 +/- 62.2, and 2161.0 +/- 243.4 pmol/mg protein, respectively) were significantly higher (P < 0.05 for all) than those of the normotensive rats. In conclusion, it has been demonstrated that the vesicle-bound catecholamines in the synaptosome-rich fraction of the urinary bladder were significantly increased in hypertensive rats. On the contrary, the synaptosomal acetylcholine concentration was significantly decreased. These findings are suggestive of increased sympathetic innervation and decreased parasympathetic innervation in the urinary bladder of the spontaneously hypertensive rat.

Nonadrenergic, noncholinergic (NANC) contraction has been demonstrated in animal urinary bladder. However, the exact nature of the NANC innervation is still unclear. 1,1-Dimethylphenylpiperazinium (DMPP), which generates action potentials in the cell body of the postganglionic neuron and causes neurotransmitter release (both acetylcholine and noradrenaline), was given intravenously (0.1-0.7 mg/kg) to 3-month-old female Wistar rats under anesthesia (n = 20). Intravesical pressure, heart rate and blood pressure of the rats were monitored on Gould polygraph. Monophasic dose-dependent contractile response was observed upon administration of DMPP in 12 of 20 rats. After total adrenergic and cholinergic blockade with atropine, guanethidine, phentolamine and propranolol, the contractile response was reduced, not completely, in the animals. At the dose of 0.7 mg/kg, the contraction was reduced to about 48% of the original response. The study provides in vivo evidence for NANC contraction in the rat urinary bladder, moreover, the neurotransmitter is released from the postganglionic neurons.

The role of peptides in mediating the nonadrenergic, noncholinergic (NANC) response of the rat urinary bladder was studied. Electrical stimulation of muscle strips from 3-month-old female Wistar rat urinary bladders in the presence of autonomic blockers (atropine 10(-6) mol/l, propanolol 10(-6) mol/l, phentolamine 10(-6) mol/l, and guanethidine 10(-6) mol/l) showed NANC contraction accounting for 60% of the maximum contractile responses at 40 Hz. Frequency-response studies showed that in the presence of alpha-chymotrypsin (2 U/ml, 30-min incubation), the NANC contractile responses to electrical stimulation at lower frequencies (3-10 Hz) were enhanced (p < 0.05; n = 9). However, no significant differences were observed at higher frequencies (20-40 Hz). With repetitive 4-Hz stimulation, alpha chymotrypsin caused a 19% increase in the NANC contractile response (p < 0.05; n = 8). It is postulated that the NANC response of the rat bladder smooth muscle is composed of an excitatory (contractile) and an inhibitory (relaxant) component. Some peptide(s) is/are responsible for mediating the inhibitory response.

Previous studies have shown that pregnancy is associated with a decrease in cholinergic function in the rabbit urinary bladder. The present study aimed at evaluating the effects of pregnancy on the autonomic function of the rat urinary bladder and to elucidate whether progesterone is responsible for such alterations. Female Wistar rats, 3 months old, were divided into four groups: (1) 2-week pregnant rats; (2) rats given daily intramuscular injections of progesterone 5 mg/kg for 2 weeks; (3) rats given intramuscular injections of vehicle for 2 weeks, and (4) controls. Cystometry showed a significant increase in bladder capacity in the pregnant rats. The wet weight of the pregnant rat bladder was also significantly increased. Histologic study revealed increased bladder wall thickness with interstitial edema and urothelium proliferative changes to a papillary configuration in these pregnant bladders. Bladder muscle strip study showed significantly reduced maximum contractile responses to acetylcholine and methoxamine in the pregnant and the progesterone groups. Muscarinic receptor binding study demonstrated reduced Bmax in the pregnant rats and rats receiving progesterone injections (control group Bmax = 57 +/- 11, pregnant group Bmax = 44 +/- 8, p < 0.05; progesterone group Bmax = 40 +/- 7, vehicle group Bmax = 58 +/- 9 fmol/mg protein, p < 0.05). The contractile response to lower concentrations (10(-6) mol/l to 10(-4) mol/l) of ATP was elevated in the pregnant rats.(ABSTRACT TRUNCATED AT 250 WORDS)