Muscarinic Receptors

Muscarinic receptors comprise five subtypes, encoded by five distinct genes.⁷ The five gene products correspond to pharmacologically defined receptors, and M₁ through M₅ are used to describe the molecular and pharmacologic subtypes. In the human bladder, the mRNAs for all muscarinic receptor subtypes have been demonstrated,⁸ with a predominance of mRNAs encoding M₂ and M₃ receptors.^8,9 These receptors are functionally coupled to G proteins, but the signal transduction systems vary.^10–12

Detrusor smooth muscle contains muscarinic receptors of mainly the M₂ and M₃ subtypes, with a predominance (70% to 80%) of M₂ receptors.^10–13 The M₃ receptors are the most important for detrusor contraction. It is generally believed that M₃ receptors are coupled to release of inositol 1,4,5-triphosphate (IP₃) and calcium release from the sarcoplasmic reticulum and that M₂ receptors are linked to inhibition of adenylyl cyclase. This may not be the case in the detrusor.¹⁴ Jezior and colleagues¹⁵ suggested that muscarinic receptor activation of detrusor muscle includes nonselective cation channels and activation of Rho kinase. Supporting a role of Rho kinase in the regulation of rat detrusor contraction and tone, Wibberley and coworkers¹⁶ found that Rho kinase inhibitors (e.g., Y-27632, HA 1077) inhibited contractions evoked by carbachol without affecting the contraction response to KCl. They also demonstrated high levels of Rho kinase isoforms (I and II) in the bladder. Schneider and associates¹³ concluded that carbachol-induced contraction of human urinary bladder is mediated by M₃ receptors and largely depends on Ca²⁺ entry through nifedipine-sensitive channels and activation of the Rho kinase pathway. The main pathway for muscarinic receptor activation of the detrusor by means of M₃ receptors may be calcium influx through L-type calcium channels and increased sensitivity to calcium of the contractile machinery produced by inhibition of myosin light chain phosphatase through activation of Rho kinase (Fig. 4-1).¹⁴

Figure 4-1 Transmitter signal pathways involved in activation of detrusor contraction by means of muscarinic M₃ receptors. ACh, acetylcholine; CIC, calcium-induced calcium release; DAG, diacylglycerol; IP₃, inositol 1,4,5-triphosphate; MLC, myosin light chain; PLC, phospholipase C; PKC, protein kinase C; RhoA, protein encoded by RAS homolog gene family member A; SR, sarcoplasmic reticulum. There seem to be differences between species in the contribution of the different pathways in contractile activation. In the human detrusor, Ca²⁺ influx is of major importance.

The functional role for the M₂ receptors has not been clarified, but it has been suggested that M₂ receptors may oppose sympathetically mediated smooth muscle relaxation, mediated by β- adrenergic receptors.¹⁷ M₂-receptor stimulation may also activate nonspecific cation channels¹⁸ and inhibit K_ATP channels through activation of protein kinase C.^19,20 In certain disease states, M₂ receptors may contribute to contraction of the bladder. In the denervated rat bladder, M₂ receptors or a combination of M₂ and M₃ mediated contractile responses, and the two receptor types seemed to act in a facilitatory manner to mediate contraction.^21–23 In obstructed, hypertrophied rat bladders, there was an increase in total and M₂ receptor density, whereas there was a reduction in M₃ receptor density.²⁴ The functional significance of this change for voiding function has not been established. Pontari and colleagues²⁵ analyzed bladder muscle specimens from patients with neurogenic bladder dysfunction to determine whether the muscarinic receptor subtype mediating contraction shifts from M₃ to the M₂ receptor subtype, as found in the denervated, hypertrophied rat bladder. They concluded that whereas normal detrusor contractions are mediated by the M₃ receptor subtype, in patients with neurogenic bladder dysfunction, contractions also can be mediated by the M₂ receptors.

