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Neurally mediated hyperactive voiding in spontaneously hypertensive rats
Brain Research 790 Ž1998. 151–159
Research report
Neurally mediated hyperactive voiding in spontaneously hypertensive rats John M. Spitsbergen a b
c,1
, David B. Clemow a,1, Richard McCarty b , William D. Steers c , Jeremy B. Tuttle a,c,)
Department of Neuroscience, Box 230, UniÕersity of Virginia Health Sciences Center, CharlottesÕille, VA 22908, USA Department of Psychology, Box 230, UniÕersity of Virginia, Health Sciences Center, CharlottesÕille, VA 22908, USA c Department of Urology, Box 230, UniÕersity of Virginia, Health Sciences Center, CharlottesÕille, VA 22908, USA Accepted 13 January 1998
Abstract The development of hypertension in spontaneously hypertensive rats ŽSHR. and hyperactive voiding in rats with urethral obstruction are characterized by abnormal smooth muscle growth, increased tissue levels of nerve growth factor ŽNGF. and altered patterns of innervation. The present study was undertaken to determine if bladder smooth muscle from SHRs contains and secretes elevated levels of NGF, and if so, whether the augmented NGF contributes to changes in bladder innervation and function without tissue hypertrophy. Voiding behavior was monitored using specially designed metabolic cages. NGF levels in tissue homogenates and conditioned cell culture media were measured by ELISA. NGF mRNA in cultured bladder smooth muscle cells ŽBSMCs. was quantified using reverse transcriptase PCR. Noradrenergic innervation was assessed by staining with glyoxylic acid and assaying norepinephrine ŽNE. content in bladders with high performance liquid chromatography. SHRs voided more frequently than WKY rats. NGF content was higher in bladders from adult SHRs when compared to Wistar–Kyoto normotensive rats ŽWKYs.. No significant difference in NGF mRNA content was observed between SHR and WKY BSMCs. However, SHR BSMCs secreted NGF at a higher rate and amount per unit mRNA than did WKY BSMCs. SHR bladders contained more NE and were more densely stained for catecholaminergic fibers than bladders from WKY rats. The results support the hypothesis that elevated NGF secretion by bladder smooth muscle is associated with hyperinnervation of bladder and hyperactive voiding in SHRs. Thus, the SHR strain may represent a genetic model to study changes in bladder function resulting from altered patterns of innervation. q 1998 Elsevier Science B.V. Keywords: Nerve growth factor; Sympathetic; Micturition; Autonomic; Bladder smooth muscle; Hypertension
1. Introduction Urethral obstruction causes smooth muscle growth, changes in morphology and physiology of afferent w29x and efferent w28x innervation, augmented nerve growth factor content w32x as well as altered bladder filling and emptying w31x. Afferent and efferent neurons that are dependent on nerve growth factor ŽNGF. and innervate the bladder, respond to elevated NGF levels with an increased cell body size, enlarged dendritic arborization and terminal sprouting w6,28,29,32x. Autoimmunity to NGF prevents the morphological and functional changes of the nervous system and urinary frequency associated with obstruction w29,30,32x. These data support a role for elevated bladder ) Corresponding author. Box 230, Health Sciences Center, University of Virginia, Charlottesville, VA 22908, USA. Fax: q1-804-982-4159. 1 First two authors share equal credit.
