Muscle-derived cell-mediated ex vivo gene therapy for urological dysfunction.

We have tested the feasibility of muscle-based gene therapy and tissue engineering for urological dysfunction using highly purified muscle-derived cells (MDC) that display stem cell characteristics. We then explored the potential use of these MDC as an alternative therapy for the treatment of impaired detrusor contractility. The MDC were genetically engineered to express the gene encoding beta-galactosidase and injected into the bladder walls of SCID mice. The injected bladders were harvested at various time-points after injection and assayed for beta-galactosidase activity; the presence of myofibers within the injected tissue was determined by detection of fast myosin heavy chain isoform (MyHCs). We have demonstrated that the injected MDC are capable of not only surviving in the lower urinary tract, but also improving the contractility of the bladder following an induced injury. Two potential mechanisms can be used to explain this finding. First, we have observed that some of the beta-galactosidase-expressing cells expressed alpha-smooth muscle actin, suggesting a differentiation into smooth muscle. Second, a stain for acetylcholine receptors (AChRs), which identifies the location of neuromuscular junctions, revealed that the myofibers derived from the doner cells became innervated into the bladder as early as 2 weeks after injection. These results suggest that gene therapy and tissue engineering based on MDC potentially can be used for urological dysfunction.

Botulinum toxin urethral sphincter injection resolves urinary retention after pubovaginal sling operation.

The management of prolonged urinary retention following pubovaginal sling surgery typically involves transvaginal urethrolysis for anatomical urethral obstruction. Brubaker [1] recently reported on urethral sphincter abnormalities as a cause of postoperative urinary retention following either Burch suspension or pubovaginal sling procedure. We report a case of functional urethral obstruction and detrusor acontractility following pubovaginal sling surgery that was successfully treated by botulinum A toxin urethral sphincter injection.

Botulinum toxin urethral sphincter injection resolves urinary retention after pubovaginal sling operation.

The management of prolonged urinary retention following pubovaginal sling surgery typically involves transvaginal urethrolysis for anatomical urethral obstruction. Brubaker recently reported on urethral sphincter abnormalities as a cause of postoperative urinary retention following either Burch suspension or a pubovaginal sling procedure. We report a case of functional urethral obstruction and detrusor acontractility following pubovaginal sling surgery that was successfully treated by botulinum A toxin urethral sphincter injection.

Protein kinase C is involved in M1-muscarinic receptor-mediated facilitation of L-type Ca2+ channels in neurons of the major pelvic ganglion of the adult male rat.

We used patch clamp recording techniques to determine if muscarinic signaling mechanisms are present in dissociated autonomic neurons obtained from the major pelvic ganglion, which provides the cholinergic innervation of the urinary bladder and other pelvic organs. The M1 specific agonist, McN-A-343 (2-30 microM) enhanced Ca2+ currents in approximately 37% of neurons (by 50-80%). This enhancement was reduced by atropine (5-10 microM) or a PKC inhibitor (bisindolylmaleimide, 50-200 nM). In responsive neurons Ca2+ currents were also enhanced by the phorbol ester, phorbol-12,13-dibutyrate (50-300 nM) and the dihydropyridine agonist Bay K 8644 (5 microM) and had kinetics of activation and inactivation as expected for L-type Ca2+ channels. We conclude that in a subpopulation of MPG neurons, M1-mediated activation of PKC phosphorylates and enhances L-type Ca2+ channel activities. This muscarinic facilitatory mechanism in MPG neurons may be the same as the M1-mediated facilitation of transmitter release reported previously at the nerve terminals in the urinary bladder.

Facilitation of transmitter release in the urinary bladders of neonatal and adult rats via alpha1-adrenoceptors.

Age-dependent changes in the effects of the alpha1-adrenoceptor agonist, phenylephrine were investigated on neurally evoked contractile responses and basal tone in smooth muscle strips from rat urinary bladder. Phenylephrine facilitated the neurogenic contractions in both neonatal and 7-month-old adult rats. However, phenylephrine increased the basal tone in adult but not neonatal rats. In adult rats, phenylephrine-induced facilitation of neurally evoked contractions occurred before and after the block of cholinergic contractions with 1 microM atropine. In adult rats, the phenylephrine facilitation was reduced at stimulation parameters (20 Hz, 80 shocks and maximal voltage) which activated muscarinic receptor mediated facilitation of acetylcholine release. The results indicate that pre-synaptic alpha1-adrenoceptors facilitate the release of both acetylcholine and the non-cholinergic non-adrenergic transmitter. In summary, alpha1-adrenoceptor-mediated facilitation is less expressed when muscarinic M1 receptor mediated facilitation is functioning; pre-junctional alpha1-adrenoceptors are present in the bladder of both neonatal and adult rats, whereas post-junctional alpha1-adrenoceptors are expressed only in older adult rats.