Muscarinic receptors may also be located on the presynaptic nerve terminals and participate in the regulation of transmitter release. The inhibitory prejunctional muscarinic receptors have been classified as M₄ in the human bladder.²⁶ Prejunctional facilitatory muscarinic receptors appear to be the M₁ subtype.²⁷ The muscarinic facilitatory mechanism seems to be upregulated in overactive bladders from chronic spinal cord transected rats. Facilitation in these preparations is primarily mediated by M₃ receptors.^27,28

Muscarinic receptors have been demonstrated on the urothelium or suburothelium, but their functional importance has not been clarified.^12,29 It has been suggested that they may be involved in the release of an unknown inhibitory factor.¹²

It is well documented^2,3 that antimuscarinic agents are effective for treatment of overactive bladder, which suggests that muscarinic receptors may be involved in its pathogenesis. Contraction of the bladder, whether voluntary or involuntary, involves stimulation of the muscarinic receptors on the detrusor by acetylcholine released from activated cholinergic nerves. However, antimuscarinic agents at clinically recommended doses have little effect on voiding contractions and may act mainly during the bladder storage phase,³⁰ during which there is normally no parasympathetic outflow from the spinal cord.³¹ Supporting this, antimuscarinic agents have been shown to reduce bladder tone during storage and to increase cystometric bladder capacity. A basal release of acetylcholine from non-neuronal (urothelial) and neuronal sources has been demonstrated in isolated human detrusor muscle.³² It has been suggested that this release, which is increased by stretching the muscle and in the aging bladder, contributes to detrusor overactivity and overactive bladder by eventually increasing bladder afferent activity during storage.³³ This may occur because of a direct effect on suburothelial afferents or stimulation of contraction of detrusor muscle cells, which already have an increased myogenic activity in the overactive detrusor.³⁴ Enhanced myogenic contractions can generate an enhanced afferent signal, contributing to urge or initiation of the micturition reflex.

Alpha-Adrenoceptors

α-Adrenoceptors may have effects on different locations in the bladder: the detrusor smooth muscle, the detrusor vasculature, the afferent and efferent nerve terminal, and intramural ganglia. The importance of the α₁-adrenoceptors in the human detrusor in the generation of lower urinary tract symptoms has not been established. Most investigators agree on that there is a low-level expression of these receptors.^35,36 In studies of the human detrusor, Malloy and coworkers³⁶ found that two thirds of the αadrenoceptor mRNAs expressed were α_1D, there was no α_1B, and one third was α_1A. In the rat detrusor, the α₁-adrenoceptor distribution was different: the α_1A-adrenoceptor was the predominant form, one third was the α_1D-adrenoceptor, and there was very little of the α_1B-adrenoceptor form. This was consistent in the different parts of the detrusor.³⁷

A change of subtype distribution may be produced by outflow obstruction. Hampel and associates³⁷ reported that there was a change in the obstructed bladder from α_1A-adrenoceptor to α_1D-adrenoceptor mRNA predominance. In humans, there is an α_1D-adrenoceptor predominance in the normal detrusor, which means that a change in a similar direction, as in the rat, would be of minor importance provided that the number of receptors did not increase. Studies by Nomiya and colleagues³⁸ confirmed the low-level expression of α-adrenoceptor mRNA in normal human detrusor, and they further demonstrated that there was no upregulation of any of the adrenergic receptors with obstruction. In functional experiments, they found a small response to phenylephrine at high drug concentrations with no difference between normal and obstructed bladders. In the obstructed human bladder, there seems to be no evidence for α-adrenoceptor upregulation or change in subtype. This finding was challenged by Bouchelouche and coworkers,³⁹ who found an increased response to α₁-adrenoceptor stimulation in obstructed bladders. Whether this would mean that the α_1D-adrenoceptors in the detrusor muscle are responsible for detrusor overactivity or overactive bladder is unclear. Based on available evidence, however, it does not seem likely that in the detrusor muscle these receptors should be an important treatment target, although α_1D-adrenoceptors located elsewhere in the bladder may be important.

In the bladder, the function of the detrusor muscle depends on the vasculature and on perfusion. Hypoxia induced by partial outlet obstruction is believed to play a major role in the hypertrophic and degenerative effects of partial outlet obstruction. Das and associates⁴⁰ investigated in rats whether doxazosin affected blood flow to the bladder and reduced the level of bladder dysfunction induced by partial outlet obstruction. They found that 4 weeks of treatment with doxazosin increased bladder blood flow in control and obstructed rats. Doxazosin treatment also reduced the severity of the detrusor response to partial outlet obstruction. Doxazosin may reduce the increase in bladder weight in obstructed animals, which may be one of the mechanisms that contributes to a positive effect on detrusor overactivity caused by the obstruction.