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 0 6 1 - 4
NGF in the quantifiable neural changes linked to voiding pathology. The spontaneously hypertensive rat ŽSHR. and its genetically normotensive control strain, the Wistar–Kyoto ŽWKY. rat, have been used to study structural and functional changes associated with hypertension. SHRs develop progressive hypertension by 10 weeks of age, while agematched WKY rats do not w16,21x. Smooth muscle hypertrophy and hyperplasia w22x, enhanced responsiveness of the sympathetic nervous system w12,15,20x and increased noradrenergic innervation of vascular tissues w11x have all been observed in SHRs. Vascular beds receiving dense innervation in adult SHRs express higher levels of NGF during development than the same vascular beds in WKYs w7x. One source of NGF for neurons innervating blood vessels is vascular smooth muscle. Cultured vascular smooth muscle cells ŽVSMCs. from SHRs have a disturbance in NGF regulation that causes heightened NGF
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output when compared to VSMCs from WKYs w27x. Elevated target NGF is thought to play a role in the vascular hyperinnervation that leads to vascular remodeling and ultimately high blood pressure in SHRs w9x. Similarities exist between the progression of changes following bladder outlet obstruction and the development of hypertension in SHRs, both of which are characterized by hyperinnervation. For example, in both cases, elevated tissue levels of NGF are observed prior to changes in innervation w10,32x. The purpose of the present study was fourfold. First, do bladder smooth muscle cells ŽBSMCs. from SHRs contain the same defect observed in VSMCs leading to elevated secretion of NGF? Second, does elevated secretion of NGF by SHR BSMCs parallel increased tissue content? Third, do SHR bladders contain increased noradrenergic innervation as is found in the vasculature? Fourth, if there are neural differences in SHRs when compared to WKYs, are the differences reflected in voiding behavior?
2. Materials and methods 2.1. Bladder smooth muscle cell (BSMC) cultures and conditions SHR and WKY breeders were maintained from breeding stock originally purchased from Taconic Farms. For all of the studies, SHR Ž300–350 g. and WKY Ž400–450 g. adult rats between 14 and 16 weeks of age were screened for resting systolic BP with tail cuff plethysmography w13x. In this procedure, rats were restrained in clear Plexiglas tubes, and ambient temperature was maintained at less than 278C. Each rat was in a restraint tube for at least 10 min before BP was recorded. Precautions were taken to ensure that rats were calm when BP recordings were made. A tail cuff with a photosensitive cell was placed around the base of each rat’s tail. Changes in pressure applied to the cuff were transduced and amplified ŽModel 29, IITC. and the output traced on a chart recorder. Systolic BP values for each rat were based on an average of three to five tracings spaced at least 2 min apart. This is an age after the development of hypertension but before the age of peak BP. An average BP of at least 150 mmHg was required for all SHR and one of not more than 130 mmHg for all WKY. Bladders were dissected from rats following CO 2 euthanasia and placed in filtered Ca2q–Mg 2q-free tyrodes buffer with antibioticsrantimycotics. Bladders were cleaned of large blood vessels, mucosa and connective tissue and cut into small pieces Žapprox. 1 mm.. Minced bladders were then incubated Ž1–2 h. in saline containing trypsin Ž1 mgrml. and collagenase ŽSigma: 1 mgrml.. Tissue was periodically triturated to break up remaining clumps. Following enzymatic digestion, cells were centrifuged, culture medium containing 10% FBS was added,