Botulinum toxin urethral sphincter injection to restore bladder emptying in men and women with voiding dysfunction.

PURPOSE: Botulinum toxin injection into the external urinary sphincter in spinal cord injured men with detrusor-sphincter dyssynergia has been reported. We expand the clinical use of botulinum toxin for a variety of bladder outlet obstructions and to decrease outlet resistance in patients with acontractile detrusor but who wish to void by the Valsalva maneuver. MATERIALS AND METHODS: Prospective treatment was performed for voiding dysfunction in 8 men and 13 women 34 to 74 years old. The reasons for voiding dysfunction included neurogenic detrusor-sphincter dyssynergia in 12 cases, pelvic floor spasticity in 8 and acontractile detrusor in 1 patient with multiple sclerosis who wished to void by the Valsalva maneuver. Using a rigid cystoscope and a collagen injection needle, a total of 80 to 100 units of botulinum A toxin (Botox) were injected into the external sphincter at the 3, 6, 9 and 12 o'clock positions. RESULTS: Preoperatively 19 of 21 patients were on indwelling or intermittent catheterization. After botulinum A injection all but 1 patient were able to void without catheterization. No acute complications, such as general paralysis or respiratory depression, occurred and none of the patients had dribbling or stress urinary incontinence. Postoperative post-void residual decreased by 71% and voiding pressures decreased on average 38%. Of the 21 patients 14 (67%) reported significant subjective improvement in voiding. Followup ranges from 3 to 16 months, with a maximum of 3 botulinum A injections in some patients. CONCLUSIONS: Urethral sphincter botulinum injection should be considered for complex voiding dysfunction. Encouraging improvement without complications were seen in most of our patients. We have expanded the use of botulinum toxin to treat pelvic floor spasticity and also women.

Smooth muscle and parasympathetic nerve terminals in the rat urinary bladder have different subtypes of alpha(1) adrenoceptors.

Neurally evoked contractions and release of (3)H- acetylcholine (ACh) during electrical field stimulation were measured in rat urinary bladder strips. The alpha(1) agonist phenylephrine (PE, 2-8 microM) increased the amplitude of neurally evoked contractions, facilitated the release of ACh and increased the baseline tone of the bladder strips. The PE-induced facilitation of the contractions did not significantly change during a prolonged exposure to PE (120 min), whereas the PE-induced rise in baseline tone gradually decreased to 65% of the initial value. Low concentrations of specific alpha(1A) antagonists, 5-methyl urapidil (5-MU), REC15/2739 and WB-4101 competitively inhibited the facilitation of the neurally-evoked contractions (pA(2:) 8.77; 9.59 and 9.62, respectively), whereas higher concentrations of 5-MU (IC(50): 48 nM) were required to suppress the PE-rise in baseline. WB-4101 (100 microM) inhibited the PE-induced facilitation of ACh release. The irreversible alpha(1B) antagonist chloroethyl-clonidine (CEC, 10-50 microM) inhibited the PE-evoked rise in base line tone, but did not affect the PE-induced facilitation of the neurally evoked contractions nor the facilitation of ACh release. However, CEC increased the area and amplitude of the neurally-evoked contractions by 261+/-33 and 47.2+/-8.4%, respectively. Atropine significantly inhibited the CEC evoked increase in area and amplitude of the electrically evoked contractions (76.5+/-4.8 and 40.8+/-3%, respectively) indicating that CEC facilitated the cholinergic responses of the electrically stimulated bladder strips. It is concluded that alpha(1A) and CEC sensitive alpha(1B) and/or alpha(1D) adrenoceptors are expressed in the rat bladder in different locations. On the cholinergic nerve terminals alpha(1A) adrenoceptors mediate prejunctional facilitation, whereas postjunctional alpha(1B)/alpha(1D) adrenoceptors mediate smooth muscle contraction.

Review article: effects of the 5-HT3 receptor antagonist alosetron on neuromuscular transmission in canine and human intestinal muscle.

