Morphology, Calcium Signalingand Mechanical Activity in Human UreterRachel V. Floyd, Ludmylla Borisova, Ali Bakran, C. Anthony Hart,* Susan Wrayand Theodor V. Burdyga†From the Physiological Laboratory, School of Biomedical Sciences, University of Liverpool (RVF, LB, SW, TVB), Royal LiverpoolUniversity Hospital (AB) and Department of Medical Microbiology and Genitourinary Medicine, University of Liverpool (AH), Liverpool,United Kingdom

Purpose: We determined the mechanisms of calcium signaling in the human ureter, and the relationship to peristalticcontractions and bundular structure in living tissue, thereby advancing the understanding of ureteral function in health andobstruction and reflux.Materials and Methods: Confocal imaging of 31 ureters was performed and simultaneous force and calcium measurementswere made. Immunohistochemistry and Western blotting were also performed.Results: Confocal imaging showed a 3-dimensional network of smooth muscle bundles with no defined longitudinal orcircular layers. Fast propagating Ca waves spread throughout the bundles, were closely associated with contraction anddepended on L-type Ca channel entry. Immunohistochemistry and Western blotting demonstrated L-type Ca channels, Cadependent K channels, sarcoplasmic reticulum Ca-adenosine triphosphatase isoforms 2 and 3, inositol triphosphate, andryanodine receptors. Modulation of Ca and K channel activity was a potent mechanism for affecting Ca and force, whereasmanipulation of the sarcoplasmic reticulum had little effect.Conclusions: To our knowledge this study represents the first measurements of Ca signals in the human ureter obtainedduring phasic contractions and in response to agonists. Results show that it is controlled by fast propagating Ca waves, whichspread rapidly between the muscle bundles, producing regular contractions, and drugs that interfere with excitability or Caentry through L-type Ca channels have profound effects on Ca signaling and contractility. These data are discussed inrelation to the treatment of patients with suspected ureteral dysfunction using Ca entry blockers.

Key Words: ureter; smooth muscle; sarcoplasmic reticulum; calcium channels; muscle contraction

T he ureteral musculature is important for renal health,as evidenced by damage done by obstruction or reflux.Aberrant smooth muscle morphology may contribute

to in-coordinate contractions in obstruction.1 To our knowl-edge there are no data on the morphology of human ureteralmuscle bundles in living tissue and the relationship of suchstructure to ureteral function, although the arrangement ofsmooth muscle bundles into longitudinal and circular layersmay not occur in human smooth muscle.2

Urinary stones are a growing clinical problem affecting12% of the population.3 Antagonism of L-type Ca channelscauses relaxation and it may be used to treat ureteralstones.4,5 However, to our knowledge no measurements ofintracellular Ca have been made in human ureters and,hence, there is no direct determination of their effects. Datafrom animal studies have shown marked species variation inCa signaling, increasing the need for studying the humanureter.6 The internal Ca store (SR) can affect ureteral excit-ability by Ca release.6–8 Profound effects on contractility

Submitted for publication October 16, 2007.Study received local ethical approval.Supported by Mersey Kidney Research (RVF).* Deceased.† Correspondence: Department of Physiology, University of Liver-

pool, Liverpool, L69 3BX, United Kingdom (telephone: 0151 7944971; FAX: 0151 794 5321; e-mail: burdyga@liv.ac.uk).

0022-5347/08/1801-0398/0THE JOURNAL OF UROLOGY®

Copyright © 2008 by AMERICAN UROLOGICAL ASSOCIATION

398

occur with SERCA inhibition.9,10 However, to our knowledgethere are no data on SR Ca in the human ureter.

We report simultaneous recordings of contractions andintracellular Ca in the human ureter to determine 1) therelationship between Ca transients and contraction, and therole of Ca entry, 2) the effect on Ca signaling and contrac-tility of L-type Ca channel modulators, 3) the contribution ofSR Ca release to contraction and 4) the effect of inhibiting Kchannels. Using confocal microscopy we determined the ar-rangement of ureteral muscle bundles and their relation tothe spread of Ca.