Beta-Adrenoceptors

In the human detrusor, the most important β-adrenoceptor for bladder relaxation is the β₃-adrenoceptor.⁴¹ This partly explains why the clinical effects of selective β₂-adrenoceptor agonists in detrusor overactivity have been controversial and largely inconclusive.^2,3 However, the β₂-adrenoceptor agonist clenbuterol inhibited electrically evoked contractions in human “unstable” but not normal bladder,⁴² which is in agreement with previous experiences in patients, suggesting that clenbuterol and other β₂-adrenoceptor agonists such as terbutaline may inhibit detrusor overactivity.^43,44

The β₃-adrenoceptor seems to be an interesting target for drugs aimed for treatment of overactive bladder. Selective β₃-adrenoceptor agonists have relaxant effects on detrusor muscle in vitro and have been effective in animal models of detrusor overactivity.^45–47 However, no proof of concept studies seems to have been performed in humans showing that this is an effective principle to treat overactive bladder.

Purinergic Receptors

In most animal species, bladder contraction induced by stimulation of nerves consists of an atropine-sensitive component and a component mediated by NANC mechanisms.⁴⁸ NANC-mediated contractions have been reported to occur in normal human detrusor,⁴⁸ even if not representing more than a few percent of the total contraction in response to nerve stimulation. However, a significant degree of NANC-mediated contraction may exist in morphologically or functionally changed human bladders, and it has been reported to occur in several disorders associated with lower urinary tract symptoms, such as bladder hypertrophy,^49–51 idiopathic detrusor overactivity,^51,52 interstitial cystitis,⁵³ and neurogenic damage⁵⁴ and in the aging bladder.⁵⁵ In these disorders, the NANC component of the nerve-induced response may be responsible for up to 40% to 50% of the total bladder contraction.

There is good evidence that the transmitter responsible for the NANC component is adenosine triphosphate (ATP)⁵⁶ acting on P2X receptors found in the detrusor smooth muscle membranes of rats⁵⁷ and humans.⁵⁸ The P2X₁receptor subtype predominated in both species. Changes in P2X receptor subtypes in bladders from patients with idiopathic detrusor overactivity have been reported.^52,59 O’Reilly and colleagues⁵² were unable to detect a purinergic component of nerve-mediated contractions in control (normal) bladder preparations, but they found a significant component in overactive bladder specimens, in which the purinergic component was approximately 50%. They concluded that this abnormal purinergic transmission in the bladder might explain symptoms in these patients. ATP was a more potent contractile agonist in bladder preparations from patients with overactive and obstructive bladders than in specimens from normal bladders,⁶⁰ a finding that was suggested to contribute to detrusor overactivity.

O’Reilly and coworkers⁶¹ confirmed that the P2X₁ receptor was the predominant purinoceptor subtype in the human male bladder. They also found that the amount of P2X₁ receptor per smooth muscle cell was greater in the obstructed than in control bladders. This suggests an increase in purinergic function in the overactive bladder arising from bladder outlet obstruction.

It has not been established whether abnormalities in the purinergic transmission in the bladder can explain overactive bladder symptoms in idiopathic detrusor overactivity in women and in men with bladder outlet obstruction. If this is the case, abnormal purinergic activation of the detrusor may explain why antimuscarinic treatment fails in a number of patients.

Vanilloid Receptors

Appropriate bladder function depends on an intact afferent signaling from the bladder to the central nervous system. This signaling conveys information about bladder filling and the status of the tissue (e.g., presence of infectious agents). The afferent nerves consist of small, slowly conducting, myelinated Aδ fibers and slowly conducting, unmyelinated C fibers. The former are excited by mechanoreceptors and convey information about bladder filling, whereas C fibers mediate painful sensations recognized by chemoreceptors. By means of capsaicin, a subpopulation of primary afferent neurons innervating the bladder and urethra (i.e., capsaicin-sensitive nerves) has been identified. It is believed that capsaicin exerts its effects by acting on specific vanilloid receptors on these nerves.⁶² Capsaicin exerts a biphasic effect; initial excitation is followed by a long-lasting blockade, which renders sensitive primary afferents (C fibers) resistant to activation by natural stimuli. In sufficiently high concentrations, capsaicin is believed to cause desensitization, initially by releasing and emptying the stores of neuropeptides and then by blocking further release.⁶³ Resiniferatoxin, an analogue of capsaicin, is approximately 1000 times more potent for desensitization than capsaicin,⁶⁴ but it is only a few hundred times more potent for excitation.⁶⁵ Capsaicin and resiniferatoxin also may have effects on Aδ fibers. It is also possible that capsaicin at high concentrations (mM) has additional, nonspecific effects.⁶⁶