BACKGROUND: Currently, therapeutic treatments for irritable bowel syndrome fail to produce significant clinical results. We hypothesized that alosetron, a selective 5-HT3 antagonist, may provide symptomatic relief in irritable bowel syndrome patients through a decrease in the amplitude of gastrointestinal contractions. AIM: To determine the in vitro effect of alosetron on neuromuscular transmission in the canine and human jejunal and colonic muscularis externa. RESULTS: Alosetron diminished electrical field-stimulated (EFS) contractions recorded from muscles of the canine and human small and large intestines. Mechanistically, the diminished EFS response could be explained by the ability of alosetron to decrease the fractional release of 14C-choline radiolabelled acetylcholine evoked by EFS from human jejunal muscle. The inhibition of EFS contractions was not limited to atropine-sensitive events, as non-cholinergic excitatory EFS evoked contractions were also inhibited. Additionally, alosetron at high concentrations (> 30 microM) directly altered bethanechol stimulated contractions. CONCLUSION: Caution must be used in the interpretation of these data because significant alterations in EFS-induced contractions were only observed with large pharmacological concentrations of alosetron, and the response was not selective for cholinergically-mediated excitatory neuromuscular transmission.

Modulation of voiding and storage reflexes by activation of alpha1-adrenoceptors.

OBJECTIVE: This paper reviews recent studies in animals that examined the effect on lower urinary tract function of alpha1-adrenoceptor agonists and antagonists. METHODS: Bladder reflexes were studied in vivo on anesthetized rats and cats using cystometrographic and electrophysiologic techniques. Neurally-evoked bladder contractions and release of acetylcholine (ACh) were also studied in rat bladder strips in vitro. RESULTS: Administration of the alpha1-adrenoceptor agonist, phenylephrine (PE) to isolated strips of rat bladder enhanced neurally-evoked bladder contractions and increased basal tone. The former effects of PE were blocked by a selective alpha1A antagonist and the latter by an alpha1B antagonist. Activation of alpha1A receptors by PE enhanced ACh release evoked by electrical field stimulation in bladder strips. PE also enhanced transmission in cat bladder ganglia. PE or noradrenaline act on alpha- and beta-adrenoceptors on urothelial cells to release nitric oxide. It is concluded that facilitatory alpha1A-adrenoceptors are located prejunctionally in the bladder, whereas alpha1B adrenoceptors are located postjunctionally. In the central nervous system of the rat and cat facilitatory alpha1-adrenergic mechanisms can modulate the sympathetic, parasympathetic and somatic outflow to the urinary tract. In addition inhibitory alpha1 adrenoceptor mechanisms have been detected in the rat spinal cord. Activation of these receptors with PE raises the intravesical pressure threshold for inducing micturition and decreases voiding frequency. CONCLUSIONS: alpha1-adrenoceptors are located at various sites in the bladder and in the neural pathways controlling lower urinary tract function. At most sites these receptors mediate facilitatory responses that enhance smooth muscle activity or facilitate storage or voiding reflexes. However, alpha1-adrenoceptor inhibitory mechanisms in the rat spinal cord, can also reduce the frequency of voiding reflexes. This effect is possibly mediated by an inhibition in the afferent limb of the micturition reflex pathway.

Concentration-dependent isoflurane effects on depolarization-evoked glutamate and GABA outflows from mouse brain slices.

The synaptic concentrations of glutamate and gamma-aminobutyric acid (GABA) are modulated by their release and re-uptake. The effects of general anaesthetics on these two processes remain unclear. This study evaluates the effects of isoflurane, a clinically important anaesthetic, on glutamate and GABA release and re-uptake in superfused mouse cerebrocortical slices. Experiments consisted of two 1.5-min exposures to 40 mM KCl in 30 min intervals. During the second exposure, different concentrations of isoflurane with and without 0.3 mM L-transpyrrolidine-2,4-dicarboxylic acid (PDC, a competitive inhibitor of glutamate uptake transporter) or 1 mM nipecotic acid (a competitive inhibitor of GABA uptake transporter) were introduced. The ratios of the second to first KCl-evoked increases in glutamate and GABA were used to determine the isoflurane concentration-response curves. The results can be described as a sum of two independent processes, corresponding to the inhibitions of release and re-uptake, respectively. The EC50 values for the inhibitions of release and re-uptake were 295+/-16 and 805+/-43 microM for glutamate, and 229+/-13 and 520+/-25 microM for GABA, respectively. Addition of PDC did not significantly affect glutamate release but shifted the re-uptake curve to the left (EC50= 315+/-20 microM). Nipecotic acid completely blocked GABA uptake, rendering isoflurane inhibition of GABA re-uptake undetectable. Our data suggest that isoflurane inhibits both the release and re-uptake of neurotransmitters and that the inhibitions occur at different EC50's. For GABA, both EC50's are within the clinical concentration range. The net anaesthetic effect on extracellular concentrations of neurotransmitters, particularly GABA, depends on the competition between inhibition of release and that of re-uptake.