METHODS

TissueWe studied the middle third segments of 31 macroscopicallyhealthy ureters from live kidney donors, which were ob-tained after informed consent and local ethical approvalwere provided. Donor age was 20 to 55 years (median 39)and 55% of donors were men. Data are presented on 24ureters. One ureter did not contract and 6 did not showstable activity. Tissues were dissected into 4 mm transverserings and loaded with cell permeant fluorescent Ca sensitiveindicators Indo-1AM or Fluo-4AM8 (Molecular Probes®) by2-hour incubations in 15 �M of indicator with Pluronic®

F-127. Tissues were rinsed in saline for 30 minutes before

Vol. 180, 398-405, July 2008Printed in U.S.A.

DOI:10.1016/j.juro.2008.02.045

MORPHOLOGY, CALCIUM SIGNALING AND MECHANICAL ACTIVITY IN HUMAN URETER 399

use. In some experiments force was measured in uretersfrom adult male rats or guinea pigs following humane sac-rifice.6,7

SolutionsTissues were superfused at 7 to 8 ml per minute and 35Cunless stated otherwise with oxygenated buffered physiolog-ical saline (pH 7.4), composed of 120 mM NaCl, 5.9 mM KCl,1.2 mM MgSO4, 2 mM CaCl2, 8 mM glucose and 11 mMHEPES. Ca-free solutions contained 2 mM egtazic acid. Con-tractility was manipulated using 20 mM caffeine, 20 �MCPA, 1 �M BayK8644, 10 �M histamine, 10 �M phenyleph-rine, 10 �M nifedipine, 10 to 100 �M carbachol, and 1 and 10mM TEA. BayK-8644 and nifedipine were dissolved in eth-anol and CPA was dissolved in dimethyl sulfoxide to a finalconcentration of 0.1%, which is a concentration shown not tohave any effects.

Confocal MicroscopyConfocal microscopy was performed using an UltraVIEW™LCI scanner of the Nipkow disc type. This was linked to afast digital camera and UltraVIEW LCI software, and at-tached to Olympus® inverted microscope with image acqui-sition from a large area of the sample at the relatively highspeed of 20 to 50 frames per second.8

Ca Transients and ForceFluo-4 or Indo-1 loaded ureteral rings were mounted on 2stainless steel hooks, of which 1 was attached to a forcetransducer.6,7 Rings were mounted horizontally and parallelto the bottom of the chamber. They were stretched with 25%to 30% of active force produced by electrical stimulationbecause spontaneous activity was rarely seen in these prep-arations. Rectangular pulses of 57 V and 100 millisecondswere used at 40-second intervals.

Sodium Dodecyl Sulfate-PolyacrylamideGel Electrophoresis and Western BlottingProteins were extracted, quantified and separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis and trans-ferred to nitrocellulose membranes.11 Membranes were probedwith primary antibodies overnight with agitation at 4C, includingBKCa� (1:1,000), BKCa� (Abcam®) (1:500), L-type Ca channel(Alomone Labs, Jerusalem, Israel) (1:200), SERCA1 (1:2,500),SERCA2 (1:1,000), SERCA3 (Affinity BioReagents™) (1:500),

FIG. 1. X-Y gray color images of Fluo-4 loaded human ureter reveashows smooth muscle bundles with different orientations and thick

forming tight morphological connections. Reduced from �60. C, 50 � 5Reduced from �60.

RyR1 (Sigma®) (1:5,000), RyR2 (Affinity BioReagents) (1:1,000),RyR3 (1:1,250) and IP3R (Chemicon®) (1:250). Immunoreactivesites were revealed using goat anti-rabbit or goat anti-mouse IgGconjugated to horseradish peroxidase and SuperSignal® WestPico substrate.