and the cells were resuspended and plated in culture dishes. For routine culture BSMCs were plated at a density of 5 = 10 3 cellsrcm2 and maintained in growth medium consisting of Dulbecco’s Modified Eagle Medium ŽDMEM; Gibco. supplemented with 10% FBS, streptomycin Ž100 m grml., penicillin Ž100 unitsrml. and amphotericin B Ž250 ngrml; AntibioticrAntimycotic; Sigma.. All cultures were maintained in a humidified chamber containing 5.0% CO 2 at 378C. Cultures were made from six different primary cell lines Ž3 WKY and 3 SHR., each derived from four bladders Ž2 male:2 female rats.. Each set of rats represented the offspring of different breeders. There were no apparent differences between cell lines derived from a particular strain. Only cultures between passages 3 and 6 were used. To confirm the smooth muscle character of the cultured cells, immunohistochemical staining with anti-smooth muscle a-actin specific antibody ŽSigma. was used as previously described w5x. To study the secretion of NGF by smooth muscle, BSMCs were plated into 12-well tissue culture plates at a density of 5 = 10 3 cellsrcm2 and maintained in serum containing medium until confluence Žentire plate covered by cells such that no part of plate was visible with cultures being checked daily.. Once confluent, the culture medium was changed to serum free medium ŽSFM. consisting of growth medium without FBS, supplemented with insulin Ž1 Urml., transferrin Ž5 m grml., and ascorbic acid Ž200 m molrl; Sigma.. Culture medium was changed 48 and 72 h after the initial change to SFM upon which fresh SFM or SFM containing 100 m M forskolin, 100 nM phorbol–12myristate–13-acetate ŽPMA: Sigma., or 1 nM platelet-derived growth factor ŽPDGF: Upstate Biotechnology. was added Žconcentrations based upon literature search.. Each culture well was then sequentially sampled Ž250 m l each time point from the same medium. at 4, 6 and 24 h after treatment. Each experimental paradigm Ž n s 8. was run in triplicate. Following 24 h of treatment, cultures were rinsed with 0.1 molrl phosphate buffered saline ŽPBS., fixed for 20 min with acetic acid:H 2 O:ethanol Ž1:1:20., rinsed with PBS and stored in 70% alcohol prior to staining with bisbenzimide ŽSigma. for determination of cell numbers. For studies in which NGF mRNA levels were examined, BSMCs were plated in 100 mm Ž78.5 cm2 . dishes and grown in serum containing medium for 2 days. Prior to reaching confluence, the medium was changed to SFM and cultures were maintained for an additional 48 h. After 48 h in SFM, fresh SFM was added to the cultures. Culture medium was sampled and cells harvested after 24 h. For each experiment, parallel cultures were established; one culture was fixed for determination of cell number, the other was used for RNA collection. Conditioned medium was collected from both cultures to be assayed for NGF protein. Values are based on five experiments Ž n s 5: mRNA; n s 10: protein.. All experiments were performed on cultures between passages 2 and 5.
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2.2. Conditioned medium NGF determination For the quantification of NGF, cell culture conditioned medium samples, stored at y408C until assayed, were applied directly to ELISA plates. A two-site ELISA for NGF was used as described previously w27x. Briefly, 96well plates ŽNunc. were incubated with a monoclonal antibody raised against NGF ŽBoehringer Mannheim.. Remaining sites were blocked with 1.0% BSA in 50 mmolrl NaCO 3 buffer. After several rinses with wash buffer, 50 m l of conditioned medium or NGF standard Žpurified murine b-NGF kindly provided by E.M. Johnson, Jr., Washington University., freshly diluted in sample buffer immediately prior to each assay, was added to each well. The following day, wells were washed and incubated with an anti-NGF antibody conjugated to b-galactosidase ŽBoehringer Mannheim.. Three 45-min washes followed, after which a fluorescent substrate solution with 4-methylumbelliferyl-8-D-galactopyranoside Ž1 mgr10 ml; Sigma. was added to the wells and allowed to incubate overnight. Plates were read on a Titertek Fluoroscan II ŽFlow Laboratories. plate reader. For each assay, a standard curve was calculated from the known NGF standard concentrations. For cultured smooth muscle cells, the rate of NGF secretion for each sample period Ž0–4, 4–6, 6–24 h. was calculated by subtracting the total amount of NGF at the start of each time period Žconcentration of NGF in conditioned media multiplied by the volume of media. from that present at the end of each sample period and divided by the time between samples. The assay was sensitive to 1.0 pgrml of NGF. Single time point samples from each well were assayed in quadruplicate.