Function, signal transduction mechanisms and plasticity of presynaptic muscarinic receptors in the urinary bladder.

Presynaptic M1 muscarinic receptors on parasympathetic nerve terminals in rat urinary bladder strips are involved in an autofacilitatory mechanism that markedly enhances acetylcholine release during continuous electrical field stimulation. The facilitatory muscarinic mechanism is dependent upon a PKC mediated second messenger pathway and influx of extracellular Ca2+ into the parasympathetic nerve terminals via L and N-type Ca2+ channels. Prejunctional muscarinic facilitation has also been detected in human bladders. The muscarinic facilitatory mechanism is upregulated in hyperactive bladders from chronic spinal cord transected rats; and the facilitation in these preparations is primarily mediated by M3 muscarinic receptors. Presynaptic muscarinic receptors represent a new target for pharmacological treatment of bladder hyperactivity. If presynaptic facilitation is restricted to the bladder and not present in other tissues then drugs acting at this site might be expected to exhibit uroselectivity.

Frequency dependence of muscarinic facilitation of transmitter release in urinary bladder strips from neurally intact or chronic spinal cord transected rats.

1. Electrical stimulation evoked release of 3H-noradrenaline (NA) and 14C-acetylcholine (ACh), as well as neurally evoked contractions were measured at various (1-40 Hz, 100 shocks) stimulation frequencies in bladder strips from neurally intact (NI) and spinal cord transected (SCT) rats. 2. The frequency response curves for ACh and NA release were shifted to the left in SCT bladder strips as compared to NI bladder strips. 3. Atropine (1 microM) depressed ACh release in NI bladder strips at high frequency stimulation (10 and 40 Hz) but not at low frequency stimulation (2-5 Hz). However, in SCT bladders, atropine depressed ACh release both at low and high frequencies of stimulation, indicating that muscarinic facilitation occurs at lower frequencies. 4. Atropine depressed the release of NA in NI bladders at only 40 Hz stimulation, but depressed release at all frequencies in SCT bladders. 5. The amplitude of neurally evoked contractions of bladder strips from NI rats was enhanced as the frequency of stimulation was increased from 1 to 40 Hz (80 shocks). The frequency response curve was shifted to the left in SCT bladders. Atropine blocked the neurally evoked contractions in SCT bladder strips to a greater extent than the contractions in NI strips indicating a cholinergic dominance in the SCT bladders. 6. Maximal contractile force of SCT bladder strips evoked by neural stimulation at 20 Hz 10 shocks and 80 shocks was significantly lower than that of NI bladder strips, whereas the release of ACh was significantly higher in SCT than NI bladders indicating a postjunctional defect in the SCT preparations. 7. It is suggested that presynaptic muscarinic facilitatory mechanisms are upregulated in the cholinergic and adrenergic nerve terminals in SCT bladders leading to a larger relative contractile response at lower frequencies of stimulation (2-5 Hz). Thus the hyperreflexic bladder occurring after spinal cord injury may be due in part to an enhancement of transmitter release at bladder postganglionic nerve terminals.

Comparison of anaesthetic and non-anaesthetic effects on depolarization-evoked glutamate and GABA release from mouse cerebrocortical slices.

