Gap junction channel activity in short-term cultured human detrusor myocyte cell pairs: gating and unitary conductances

Abstract

. Gap junction channel activity in short-term cultured human detrusor myocyte cell pairs: gating and unitary conductances. Am J Physiol Cell Physiol 291: C1366 -C1376, 2006. First published August 9, 2006 doi:10.1152/ajpcell.00027.2006.-Several independent lines of investigation indicate that intercellular communication through gap junctions modulates bladder physiology and, moreover, that altered junctional communication may contribute to detrusor overactivity. However, as far as we are aware, there are still no direct recordings of gap junction-mediated intercellular currents between human or rat detrusor myocytes. Northern and Western blots were used to identify connexin expression in frozen human bladder tissue and short-term cultured human detrusor myocytes. Double whole cell patch (DWCP) recording revealed that human detrusor myocyte cell pairs were well coupled with an average junctional conductance of 6.5 Ϯ 4.6 nS (ranging from 0.1 to 15 nS, n ϭ 22 cell pairs). Macroscopic gap junction channel currents in human detrusor myocytes exhibited voltage dependence similar to homotypic connexin43. The normalized transjunctional conductance-voltage (G j-Vj) relationship was symmetrical and well described by a two-state Boltzmann relation (G min Ϸ 0.33, V 0 ϭ 63.6 mV, Z ϭ 0.117 or equal to 2.95 gating charges), suggestive of a bilateral voltage-gated mechanism. In symmetric 165 mM CsCl, the measured single-channel slope conductance was ϳ120 pS for the fully open channel and ϳ26 pS for the major substate. Occasionally, other subconductance states were also observed. The single-channel mean open time declined with increasing V j, accounting for the V j-dependent decline of macroscopic junctional current. Qualitatively similar electrophysiological characteristics were observed in DWCP of freshly isolated rat detrusor myocytes. These data confirm and extend previous observations and are consistent with reports in other smooth muscle cells types in which Cx43-mediated intercellular communication has been identified. bladder function; intercellular communication; smooth muscle

Connexin 43 Hemichannels Are Permeable to ATP

“…Analysis of channel kinetics showed that the 26 pS state had much longer open times (31 Ϯ 14 ms, n ϭ 27 patches) and higher open probability (P o , 0.7 Ϯ 0.03) than the fast-gating state in cell-attached patches (P o , 0.006 Ϯ 0.003; p Ͻ 0.01, Student's t test). The substate opening in inside-out patches had a conductance (26 pS) similar to that of the substate of cell-cell channels (26 pS) (Wang et al, 2006). Twenty-six picosiemen openings reversed at the membrane potential near 0 mV (Fig.…”

Detrusor myocyte activity and afferent signaling

“…Cx45 37 and Cx43 40,41 appear to be the most prominent Cxs expressed in human detrusor smooth muscle tissue and cultured cells. 39 Observations in tissue biopsies from patients with neurogenic DO 40 and urgency symptoms 41 clearly demonstrated an increase in the presence of Cx43-derived gap junction channels in detrusor muscle. These clinical data are consistent with experimental observations in a rodent model of partial urethral outlet obstruction, 42 which documented a dramatic increase of Cx43 mRNA levels associated with bladder hypertrophy and overactivity following 6 weeks of obstruction.…”

“…35 -38 The small size of the junctional plaques observed likely accounts for the difficulty in detecting these structures in the first place. Wang et al 39 used double whole-cell patch clamp recordings together with Northern and Western blot techniques to characterize gap junction channel properties, and to evaluate Cx43 expression in human detrusor cells. The presence of Cx43 mRNA and protein in cultured detrusor myocytes, and also in situ, was demonstrated.…”

“…The presence of Cx43 mRNA and protein in cultured detrusor myocytes, and also in situ, was demonstrated. Wang et al 39 found human detrusor cells to be well coupled and concluded that gap junctions could have an important role in the initiation, maintenance, and modulation of detrusor tone.…”

Changes of gap junctional cell-cell communication in overactive detrusor in rats

“…One of the main causes is partial bladder outflow obstruction (PBOO), in which overactivity could result neurogenically and/or myogenically from changes in the detrusor innervation and myocyte excitability. Recently, changes in cell-cell communication have been proposed to be one of the possible mechanisms (3,8), and Cx43 plays an important role in the development of DO (6,25). However, the functional changes of cell connectivity in DO are not well elucidated, and there is considerable controversy between reports on changes in cell-cell contact in DO after PBOO.…”