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Triton w X-100, 0.02% sodium azide, 0.1 m grml pepstatin A, 5 m grml aprotinin, 0.5 m grml antipain, 167 m grml benzamidine, 5.2 m grml phenylmethyl sulfonyl fluoride using 6-ml polypropylene tubes. Samples were then basetreated to pH 11 using 4 M NaOH. After centrifugation at 13,000 = g Ž48C, 15 min., the supernatant was acid-treated to pH 3 using glacial acetic acid followed by centrifugation at 13,000 = g Ž48C, 30 min.. The supernatant was then neutralized to pH 7 using 10 M NaOH. After a final centrifugation at 13,000 = g Ž48C, 15 min. the supernatant was assayed using the previously described ELISA for NGF. The samples were vortexed and left undisturbed for 5 min before each centrifugation. 2.4. Determination of cell number Fixed cultures that had been stored in alcohol were rinsed 1 = 5 min with distilled water, 1 = 5 min in PBS followed by incubation in PBS containing 0.5 m grml bisbenzimide ŽHoechst 33258 Dye. for 15 min. Stained nuclei were visualized with a 20 = objective under ultraviolet illumination. The cell number value for each 3.8 cm2 culture well of a 12-well plate consisted of the mean number from nine separate 250,000 m m2 areas each separately framed and counted using NIH Image software. The cell number value for each 100 mm Ž78.5 cm2 . dish consisted of the mean number from 24 separate areas. Each of the areas were the same for every wellrplate. Due to the possible effects of cell number on NGF secretion in a given experiment, rates of NGF secretion were expressed as fg NGF hy1 100 cellsy1 . 2.5. NGF mRNA determination
2.3. Bladder tissue NGF determination Bladders were dissected out of male rats Ž n s 8 WKY and 8 SHR. and frozen at y808C. Bladders were prepared based upon a protocol developed by Zettler et al. w35x. Frozen bladders were finely pulverized in liquid N2 and prepared for 1 min by Polytron w tissue disruption at 1:40 Žwrv. in a high saltrhigh detergent buffer: 100 mM Tris–HCl, 1 M NaCl, 2% BSA, 4 mM EDTA, 2%
Competitive, quantitative reverse transcriptase polymerase chain reaction ŽcqRT–PCR. was used to analyze tissue culture mRNA levels as previously described w23x. In brief, total RNA was extracted using TRIzole ŽGibco., according to manufacturer’s instructions. The amount of NGF mRNA in the isolate was determined with cqRT–PCR utilizing an internal, competitive cRNA standard w23x. First-strand cDNA synthesis was accomplished using Mal-
Table 1 Bladder NGF and norepinephrine content
Body weight Bladder weight Ratio of bladder to body weight NGFrbladder NGFrmg bladder weight NErmg bladder weight NErm g bladder protein
WKY
SHR
430 " 30 g 134 " 8 mg 0.032% 1.8 " 0.08 pgrbladder 0.014 " 0.001 pgrmg 35.7 " 2.2 pgrmg 2.2 " 0.11 pgrm g
328 " 20 g) 100 " 5 mg) 0.031% 3.4 " 0.22 pgrbladder) 0.034 " 0.002 pgrmg) 58.6 " 2.9 pgrmg) 5.30 " 0.32 pgrm g)
Bladder nerve growth factor ŽNGF. content was determined using an acidrbase-based protocol to extract NGF protein into a supernatant which was then analyzed with an ELISA. Norepinephrine ŽNE. content was determined with high-performance liquid chromatography with electrochemical detection. The asterisks Ž). denote significant differences between strains.
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Table 2 NGF mRNA and protein in BSMCs
Total RNA Žpg RNAr100 cells. NGF mRNArtotal RNA Žpg mRNArm g total RNA. NGF mRNA Žpg mRNAr100 cells. NGF secretion Žfg hy1 100 cellsy1 . NGF proteinrmRNA Žfg NGF hy1 pgy1 mRNA.
WKY BSMCs
SHR BSMCs
4.7 = 10 3 " 1.6 = 10 3 2.6 " 1.0 1.8 = 10y2 " 0.7 = 10y2 4.0 " 0.4 223 " 22
7.0 = 10 3 " 1.5 = 10 3 2.2 " 1.1 2.2 = 10y2 " 1.4 = 10y2 9.5 " 1.0) 431 " 46)
Cells were plated in 100 mm tissue culture dishes at a density of 5 = 103 cellsrcm2 . Cultures were maintained in normal growth medium for 48 h, then serum free medium ŽSFM. for 48 h, followed by SFM for 24 h after which the medium was sampled for NGF. Preconfluent, parallel cultures were either fixed for cell counting, or RNA was harvested using TRIzol reagent. The asterisks Ž). denote significant differences between strains.