ImmunohistochemistryTissue microarrays containing formalin fixed, paraffin embed-ded samples of 16 ureters were produced.11 Microarrays con-tained rat brain, skeletal muscle, heart and uterus controltissues from nonpregnant animals. Immunohistochemistrywas performed as described previously with an additional 20-minute antigen retrieval step in boiling 10 mM sodium citrate(pH 6).11 Antibodies were diluted, including BKCa� (BD Trans-duction Laboratories™) (1:50), BKCa� (1:100), L-type Ca chan-nel (1:100), SERCA1 (1:100), SERCA2 (1:200), SERCA3 (1:100), RyR1 (1:50), RyR2 (1:1,000), RyR3 (1:300), IP3R (1:100)and histamine H1 receptor (Affinity BioReagents) (1:200). Im-age capture was done using an Olympus BX51 microscope witha PL-A662 FireWire camera (PixeLINK™) using a 20� objec-tive.

Results are expressed as the mean � SE. Data wereanalyzed for statistical difference with Student’s t test withsignificance considered at p �0.05.

RESULTS

ArchitectureConfocal imaging showed a 3-dimensional network of inter-lacing muscle bundles of varying thicknesses and orienta-tion with no distinct separation into longitudinal or circularlayers (fig. 1, A and B). Bundle thickness was 15 to 150 �min 6 observations in different ureters and 50 to 100 �m gapscontaining blood vessels and connective tissue separated thebundles (fig. 1, A). Light microscopy also showed musclebundles lying in numerous orientations in the connectivetissue matrix (figs. 2 to 5). Myocytes were approximately 100�m long and 3 to 5 �m thick, and they had contact areas ofvariable sizes (fig. 1, B and C). Areas of close apposition,which may form junctional structures between myocytes,could be seen as a series of protrusions on their lateral sides(fig. 1, C).

Propagating Ca TransientsPropagating Ca transients (intercellular Ca waves) evokedby electrical stimulation generated phasic contractions in

man ureteral muscle bundle morphology. A, 300 � 400 �m imagees. Reduced from �20. B, 110 � 110 �m image demonstrates cells

ls huness

0 �m image shows cells forming tight morphological connections.

chan

MORPHOLOGY, CALCIUM SIGNALING AND MECHANICAL ACTIVITY IN HUMAN URETER400

which the rising phase was preceded by an increase in Ca(fig. 6, A and B). The Ca transient had a rapid upstroke,short plateau and slow relaxation phase. Time to peak,relaxation half-time and duration at 50% of the peak Catransient were 240 � 30, 1,100 � 20 and 1,600 � 35 milli-seconds, respectively, in 7 observations in different ureters.Removal of external Ca abolished propagating Ca transients(fig. 6, C). Decreasing perfusate temperature to 23C pro-longed the plateau component of the Ca transient by 1.9 �0.1 times and increased the amplitude of contractions 1.6 �0.05 times in 7 observations in different ureters (fig. 6, D).

Force and Ca transients were measured in multicellularpreparations (fig. 6, E and F). Control paired recordingsshowed no adverse effect of Indo-1 on ureteral contractions(fig. 6, E). Reproducible Ca transients and contractions could

FIG. 2. L-type Ca channel expression and functional role in humanchannels �1C in human ureter (HU) compared to rat heart (�ve).channel (brown areas) in ureter, which was localized to smooth mushematoxylin. Scale bar represents 100 �m. C, effect of 1 �M of Ca�M nifedipine on phasic contractions and signaling.

be achieved during 1 to 2 hours. These data point to a critical

role for Ca in human ureteral contraction, which in turndepends on membrane excitability. In the next set of exper-iments we investigated which ion channels controlled the Catransient.

