Hyperpolarization-activated cyclic nucleotide-gated cation channels (HCN channels) carry the voltage-gated inward rectifying (Ih) currents activated during hyperpolarization.  HCN channels typically open at potentials more negative to −50 mV for regulating regulate membrane resistance and intrinsic membrane excitability. Although the expression of HCN channels in human bladder is reported by several groups, their functional role is unclear. Here we investigated the effect of HCN blocker, ZD7288 on the nerve evoked contractions of human detrusor. ZD7288 is characterized by its high affinity with a reported IC50 of 200nM for HCN channels expressed in heart.  HCN channels are gated by cyclic adenosine monophosphate (cAMP), which is the intracellular second messenger for the two major classes of drugs approved for treating overactive bladder (OAB), namely beta 3 receptor agonists and muscarinic receptor antagonists. It is likely that drug treatment for OAB also affects the activity of HCN channels expressed in bladder to ultimately influence the detrusor contractility.  HCN channels exist in 4 isoforms which differ in activation kinetics and sensitivity to cAMP.

Bladder from 3 deceased organ donors was obtained after ethical approval from the institutional committee. Urothelium intact and urothelium denuded detrusor strips were mounted in 37°C organ bath constantly gassed with 95% oxygen-5% carbon dioxide. Strips were stretched to 1g and equilibrated for 1h before isometric tension studies. Nerve-evoked contractions (tetrodotoxin-sensitive) were generated by electrical field stimulation (EFS: 5 ms pulses, 0.1-32Hz, 2s train at 20V) before and after addition of ZD7288 in nanomolar and micromolar range  (10nM or 100 µM) or Neostigmine (1µM) separately and together. EFS frequency response curve were generated by stimulating at 0.1, 0.5, 1, 2, 4, 8, 16, and 32Hz (one stimulation at each frequency) at 15s intervals. Peak contractile responses after the addition of different drugs were normalized to the peak contractile response evoked by 32Hz stimulation in absence of any drug (control). A portion of bladder tissue was also preserved for double immunostaining of HCN channel isoforms,  HCN1 and HCN4 with neuronal markers, Calcitonin gene related peptide (CGRP) and choline acetyl transferase (ChAT).

Addition of ZD7288 in micromolar range (100µM) to urothelium intact or denuded strips significantly inhibited the contractions evoked at frequencies ≥8Hz (p<0.05; Two-way ANOVA). After the addition of ZD7288 in nanomolar range (10nM), the peak contractile response of urothelium intact strips at frequencies of 4-16 Hz was significantly higher than urothelium denuded strips.  In addition, prior incubation of strips with ZD7288 (10nM) also significantly enhanced the contractile response evoked by EFS ≥8Hz in presence of Neostigmine(1µM) (n=3; p<0.05; Fig.1), suggesting that the blockade of HCN channels at pre-junctional sites in cholinergic nerves augments the detrusor contractility.  Indeed, double immunostaining of the separated mucosa (urothelium and sub-urothelium) and detrusor sections of human bladder revealed a co-localization of the HCN channel isoform HCN4 with ChAT in suburothelium and detrusor and the co-localization of HCN1 isoform with CGRP  in urothelium, sub-urothelium and detrusor.

Enhancement of the response elicited by Neostigmine in presence of HCN blockade by  ZD7288 (10nM) suggests that HCN channels expressed on cholinergic nerves constrain the detrusor contractility by modulating the evoked release of acetylcholine released at EFS frequency ≥8Hz. Unlike the pacemaking role of HCN channels in heart and upper urinary tract, these findings suggest that HCN channels expressed in different cells of human bladder are likely to perform a non-pacemaking role.  We postulate that inward rectifying currents carried by HCN channels will set the resting membrane potential more negative than -70mV to reduce the steady Ca2+ influx through increased inactivation of the low voltage gated N-type and T-type and Ca2+ channels.  Consequently, blockade of HCN channels at prejunctional sites in bladder will increase the neuroexocytosis of neurotransmitters and the blockade of HCN channels at post-junctional sites will increase the temporal integration of excitatory and inhibitory post-junctional potential evoked by EFS. Importantly, detrusor myocytes obtained from OAB patients are reported to have higher T-type Ca2+ current density, whose function can be modulated by HCN channel activators such as beta 3 agonists. These ex vivo functional studies with HCN blocker are consistent  with the expression of HCN 1 and 4 channel isoforms in cholinergic and peptidergic nerves innervating urothelium, lamina propria, interstitial cells and smooth muscle.  Co-localization of faster activating HCN1 isoform with CGRP and of the slower activating HCN4 isoform with ChAT is presumably linked to the differences in the functional role of sensory and the efferent nerves. In addition, strong staining of HCN4 isoform with ChAT in suburothelium and detrusor may explain the enhanced contractile response of urothelium intact strips with HCN blocker.  Compared to the findings with ZD7288 (100µM), our findings obtained with ZD7288 (10nM) are selective for HCN channels, when considered in light of the reported blockade of Na+ channels in cell bodies of afferent neurons by ZD7288 with IC50 of 1.17µM (3) and the nanomolar affinity of ZD7288 for HCN in heart

Collectively, this evidence suggests that HCN channels expressed in bladder serve a non-pacemaking role of constraining the human detrusor contractility through the modulation of spontaneous activity and the evoked release of neurotransmitters. Findings suggest that agents capable of selectively opening and closing HCN channels in bladder will be therapeutic candidates for OAB and UAB, respectively.

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