oney-murine leukemia virus RT ŽGibco. in a mixture of internal standard Ž1 of 4 different bracketing amounts., native mRNA, and the downstream NGF mRNA primer resulting in four separate competitive RT–PCRs. The RT reaction products were used for PCR amplification. Amplified cRNA was analyzed with gel electrophoresis on a 1.3% agaroser0.5 = Tris–borate gel stained with ethidium bromide Ž10 mgrml.. Band intensities were measured from digitized images using an EagleEye system ŽStratagene. and NIH Image software. The ratio of native cDNA to the standard cDNA was plotted Žlogrlog. against the amount of cRNA standard added Ž50, 10, 5, 1 pgrml. at the beginning of the cqRT–PCR. The amount of native NGF mRNA was calculated using linear regression w23x. 2.6. Glyoxylic acid staining Bladders were removed from male SHR Ž n s 5. and WKY Ž n s 5. rats, frozen in cryomolds in a cryostat
Žy308C. and sectioned lengthwise from dome to base at 20 m M. The entire bladder was serially sectioned and kept in order from the first to last slice. As each slide was filled with bladder sections, it was dipped in buffered saline Ž3 = 1 s. containing 200 mM sucrose, 236 mM KH 2 PO4 and 1% glyoxylic acid ŽpH 7.4.. Slides were air dried for 10 to 15 min and placed in an 808C oven for 5 min. After heating, slides were coverslipped with mineral oil, heated on a hot-plate for 90 s to remove bubbles and viewed under ultraviolet illumination on a microphot TMD microscope ŽNikon.. To determine if innervation differed in bladders from SHR and WKY rats, two methods of quantification were used. First, we calculated the percentage of sections containing stained fibers Ž200 = magnification., as well as the percentage of sections containing fibers in the bladder base, bladder body and bladder dome. The entire area of every section of every bladder was examined. It is important to note that perivascular staining was clearly evident and not counted as stained fibers. Second,
Fig. 1. NGF secretion by bladder smooth muscle cells (BSMCs): The effect of 1 nM PDGF, 100 nM PMA or 100 m M forskolin on BSMCs under confluent, serum free conditions quantified from conditioned samples taken 4, 6, and 24 h from the start of each experiment. Cultures were derived from bladders of adult WKYs ŽA. and SHRs ŽB.. Nerve growth factor content of culture medium was determined using an ELISA. The asterisks Ž). denote significant differences from control. The solid circles Žv . represent significant differences between strain control values.
J.M. Spitsbergen et al.r Brain Research 790 (1998) 151–159
to examine fiber density and fiber branching, we took photographs Ž20 = objective. of stained fibers. We then projected these images onto a screen, superimposed a grid onto these and counted the number of times fibers crossed the grid lines, giving an estimate of the extent of fiber length and branching in the sections. For quantification, one picture was taken from each of 15 sections from each bladder. Because of the size differences between SHR Žmean s 50 sectionsrbladder. and WKY Žmean s 63 sectionsrbladder. bladders, sections used for pictures were approximately selected in 6.7% serial increments through
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the bladder, i.e., for a bladder with 50 sections the first section used for pictures was the 3rd section while the 4th section was used for a bladder with 63 sections. Because of the high amount of variability in staining density in a specifically defined area of a given section, the picture taken for a section was of the most densely stained area for that section. 2.7. Norepinephrine (NE) analysis For measurement of NE in tissue, male bladders Ž n s 6 WKY and n s 6 SHR. were removed from rats following
Fig. 2. Sympathetic fiber staining in bladder: Bladders were removed from adult SHRs and WKYs. Sections were cut at 20 m M and stained for catecholamine containing fibers using glyoxylic acid. Two methods were used to quantify bladder staining. First, the percent of sections containing positively stained fibers were calculated, as well as the percent of sections containing fibers in the bladder base, bladder body and bladder dome ŽA: the asterisks Ž). denote significant differences between strains.. Second, to examine fiber density and fiber branching, we took photographs Ž20 = . of particular regions containing fibers and superimposed a grid onto these. We then calculated the number of times fibers crossed the grid lines for a given measured area and compared the frequency distributions for WKY and SHR bladders ŽB: the asterisk Ž). denotes that the frequency distributions are significantly different..