1. Investigation with substances that are similar in structure, but different in anaesthetic properties, may lead to further understanding of the mechanisms of general anaesthesia. 2. We have studied the effects of two cyclobutane derivatives, the anaesthetic, 1-chloro-1,2,2-trifluorocyclobutane (F3), and the non-anaesthetic, 1,2-dichlorohexafluorocyclobutane (F6), on K+-evoked glutamate and gamma-aminobutyric acid (GABA) release from isolated, superfused, cerebrocortical slices from mice, by use of h.p.l.c. with fluorescence detection for quantitative analysis. 3. At clinically relevant concentrations, the anaesthetic, F3, inhibited 40 mM K+-evoked glutamate and GABA release by 72% and 47%, respectively, whereas the structurally similar non-anaesthetic, F6, suppressed evoked glutamate release by 70% but had no significant effects on evoked GABA release. A second exposure to 40 mM KCl after a approximately 30 min washout of F3 or F6 showed recovery of K+-evoked release, suggesting that F3 and F6 did not cause any non-specific or irreversible changes in the brain slices. 4. Our findings suggest that suppression of excitatory neurotransmitter release may not be directly relevant to the primary action of general anaesthetics. A mechanism involving inhibitory postsynaptic action is implicated, in which a moderate suppression of depolarization-evoked GABA release by the anaesthetic may be consistent with the enhancement of postsynaptic GABAergic activities.

Role of L- and N-type Ca2+ channels in muscarinic receptor-mediated facilitation of ACh and noradrenaline release in the rat urinary bladder.

1. 3H-Noradrenaline (NA) and 14C-acetylcholine (ACh) released by electrical field stimulation were measured simultaneously in strips from the body of rat urinary bladder. 2. omega-Conotoxin GVIA (omega-CgTX; 20-100 nM) suppressed the non-facilitated transmitter release evoked by intermittent stimulation (IS), whereas nifedipine (1 microM) did not affect release. 3. Continuous electrical stimulation (CS) facilitated NA and ACh release via an atropine-sensitive mechanism. omega-CgTX reduced the facilitated release of NA (44% depression) but did not affect ACh release. Nifedipine depressed ACh release (43%) but not NA release. Combined administration of nifedipine and omega-CgTX (20 nM) produced a greater suppression of NA and ACh release (86 and 91%, respectively). 4. Maximal muscarinic facilitation of NA (5-fold) and ACh (17-fold) release occurred following administration of eserine, an anticholinesterase agent. Release of both NA and ACh was depressed by nifedipine (70 and 83%, respectively) but not by omega-CgTX. Combined application of omega-CgTX and nifedipine elicited a further depression of NA (95%) but not ACh release. 5. When NA and ACh release was facilitated with phorbol dibutyrate (0.5 microM), nifedipine inhibited ACh (67%) but not NA release, whereas omega-CgTX inhibited NA (73%) but not ACh release. Combined administration of both Ca2+ channel blockers did not elicit greater inhibition. 6. Bay K 8644, the L-type Ca2+ channel activator, increased ACh release in a dose-dependent manner (up to 5-fold) but did not significantly change NA release. 7. Both omega-CgTX (20-100 nM) and nifedipine (100 nM-1 microM) significantly decreased (50-80%) the neurally evoked contractions of the bladder strips. 8. It is concluded that L-type Ca2+ channels play a major role in muscarinic facilitation of NA and ACh release in the urinary bladder but are not essential for non-facilitated release. Other types of Ca2+ channels, including N-type, are involved to varying degrees in non-facilitated and facilitated release under different experimental conditions.

M1 muscarinic receptor-induced facilitation of ACh and noradrenaline release in the rat bladder is mediated by protein kinase C.

1. [3H]Noradrenaline (NA) AND [14C]acetylcholine (ACh) released by electrical field stimulation were measured simultaneously in strips from the body of rat urinary bladder. 2. [3H]NA and [14C]ACh release was greater during continuous stimulation (CS; 10 Hz, 100 shocks) or in the presence of eserine than during intermittent train stimulation (IS; 10 Hz, 10 shocks every 5 s, 10 times). Atropine (1 microM) or pirenzepine (0.05-0.1 microM) blocked the CS- or eserine-facilitated release. 3. The protein kinase C (PKC) activator phorbol dibutyrate (PDB; 0.05 and 0.5 microM) increased the release of both [3H]NA and [14C]ACh in a concentration-dependent manner. Atropine blocked the PDB-induced facilitation of ACh release but not the facilitation of NA release. 4. The protein kinase A (PKA) activator 8-Br-cAMP did not affect ACh release but enhanced NA release. 5. The PKC inhibitor H-7 (50-100 microM) inhibited the CS- or eserine-facilitated release of both ACh and NA, but did not affect the non-facilitated release evoked by IS. H-7 also inhibited 0.5 microM PDB-induced facilitation of ACh release but not NA release. 6. Down-regulating PKC by pretreatment for 30 min with 5 microM PDB decreased the facilitated release of ACh and the eserine-induced facilitation of NA release. 7. Electrically evoked contractions of the bladder strips exhibited a biphasic response to PDB (2.5 microM), which consisted of an initial enhancement of the peak amplitude and area followed after 20 min by an inhibition of contractions. H-7 inhibited the electrically evoked contractions in a dose-dependent fashion. 8. It is concluded that a phospholipase C-PKC signal transduction pathway is essential for muscarinic receptor-induced facilitation of ACh and NA release but is not involved in the non-facilitated release of transmitters in the rat urinary bladder.

