Ex Vivo Analysis of Mechanically Activated Ca2+ Transients in Urothelial Cells

Summary

This protocol describes a methodology to assess the function of mechanically activated ion channels in native urothelial cells using the fluorescent Ca²⁺ sensor GCaMP5G.

Abstract

Mechanically activated ion channels are biological transducers that convert mechanical stimuli such as stretch or shear forces into electrical and biochemical signals. In mammals, mechanically activated channels are essential for the detection of external and internal stimuli in processes as diverse as touch sensation, hearing, red blood cell volume regulation, basal blood pressure regulation, and the sensation of urinary bladder fullness. While the function of mechanically activated ion channels has been extensively studied in the in vitro setting using the patch-clamp technique, assessing their function in their native environment remains a difficult task, often because of limited access to the sites of expression of these channels (e.g., afferent terminals, Merkel cells, baroreceptors, and kidney tubules) or difficulties applying the patch-clamp technique (e.g., the apical surfaces of urothelial umbrella cells). This protocol describes a procedure to assess mechanically evoked Ca²⁺ transients using the fluorescent sensor GCaMP5G in an ex vivo urothelial preparation, a technique that could be readily adapted for the study of mechanically evoked Ca²⁺ events in other native tissue preparations.

Introduction

Epithelial cells in the urinary tract are subjected to mechanical forces as the urinary filtrate travels through the nephrons, and urine is pumped out of the renal pelvis and travels through the ureters to be stored in the urinary bladder. It has been long recognized that mechanical forces (e.g., shear stress and stretch) exerted by fluids on the epithelial cells that line the urinary tract regulate the reabsorption of protein in the proximal tubule and of solutes in the distal nephron^1,2,3,4,5,<....

Protocol

Care and handling of the animals were carried out in accordance with the University of Pittsburgh Institutional Animal Care and Use Committee. Female, 2-4-month-old C57Bl/6J mice were used for the present study. The mice were obtained commercially (see Table of Materials).

1. Equipment assembly and setup

1. Perform Ca²⁺ imaging with an upright microscope equipped with a high-resolution camera and a stable light source (see Table of Materials).
   1. Acquire the images with a microscope compatible software that permits direct control of the camera, light source, and....

Results

The present protocol describes a technique to assess mechanically evoked Ca²⁺ transients in umbrella cells using the fluorescent Ca²⁺ sensor GCaMP5G. Adenoviral transduction was employed to express GCaMP5G in urothelial cells due to its high efficiency and because it produces an elevated level of expression. Fluorescent images of stained cryosections from a transduced bladder are shown in Figure 2D. For these experiments, GCaMP5G expression is highest in the umbrella ce.......

Discussion

All organisms, and seemingly most cell types, express ion channels that respond to mechanical stimuli^{20,33,34,35,36,37}. The function of these mechanically activated channels has been predominantly assessed with the patch-clamp technique. However, due to accessibility issues, patch-clamp studies of mechanically activated ion c.......

Materials

Name	Company	Catalog Number	Comments
20x Objective	Olympus	UMPlanFL N
24 G ¾” catheter	Medline	Suresite IV slide
4x Objective	Olympus	UPlanFL N
Analog/digital converter	Molecular Devices	Digidata 1440A
Anti-GFP antibody	Abcam	Ab6556
Beam splitter	Chroma	T495lpxr
Bipolar temperature controller	Warner Instruments	TC-344B
CaCl2	Fluka	21114-1L	1 M solution
cellSens software	Olympus		Imaging software
CMOS camera	Hamamatsu	ORCA fusion
Donkey anti-rabbit conjugated to Alexa Fluor 488	Jackson ImmunoResearch	711-545-152
Excel	Microsoft Corporation
Filter	Chroma	ET470/40X
Glass capillaries Corning 8250 glass	Warner Instruments	G85150T-4
Glucose	Sigma	G8270
HEPES	Sigma	H4034
Inline heater	Warner Instruments	SH-27B
KCl	Sigma	793590
Light source	Sutter Instruments	Lambda XL
Manifold pump tubing	Fisherbrand	14-190-510	ID 1.52 mm
Manifold pump tubing	Fisherbrand	14-190-533	ID 2.79 mm
MgCl2	Sigma	M9272
Mice	Jackson Lab	664	2-4 months old female C57BL/6J
Microforge	Narishige	MF-830
Micromanipulator	Sutter Instruments	MP-285
Microscope	Olympus	BX51W
Mounting media with DAPI	Invitrogen	S36964	Slowfade Diamond Antifade with DAPI
NaCl	Sigma	S7653
pClamp software	Molecular Devices	Version 10.4	Patch-clamp electrophysiology data acquisition and analysis software
Peristaltic pump	Gilson	Minipuls 3
Piezoelectric actuator	Thorlabs	PAS005
Pipette holder	World Precision Instruments
Pipette puller	Narishige	PP-830
Quick exchange heated base with perfusion and adapter ring kit	Warner Instruments	QE-1	Quick exchange platform fits 35 mm dish
Rhodamine-phalloidin	Invitrogen	R415
Sigma-Plot	Systat Software Inc	Version 14.0	Scientific graphing and data analysis software
Silicone elastomer	Dow	Sylgard 184
Single channel open-loop piezo controller	Thorlabs	MDT694B
Square grid holder pad	Ted Pella	10520
Suture	AD Surgical	S-S618R13	6-0 Sylk
Teflon mounting rod	Custom made		Use to mount the piezoelectric actuator in the micromanipulator
Tubing	Fisher Scientific	14171129	Tygon S3 ID 1/16 IN, OD 1/8 IN
USB Digital I/O device	National Instruments	NI USB-6501