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CO 2 euthanasia and snap frozen on dry ice. Frozen samples were weighed and homogenized in 4 = volume of cold 0.2 N perchloric acid. Fifty microliters of homogenate were removed for determination of protein according to manufacturer’s instructions: Bio-Rad protein assay kit. The remaining homogenate was centrifuged at 15,000 rpm for 30 min at 48C. The resulting supernatant was removed and NE and other catechols measured by batch alumina extraction followed by high-performance liquid chromatography with electrochemical detection w14x. 2.8. Voiding studies Rats used for voiding studies were housed in individual cages with free access to food and water Ž30 adult rats: 11 F, 18 M and 10 4-week old rats: 5 F, 5 M for both WKY and SHR.. Animals were kept on a 12-h lightrdark cycle starting at 7:00 AM. Voiding studies were run in mid-light cycle Ž6-h time period: 10:00 AM–4:00 PM.. For the measurement of voiding frequency, SHR and WKY rats were placed in individual metabolic cages ŽNalge. with unrestricted food and water. A graduated cup was attached to an FT03 force transducer ŽGrass. and positioned to collect all urine during the sample period. Void events were amplified using a model 7D amplifier and traced on a polygraph ŽGrass.. Previous testing allowed comparisons between volume of individual voids and the magnitude of graph deflections. 2.9. Statistical analyses Comparisons between two variables in different strains were made using Student’s t-test for independent samples, while comparisons within the same strain were made using Student’s paired t-test. Analysis of NGF mRNA levels and glyoxylic acid staining were made using the Mann–Whitney U-test. Analysis of nerve fiber frequency distributions within bladder sections was performed using the Kolmogorov–Smirnov test. Values shown represent the mean " S.E.M. For all comparisons significance was set to p - 0.05.
3.2. NGF mRNA and protein in cultured BSMCs NGF mRNA levels and the rate of NGF secretion were examined in preconfluent cultures. Although all of the cultures were plated at the same density, SHR BSMCs Ž10 = 10 3 cellsrcm2 . proliferated more rapidly resulting in a greater preconfluent cell number than WKY BSMCs Ž6 = 10 3 cellsrcm2 .. There was no difference between strains in the amount of total RNA or NGF mRNA, but the per cell rate of NGF secretion was significantly higher in SHR than WKY preconfluent cultures ŽTable 2.. Furthermore, SHR BSMCs secreted a significantly greater amount of NGF per unit NGF mRNA ŽTable 2. suggesting that SHR BSMCs are more efficient at translating their mRNA into protein. 3.3. Pharmacology of NGF secretion by BSMCs SHR BSMCs Ž6.3 = 10 4 cellsrcm2 . grew to a greater packing density than did WKY BSMCs Ž3.2 = 10 4 cellsrcm2 . when allowed to grow to confluence. Preconfluent cultures secreted NGF at a higher rate than did confluent cultures. Confluent cultures of SHR BSMCs Ž2.3 " 0.3; 3.5 " 0.6; 2.3 " 0.2. secreted NGF at a higher rate than did WKY BSMCs Ž1.2 " 0.2; 1.6 " 0.3; 0.9 " 0.2 fg hy1 100 cellsy1 . between 0–4, 4–6 and 6–24 h, respectively ŽFig. 1.. Treatment of confluent cultures with PDGF caused a significant increase in NGF secretion between 0–4 h in SHR BSMCs, while increasing NGF secretion between 4–6 h in WKY BSMCs. Direct activation of protein kinase C ŽPKC. signaling pathways with the phorbol ester PMA had no effect on the rate of NGF secretion for either SHR or WKY BSMCs. Stimulation of cAMP-dependent pathways with forskolin decreased the rate of NGF secretion between 4–6 and 6–24 h for both SHR and WKY BSMCs ŽFig. 1.. 3.4. Noradrenergic staining of the bladder Analysis of glyoxylic acid-stained bladder sections revealed that a greater percentage of SHR bladder sections
3. Results
Table 3 Sympathetic nerve fiber density
3.1. NGF and norepinephrine (NE) content of SHR and WKY bladders
Mean grid crossings per quantified area
WKY
SHR
Total Base Body Dome
19.4"1.2 27.2"2.7 16.3"1.3 11.5"2.6
26.7"0.8) 35.6"2.9) 21.4"1.6) 16.6"3.8
Total body and bladder weights were less in SHRs when compared to WKYs. However, the ratio of bladder weight to body weight was identical between the two strains ŽTable 1.. The concentration of NGF calculated as per whole bladder or per mg of bladder wet weight was higher in SHRs ŽTable 1.. The amount of NE determined as either per mg of bladder weight or per m g bladder protein was higher in SHRs ŽTable 1..