Ca ChannelsIn 8 observations in different ureters Western blotting re-vealed an approximately 200 kDa band, which is the pre-dicted molecular weight of L-type Ca channels (fig. 2, A).Immunohistochemical studies revealed the �1C subunit insmooth muscle cell membranes in 16 observations in differ-ent ureters with rat heart as the positive control (fig. 2 B).Immunoreactivity was ablated by omission of primary anti-body or pre-incubation with antigenic peptide. Figure 2, C

er. A, Western blotting shows quantitative expression of L-type Camunohistochemistry reveals positive staining for �1C L-type Ca

ells and urothelium. Blue-purple areas indicate nuclei stained withnel opener BayK-8644 and blockade of L-type Ca channels with 10

uretB, imcle c

shows the effect of the L-type Ca channel opener BayK-8644

le ces an

MORPHOLOGY, CALCIUM SIGNALING AND MECHANICAL ACTIVITY IN HUMAN URETER 401

(1 �M) and blocker nifedipine (10 �M) on Ca transients andphasic contractions. In 6 observations in different uretersBayK-8644 produced a time dependent, significant increasein the amplitude (1.37 � 0.09 times) and duration (1.66 �0.21 times) of contractions, and amplitude (1.22 � 0.3 times)and duration (1.51 � 0.12 times) of Ca transients. In 5observations in different ureters nifedipine caused inhibi-tion (mean 16 � 0.35 minutes).

K ChannelsIn 8 observations in different ureters Western blotting re-

FIG. 3. BKCa and Kv expression and functional role in human uretersubunits in human ureter (HU) compared to rat uterus (�ve). B,(brown areas) in human ureter, which was localized to smooth musc�m. C, effect of BKCa blockade by 1 mM TEA on phasic contraction

vealed bands of approximately 110 and 30 to 35 kDa, corre-

sponding to the BKCa� and BKCa� subunits, respectively(fig. 3, A). Positive immunoreactivity corresponding to theBKCa� and BKCa� subunits was predominantly localized atthe plasma membrane of ureteral smooth muscle with dif-fuse staining in the cytosol in 16 observations in differentureters (fig. 3, B). The rat uterus control showed similarmembrane specific localization of BKCa protein. In 6 obser-vations in different ureters TEA (1 mM) blocked mainlyBKCas. It significantly increased the amplitude (1.29 � 0.08times) and duration (1.65 � 0.11 times) of contractions, andamplitude (1.16 � 0.1 times) and duration (1.52 � 0.15

estern blotting shows quantitative expression of BKCa� and BKCa�nohistochemistry reveals positive staining for BKCa� and BKCa�

lls only with no expression in urothelium. Scale bar represents 100d Ca signaling.

. A, Wimmu

times) of Ca transients (fig. 3, C). Increasing TEA to 10 mM

in u0 �M

MORPHOLOGY, CALCIUM SIGNALING AND MECHANICAL ACTIVITY IN HUMAN URETER402

to also block Kvs in 14 observations in different ureters pro-duced larger, significant increases in amplitude and duration(1.76 � 0.25 and 1.77 � 0.11 times, respectively, p �0.01).

SRWestern blotting of rat skeletal muscle, heart or brain re-vealed bands of 110 kDa for SERCA1-2 and 97 kDa forSERCA3 in 8 observations in different ureters (fig. 4, A). Inthe ureter SERCA1 protein was not detected but clear bandsfrom SERCA2 and 3 were seen. Immunohistochemistry re-vealed extensive staining with SERCA2 and 3 antibodies in16 observations in different ureters (fig. 4, B). SERCA1 wasnoted in skeletal muscle but it was absent in the ureter (fig.4, B), consistent with Western blotting.

SERCA inhibition with CPA showed a small, nonsignifi-cant potentiation of force and a sustained increase in base-line Ca in 9 observations in different ureters (fig. 4, C). To