Prejunctional facilitatory alpha 1-adrenoceptors in the rat urinary bladder.

1. The effect of activation of alpha 1-adrenoceptors on acetylcholine (ACh) release and neurally evoked contractile responses induced by electrical field stimulation was investigated in smooth muscle strips from the rat urinary bladder. 2. Neurogenic contractions were facilitated by the alpha 1-adrenoceptor agonists, phenylephrine (PE) (2-128 microM) and methoxamine (2-128 microM) in a dose-dependent manner. These agents also increased small amplitude spontaneous contractions of bladder strips and in 10% of strips increased basal tone. However, contractions elicited by exogenous ACh (1-10 microM) were not affected by alpha 1-agonists. 3. The magnitude of the PE facilitation was higher at lower frequencies (1-5 Hz) or at submaximal intensities of stimulation and at lower Ca2+ concentrations (0.5-1 mM). The selective alpha 1-adrenoceptor antagonist, terazosin (TRZ) (0.05-1 microM), competitively inhibited (pA2 value: 8.6) the PE facilitation of the neurally evoked contractions but not the PE induced increase of spontaneous contractions. 4. [3H]-noradrenaline (NA) and [14C]-ACh release evoked by electrical field stimulation were increased (140% and 173%, respectively) by 2 microM PE. TRZ (0.05-0.1 microM) blocked the PE facilitation of ACh release but not the facilitation of NA release. TRZ alone did not alter the release of ACh or NA nor the amplitude of the neurogenic contractions. 5. PE (2 microM) did not alter the basal release of ACh but did increase (by 180%) the basal release of NA. Desipramine (2 microM) blocked this effect of PE and also the PE-facilitation of evoked ACh and NA release. 6. It is concluded that cholinergic terminals in the rat urinary bladder exhibit alpha 1-adrenoceptors which can facilitate the release of transmitter. However, under the conditions of our experiments it appears that cholinergic transmission is not modulated by alpha 1 adrenergic mechanisms. Further studies are necessary to determine whether these receptors can be activated by endogenous noradrenaline released within the bladder.

M1 muscarinic receptor-mediated facilitation of acetylcholine release in the rat urinary bladder.

1. Release of [3H]ACh in response to electrical field stimulation (10 Hz) was measured in strips of rat urinary bladder and cardiac atrial tissues previously incubated with [3H]choline. 2. The volley output of [3H]ACh release was positively correlated with frequency of stimulation in the urinary bladder but negatively correlated in the atrium. 3. The quantity of [3H]ACh release was influenced by the pattern and duration of stimulation. Continuous stimulation (CS) with trains of 100 shocks released 10 times larger amounts of ACh than the same number of shocks presented as short trains of intermittent stimulation (IS): ten shocks per train with 5 s inter-train intervals. 4. The facilitation of transmitter release was antagonized completely by the administration of atropine (1 microM) or pirenzepine (0.05 microM), a selective M1 antagonist. Eserine, an anticholinesterase agent, markedly facilitated ACh release induced by CS and IS. This effect was blocked by atropine. 5. Release of ACh from atrial strips did not exhibit CS-induced facilitation. Eserine decreased IS- and CS-evoked ACh release in the atrium. 6. It is concluded that continuous stimulation of postganglionic cholinergic nerves in the rat urinary bladder leads to the activation of M1 muscarinic, facilitatory presynaptic receptors which enhance the release of ACh. Presynaptic facilitation may be an important mechanism for modulating neural input to the bladder during micturition.

Inhibitory effect of neuropeptide Y on adrenergic and cholinergic transmission in rat urinary bladder and urethra.