Bladders were serially sectioned lengthwise from dome to base at 20 m M and stained with glyoxylic acid, thereby marking catecholamine containing nerves. An estimate of fiber density and branching was examined Ž20=. by counting the nerve fiber crossings of a superimposed grid. For quantification, one viewing area was quantified from each of 15 sections from each bladder. Areas were counted in the bladder dome, body and base.
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contained positively-stained fibers in the dome, body and base than did WKY sections ŽFig. 2A.. Nerve fiber grid crossings of a superimposed grid revealed that regions exhibiting positively stained nerve fibers contained a higher density of fibers in SHR bladders when compared to WKY bladders. Not only were grid crossings higher for SHRs in
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measured areas of the bladder dome, body and base ŽTable 3. but the whole bladder frequency distribution of grid crossings per measured area was significantly different between SHRs and WKYs ŽFig. 2B.. 3.5. Voiding frequency and Õolume in SHR and WKY rats Voiding frequency and volume were examined in 4week old and adult rats. Both 4-week old and adult SHR rats voided significantly more frequently ŽFig. 3A. but at a reduced volume per void ŽFig. 3B. than did age-matched WKYs. No difference between strains was observed in the total volume voided during the 6-h experimental time period ŽFig. 3C.. Due to the design of the experimental apparatus, only approximate measurements of water intake were possible. No differences were observed between strains.
4. Discussion
Fig. 3. Void frequency and Õolume: SHRs and WKYs were placed in individual metabolic cages for a period of 6 h with unrestricted food and water. A graduated cup was attached to a force transducer and positioned to collect all urine during the sample period. Void events were amplified and traced on a polygraph. Previous testing allowed comparisons between volume of individual voids and the magnitude of graph deflections. Mean voiding frequency ŽA., void volume ŽB., and total volume ŽC. are shown. The asterisks Ž). denote significant differences between strains.
Our results show that BSMCs from SHRs contain a similar defect to that found in VSMCs such that cultured SHR BSMCs secrete an elevated level of NGF compared to normotensive WKY BSMCs. SHR bladder tissue NGF content is also elevated when compared to WKY bladders. SHR bladders contain higher concentrations of NE and NGF-dependent catecholamine nerve fibers, suggesting they are hyperinnervated. Disturbances in SHR bladder NGF production and neural innervation are concomitant with SHRs hyperactive voiding behavior and may be an underlying causal factor. Under both preconfluent and confluent conditions, SHR BSMCs secrete more NGF than WKY BSMCs. This finding mirrors results from studies in cultured VSMCs, in which SHR VSMCs secrete NGF at a greater rate than WKY VSMCs w27x. Although the rate of NGF secretion by BSMCs is more than 20 times that of the rate observed for VSMCs, the ratio of NGF secretion by BSMCs from SHRs and WKYs is almost identical to that of VSMCs. It is tempting to suggest that the differences in NGF production between strains by BSMCs and VSMCs in culture reflect differences in innervation in vivo. The effect of PDGF, PMA or forskolin on NGF secretion from VSMCs is markedly different in cultures from SHRs and WKYs w34x. We tested these treatments in the present study to determine if the regulation of NGF secretion is similar in bladder and vascular smooth muscle and to determine if the alterations in regulation found in SHR VSMCs exist in BSMCs. Direct activation of PKC signaling pathways with PMA causes a dramatic increase in the rate of NGF secretion in VSMCs from both SHR and WKY rats that returns to control levels between 8 and 24 h in WKY VSMCs but remains significantly elevated in SHR VSMCs w34x. In contrast, the present study shows