FIG. 4. Expression and functional role of SERCA pump isoforms 1ureter (HU) vs rat skeletal muscle, heart and brain (�ve). B, immareas), which was localized to smooth muscle cells with no expressionbar represents 100 �m. C, effect of SERCA pump blockade using 2

study the functional role of SR the effects of histamine (9

observations in different ureters), carbachol (6 observationsin different ureters) and phenylephrine (5 observations indifferent ureters) in Ca-free solution were investigated.None produced significant Ca release or contraction. Figure5, A shows the effects of histamine (10 �M) in 9 observationsin different ureters. Because the effects of histamine havebeen shown to be marked in the guinea pig ureter,6 werepeated the experiments with histamine in rat and guineapig ureters (fig. 5, A). Figure 5, A shows that only guinea pigureter responded to histamine with significant potentiationof phasic contractions evoked by electrical field stimulation.Immunohistochemical studies revealed that H1 histaminereceptors were expressed in smooth muscle bundles andurothelium in the human ureter in 16 observations in dif-ferent ureters with rat heart as the positive control (fig. 5,B). IP3R was abundant in the smooth muscle and urothelialcell layers in 16 observations in different ureters with skel-

in human ureter. A, Western blotting shows expression in humanistochemistry reveals positive staining for SERCA 2 and 3 (brownrothelium. SERCA 1 was not detected in any ureteral tissues. ScaleCPA on phasic contractions.

to 3unoh

etal muscle as the positive control (fig. 5, B).

y RyR

MORPHOLOGY, CALCIUM SIGNALING AND MECHANICAL ACTIVITY IN HUMAN URETER 403

RyR3 was the only isoform expressed in the human ure-ter (fig. 5, C). Abundant RyR3 immunolocalization was notedin smooth muscle and urothelium. Rat skeletal muscle,heart and small intestine showed positive expression ofRyR1, 2 and 3, respectively, on immunohistochemistry in 16observations in different ureters. However, in 8 observationsin different ureters there was no detectable Ca release orcontraction evoked by caffeine (data not shown).

DISCUSSION

To our knowledge this is the first account of Ca signals inhuman ureter, and the elucidation of the role of SR and ionchannel distribution. We found that fast propagating Cawaves, which spread rapidly between the muscle bundles,producing regular contractions, and drugs that interferewith excitability or Ca entry have profound effects on Casignaling and contractility. However, SR has little role inmodulating contraction.

Ureteral responses to field stimulation were highly regu-lar and maintained, comparable to those found in vivo.12–14

As in animal tissue, loading with Indo-1 did not effect con-

FIG. 5. SR Ca release channel expression and histamine functionaelectrical field stimulation in human, rat and guinea pig ureter. B, imexpression (brown areas) in human ureter, which was localized to smcells and urothelium. Scale bar represents 100 �m. C, RyR3 was onlbar represents 100 �m.

tractility.6,7 Thus, we conclude that our methodology is suit-

able for determining the effects of modulators of Ca signalsand contractility in the human ureter.

To our knowledge we present the first images of theureter using live tissue, enabling unique structural informa-tion on the musculature without fixation artifacts and de-terminations of Ca signals in individual cells or bundles. Wefound a mesh of interconnecting muscle bundles of differentorientations in the ureteral wall and no demarcation intolongitudinal and circular layers, in agreement with oth-ers.2,14 Oblique or perpendicular branches were given off,connecting the musculature and aiding Ca signal propaga-tion. Disturbance of this arrangement, eg with obstructionor diabetes, could disturb the spread of Ca signals and,hence, peristalsis.

The velocity of impulse conduction calculated for the humanureter is 1.0 to 4.5 cm per second,12,15 which suggests thataction potentials govern the fast propagating Ca waves seenwith confocal microscopy. However, technical limitations pre-clude more accurate measurements. Consequently gap junc-tions are important for synchronizing phasic contraction andensuring rapid electrical communication between the networks

ct. A, effect of 10 �M histamine on phasic contractions evoked byohistochemistry reveals positive staining for histamine H1 receptormuscle cells and urothelium. IP3R was detected in smooth musclefound in human ureter smooth muscle cells and urothelium. Scale

l effemunooth

of muscle bundles, which have limited areas of contact.16

deleCa t

MORPHOLOGY, CALCIUM SIGNALING AND MECHANICAL ACTIVITY IN HUMAN URETER404

Our data show a close relationship between the parame-ters of the Ca transient and the ensuing contraction. Thus,changes in force as a consequence of several manipulationsfollowed the change in [Ca]. Data on animal ureters haveshown that the parameters of the action potential determinethe changes in Ca and can explain the changes in contractileactivity.7,8 Thus, for example, figure 6, D shows an augmen-tation of force and Ca as temperature was decreased. Wehave previously reported that this is due to marked prolon-gation of the action potential.8