Capsaicin and resiniferatoxin have been used successfully to treat bladder function disturbances.^2,3 They are known to bind to the TRPV1 (VR1) receptor, a nonselective cation channel, on the peripheral terminals of nociceptive neurons, but the role of vanilloid receptors in normal bladder function and in the pathogenesis in detrusor overactivity and autonomic dysreflexia has not been established.

Birder and associates⁶⁷ investigated bladder function in conscious mice lacking the TRPV1 receptor. TRPV1 receptor–knockout mice seem to have increased voiding frequency compared with wild-type mice. The TRPV1 knockouts also had an increased frequency of nonvoiding bladder contractions. In vitro, stretch-induced ATP release was decreased in bladders from TRPV1-knockout mice, and hypo-osmolality-induced ATP released from cultured urothelial cells was reduced.⁶⁷ The investigators suggested that TRPV1 receptors participate in normal bladder function, are essential for normal mechanically evoked purinergic signaling by the urothelium, and are involved in ATP release. Experimental and clinical evidence that capsaicin-sensitive afferents may be involved also in idiopathic detrusor overactivity has been presented.^68,69

Calcium Channels

Calcium is a key component for cell function in many cells. In smooth muscle, increased intracellular calcium concentrations activate the contractile mechanisms, and in nerve terminals, calcium influx in response to action potentials is an important mechanism for neurotransmitter release. Calcium channels can be divided into at least four different subtypes: L, N, P, and Q channels. The calcium channels present in smooth muscles are L-type (dihydropyridine-sensitive) calcium channels and seem to be involved in contraction of the human bladder irrespective of the mode of activation.⁷⁰ A decrease of the membrane potential (i.e., depolarization) increases the open probability for calcium channels, thereby increasing the calcium influx. The channels depend on the membrane potential and are called voltageoperated calcium channels. Elevated intracellular calcium levels are also believed to initiate release of calcium from intracellular stores—calcium-induced calcium release.^71,72 Regulation of the intracellular calcium concentration in smooth muscle cells is a conceivable way to modulate bladder contraction. Dihydropyridines (e.g., nifedipine) have a potent inhibitory effect on isolated detrusor muscle. Inhibitory effects have also been demonstrated on experimentally induced contractions under in vivo conditions in rats and clinically in patients with detrusor overactivity.⁴⁸ However, therapeutically, there is no evidence that calcium antagonists have any useful effects in the treatment of overactive bladder or detrusor overactivity.^2,3

Potassium Channels

Potassium channels represent another mechanism for modulating the excitability of smooth muscle cells. Under normal conditions, the resting membrane potential in smooth muscle cells is determined predominantly by the membrane conductivity for potassium ions. Increased potassium conductivity lowers the membrane potential by increasing the potassium efflux. This increases the threshold for opening of voltage-operated calcium channels and initiation of contraction. There are several different types of K⁺ channels, and at least two subtypes have been found in the human detrusor, ATP-sensitive K⁺ channels (K_ATP) and large-conductance calcium-activated K⁺ channels (BK_Ca). Studies on isolated human detrusor muscle and on bladder tissue from several animal species have demonstrated that K⁺-channel openers reduce spontaneous contractions and contractions induced by carbachol and electrical stimulation.⁶ However, the lack of selectivity of the available K⁺-channel openers for the bladder versus the vasculature has limited the use of these drugs. No effects of cromakalim or pinacidil on the bladder were found in studies on patients with spinal cord lesions or detrusor overactivity secondary to outflow obstruction.^73,74 Some new K⁺-channel openers have been developed and claimed to have selectivity for the bladder.⁶ However, there is no evidence that K⁺-channels openers are an option for treatment of overactive bladder or detrusor overactivity.^2,3