The effects of porcine neuropeptide Y (NPY) on electrically evoked release of [3H]norepinephrine ([3H]NE) and [3H]acetylcholine ([3H]ACh) were investigated in isolated preparations of the rat lower urinary tract. In the urethra, NPY (0.02-0.5 microM) decreased the release of [3H]NE in a dose-dependent manner (10-53%). In the bladder base the inhibitory effect of NPY on [3H]NE release was not dose dependent. A low concentration (0.1 microM) decreased the release (38%), whereas a high concentration (0.5 microM) had no effect. However, in atropine-treated preparations, 0.5 microM NPY elicited a significant inhibition (43%). These observations suggest that 0.5 microM NPY elicits two opposing actions: a direct inhibitory action on adrenergic terminals and an indirect disinhibitory action to eliminate heterosynaptic cholinergic inhibition of [3H]NE release. In both tissues the action of NPY on [3H]NE release was not significantly modified by the alpha-adrenergic blocking agent yohimbine (1 microM). [3H]ACh release in the bladder body was not altered by 0.1 microM NPY but was suppressed (39%) by 1 microM NPY. The effect of NPY (1 microM) on [3H]ACh release was dependent on the frequency of stimulation. NPY suppressed the release at 2-Hz stimulation but had no significant effect at 20 Hz. These results suggest that NPY may have an important role in the neural regulation of the lower urinary tract by exerting differential effects on the release of cholinergic and adrenergic transmitters via autoinhibition and heterosynaptic interactions.

A comparison of inhibitory effects of neuropeptide Y on rat urinary bladder, urethra, and vas deferens.

The effects of human and porcine neuropeptide Y (NPY) on electrically induced contractions of smooth muscle strips from rat urinary bladder, urethra, and vas deferens were investigated. NPY (10 nM-10 microM) inhibited to the contractile response in all preparations. The magnitude of inhibition by NPY was dependent on frequency of stimulation in each organ, the inhibition being in general much greater (80-100%) at low frequencies (2-5 Hz) than at high frequencies (30-40% at 10-100 Hz). The vas deferens and urethra exhibited nearly maximal inhibition (90-100%) over a broader range of stimulus frequencies (1-20 Hz), while the bladder exhibited a more prominent inhibition at frequencies of stimulation below 2 Hz. When tested at 20 Hz stimulation the urethra and vas deferens were very sensitive (70-90% inhibition) to both types of NPY, whereas bladder strips were much less sensitive to NPY and the effect differed with the two types of NPY (16% inhibition with human NPY and 39% inhibition with porcine NPY). In the urinary bladder, NPY inhibited the cholinergic component of the contractile response, while in the urethra adrenergic transmission was primarily affected. These studies suggest that NPY, which is present in both cholinergic and adrenergic neurons in the pelvic ganglia, may have an important role in the neural control of the lower urinary tract by participating in autoinhibition at autonomic nerve terminals as well as in the heterosynaptic interactions between the cholinergic and adrenergic pathways.

Modulation of release of [3H]acetylcholine in the major pelvic ganglion of the rat.

Cholinergic modulation of [3H]acetylcholine release evoked by electrical stimulation was studied in the rat major pelvic ganglion, which was prelabeled with [3H]choline. Acetylcholine (ACh) release was independent of the frequency of stimulation; 0.3 Hz produced the same volley output as 10 Hz. Tetrodotoxin (1 microM) or omission of Ca2+ from the medium abolished ACh release. The M1 receptor agonist (4-hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbanilate chloride (McN-A 343, 50 microM) increased release (by 136%), whereas the M2 muscarinic agonist oxotremorine (1 microM) decreased ACh release (by 22%). The muscarinic antagonists, atropine (1 microM) or pirenzepine (M1 selective, 1 microM), did not change ACh release. However, pirenzepine (1 microM) blocked the facilitatory effect of McN-A 343, and atropine (1 microM) blocked the inhibitory effect of oxotremorine. The cholinesterase inhibitor physostigmine (1-5 microM), the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP, 10 microM), and the nicotinic antagonist D-tubocurarine (50 microM) did not change ACh release. 4-Aminopyridine, a K+ channel blocker, significantly increased the release (by 146%). Seven days after decentralization of the major pelvic ganglion, the evoked release of ACh was abolished. It is concluded that release of ACh occurs from the preganglionic nerve terminals rather than from the cholinergic cell bodies and is not modulated by actions of endogenous ACh on either muscarinic or nicotinic autoreceptors. These data confirm and extend previous electrophysiological findings indicating that synapses in the major pelvic ganglion have primarily a relay function.