Function and regulation of elemental Ca2+ signaling in urethral smooth muscle

Summary

Principal Investigator: Ronghua Zhuge
Abstract: DESCRIPTION (provided by applicant): Ca2+ signals and ion channels are essential in controlling the contraction of smooth muscle in several tissue types and organs. However, their functions and underlying mechanisms in smooth muscle from urethra, an organ critical for maintaining urinary continence, are poorly understood. The present proposal seeks to understand highly localized and short-lived Ca2+ transients ("Ca2+ sparks") that result from the spontaneous opening of type 2 ryanodine receptors (RyR2) in the sarcoplasmic reticulum, and their ion channel targets in the plasma membrane in urethral smooth muscle (USM). Our preliminary studies revealed that (1) Ca2+ sparks in USM only activate ANO1, a Ca2+-activated Cl- (ClCa) channel, to produce spontaneous transient inward currents (STICs) and depolarize the membrane sufficiently to turn on L-type voltage-dependent Ca2+ channels;(2) USM from knock-in mice of RyR2 R176Q mutation (which causes catecholaminergic polymorphic ventricular tachycardia in human) generates less force when exposed to caffeine, a RyR agonist, and phenylephrine (PE), an alpha1-adrenergic receptor agonist, than in normal RyR2 mice;(3) genetic deletion of ANO1 causes Ca2+ sparks unable to activate STICs, and decreases the urethral contraction upon stimulation by caffeine in neonatal mice;and (4) PE increases STICs, and nitric oxide (NO), a gas relaxant, inhibits STICs. Thus, we propose that RyR2 and ANO1 in USM are essential for governing urethral myogenic and neurogenic tone, and their malfunction may result in urethral dysfunction and urinary incontinence. To test this central hypothesis we will employ an integrated approach using high-speed Ca2+ imaging with simultaneous patch-clamping, 2D and 3D protein localization, single cell shortening and tissue contraction bioassays, in vivo urodynamics tests, and transgenic (knock-in and conditional knockout) mice. Specifically, we will establish that in USM Ca2+ sparks act as a contractile mechanism, rather than a relaxing mechanism as in bladder and vascular smooth muscle, by controlling global [Ca2+]I, membrane potential, and urethra tone using RyR2 R176Q mutant mice and normal mice (Aim 1). Systemic ANO1-/- mice die very young, so it has been difficult to study the role of ANO1 in urethra in mature mice and in vivo. We have obtained a line of smooth muscle specific ANO1-/- mice which live to maturity. With this knockout line, we will establish that in mature mice ANO1 is critical for the maintenance of urethral contraction and pressure and its deletion likely leads to urinary incontinence (Aim 2). We will further uncover the mechanisms underlying activation of ANO1s by RyR2s with 3D imaging, channel biophysics and reaction-diffusion modeling (Aim 2). Finally, building upon our preliminary results on the effects of PE and NO, we will establish that PE and NO differentially modulate RyR2 and ANO1, resulting in the contraction and relaxation of urethra, respectively (Aim 3). We expect that these studies will not only significantly advance our understanding of the roles of RyR2 and ANO1 in urethral physiology, but also identify novel molecular targets for developing effective and specific treatments for urinary incontinence.
Funding Period: 2013-08-15 - 2014-06-30
more information: NIH RePORT

Research Grants

Detail Information

Research Grants31

  1. TRP Channel-Dependent Regulation of Arterial Tone
    Scott Earley; Fiscal Year: 2013
    ..abstract_text> ..
  2. Strategies for Improved Shock Wave Lithotripsy
    JAMES ALEXANDER MCATEER; Fiscal Year: 2013
    ..and the session can be ended * Determine the mechanism by which cavitation within a vessel causes hemorrhage * Develop numerical models to understand the role of cavitation and non-cavitational mechanisms in causing tissue damage ..
  3. KCNQ Channels and Vasoconstrictor Signal Transduction
    Kenneth L Byron; Fiscal Year: 2013
    ....
  4. Mechanisms/Treatments of Lower Urinary Tract Dysfunction After Spinal Cord Injury
    ANTHONY JOHN KANAI; Fiscal Year: 2013
    ..We are confident that our experience and unique approaches will lead to a very interactive and fruitful program. ..
  5. Excitation contraction coupling in bladder smooth muscle
    Robert S Moreland; Fiscal Year: 2013
    ..The studies proposed in this application will provide this missing information. If we can learn how normal bladder is controlled, we can then design experiments to study and hopefully repair abnormal bladder ..
  6. Vermont Center on Behavior and Health
    Stephen T Higgins; Fiscal Year: 2013
    ..S. public health. ..
  7. PSD95 scaffolding of vascular K+ channels in hypertension
    Sung W Rhee; Fiscal Year: 2013
    ....
  8. Persistent Calcium Sparklets in Arterial Smooth Muscle
    Luis F Santana; Fiscal Year: 2013
    ..This work will generate fundamental information on the mechanisms by which AKAP150 and CaV1.2 channels control of excitability, gene expression, and EC coupling in vascular smooth muscle under physiological and pathological conditions. ..
  9. MITOCHONDRIAL ENCEPHALOMYOPATHIES AND MENTAL RETARDATION
    Salvatore DiMauro; Fiscal Year: 2013
    ....
  10. Pregnancy/NO Induced Changes in UAE Ca2+Signaling
    Ian M Bird; Fiscal Year: 2013
    ..Having more complete knowledge of how cell function is regulated in pregnancy will certainly take us one step closer to overcoming this potentially devastating disease that effects so many in this country and beyond. ..
  11. Interactive Signaling Modules in Vascular Inflammation
    Linda H Shapiro; Fiscal Year: 2013
    ..abstract_text> ..
  12. Regulatory Mechanisms In Intestinal Motility
    Kenton M Sanders; Fiscal Year: 2013
    ..The investigative team is highly synergistic and collaborative, and the PPG has a long track-record of productivity and novel discovery ..