To our knowledge our data provide the first immunohis-tochemical evidence for L-type Ca channels in the ureter ofany species and show them to be the mechanism underlyingCa entry for contraction in the human ureter. Ca transientsand phasic contractions rapidly fail when this entry is pre-vented. Furthermore, the Ca channel opener BayK-8644significantly increased transients and contraction. Nifedi-pine and related compounds are used to ease the passage ofureteral stones4,17 and they may do better than adrenergicreceptor blockade.18 Thus, a good understanding of themechanisms involved in the contraction and relaxation ofthe ureter is clinically relevant and our data suggests thattheir effects are related to decreased peristalsis.12,18

Experiments with TEA support a role for BKCas and Kvs inthe human ureter. Previously De Moura and De Lemos Neto

FIG. 6. Ca transients and force in human ureter. A, pseudo color imCa transients evoked by electrical stimulation. B, force (i), propagastimulation in intact human ureter, and overlay of normalized Ca trCa and 2 mM egtazic acid) from external medium on force and Caon force and Ca transients. E, paired ureteral samples show absentin untreated sample (i) and in sample after Indo-1 loading (ii), and

reported TEA enhanced electrically evoked contractions.19 Our

data confirm this and also show BKCa distribution. De Mouraand De Lemos Neto also noted that K channel openers (chro-makalin) inhibited contractions but glibenclamide, a blocker ofK adenosine triphosphate channels, had no effect. Our datashow that the effects of K channel modulation on ureteralcontractions are mediated by changes in [Ca] in the myocytes.Studies in animal models suggest that these effects of TEA onCa are due to prolongation of the action potential.8

Surprisingly we found little role for SR in modulatingcontractions. Thus, SERCA inhibition produced only slighteffects, IP3R stimulation had little effect and caffeine, whichis an RyR agonist, had no effect. Thus, although the ureteralSR is important in animals,6 our data do not show this butthey suggest similarities to uterine smooth muscle.9,20

CONCLUSIONS

We describe protocols for studying Ca signaling, contractil-ity and live imaging in the human ureter. Regular Ca tran-sients underlie peristaltic activity and they are controlledpredominately by excitability modulating Ca entry and tran-sient duration. The SR contribution to excitability and con-traction is limited and further investigation of the mecha-nism of agonist action on human ureter is warranted. These

hows Fluo-4 loaded ureteral ring during generation of propagatingintercellular Ca transient or Fluo-4 signal (ii) evoked by electricalnt and evoked phasic contraction (iii). C, effects of Ca removal (zero-4) in ureter. D, effect of decreasing temperature from 35C to 23Cterious effects due to Indo-1 loading, including phasic contractionsransients in sample after Indo-1 loading (iii).

age sted

ansie(Fluo

studies may inform the development of evidence based man-

MORPHOLOGY, CALCIUM SIGNALING AND MECHANICAL ACTIVITY IN HUMAN URETER 405

agement for the pathology of ureteral dysfunction and aconsequent decrease in kidney damage.

ACKNOWLEDGMENTS

Primary antibody to RyR3 was provided by Vincenzo Sor-rentino, Italy. Chemicals were obtained from Sigma unlessotherwise indicated.

Abbreviations and Acronyms

BKCa � Ca dependent K channelCPA � cyclopiazonic acid

IP3 � inositol triphosphateKv � voltage dependent K channel

RyR � ryanodine receptorSERCA � SR Ca-adenosine triphosphatase

SR � sarcoplasmic reticulumTEA � tetraethylammonium

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