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that treatment of cultures with PMA has no effect on the rate of NGF secretion in either SHR or WKY BSMCs. The cytokine PDGF causes a dramatic increase in the rate of NGF secretion in both SHR Ž3200% of control. and WKY VSMCs Ž1300% control.. This effect is blocked by PKC down-regulation w34x. In BSMCs, PDGF causes only a doubling of the rate of NGF output and is equally effective in cells from SHRs and WKYs. The only difference between strains is that elevated NGF output occurs at an earlier time point in SHR BSMC cultures ŽFig. 1.. The limited effect of PDGF, which acts in part through activation of PKC signaling pathways w8x, and the lack of effect of PMA on NGF secretion by BSMCs may indicate that PKC signaling pathways play less of a significant role in the regulation of NGF production in BSMCs as compared to VSMCs. However, this may not always be the case. PDGF and PMA increase NGF output from Wistar BSMCs 200–400% in the presence of 5% horse serum w24x. Furthermore, control of BSMC NGF secretion by PKC signaling pathways is strongly suggested in culture conditions where mechanical load is applied w3,23x. Moreover, an unknown defectŽs. in mechanical signal transduction leads to elevated NGF output in stretched SHR BSMC Ž1400% control. cultures when compared to WKY BSMC Ž165% control. cultures w3x suggesting there may be multiple irregularities in the regulation of NGF output in SHR smooth muscle. The effect of serum suggests culture conditions and cellular phenotype have an influence on the regulation of NGF secretion. b-Adrenoceptor agonists generally inhibit while aadrenoceptor agonists activate NGF secretion from smooth muscle. Forskolin mimics b-adrenergic stimulation by triggering cAMP elevations by directly activating adenylate cyclase w19x and thus protein kinase A ŽPKA. signaling pathways w17x. Forskolin causes a normal decrease in the rate of NGF secretion by WKY VSMCs, but actually increases the rate of NGF secretion by SHR VSMCs w34x. Forskolin potently inhibits NGF secretion in both SHR and WKY BSMCs suggesting that the defects in adrenergic regulation of SHR VSMC NGF production are not found in BSMCs. The observation that there is little difference in the response of SHR and WKY BSMCs to treatment with PDGF and PMA or following treatment with forskolin may indicate that the abnormal NGF secretion measured in cultured SHR BSMCs is not due to alterations in PKC or PKA signaling pathways. Other mechanisms, such as phospholipase-A2 linked signaling pathways w2x, associated with NGF output may be relevant to altered NGF secretion in SHR BSMCs. Hypertension in SHRs is characterized by an elevation in vascular bed NGF levels associated with noradrenergic hyperinnervation as the disease progresses w7,36x. A similar phenomenon occurs following bladder outlet obstruction. NGF content of obstructed bladders increases dramatically w32x. In a time-dependent manner, elevated NGF is followed by morphological changes in innervation that
include hypertrophy of afferent and efferent neurons supplying the bladder w28,29,32x. Bladder decentralization does not prevent the neuronal size increases induced by obstruction w28x. Enhanced spinal reflex discharges appear after obstruction which are linked to an increase in voiding frequency w31x. Afferent thresholds are lowered by exogenous NGF w18x. Perhaps elevated secretion of NGF by SHR bladder smooth muscle results in bladder hyperinnervation, similar to that of SHR vascular smooth muscle, and bladder hyperinnervation in the SHR is associated with voiding dysfunction resembling that observed with obstructed, hyperinnervated bladders. The present findings demonstrate that this may indeed be the case. Cultured BSMCs from SHRs secrete more NGF than WKY BSMCs, and there is an elevated level of NGF in SHR bladder tissue. SHR bladders exhibit a higher concentration of NE and a greater density of catecholaminergic fibers than bladders from WKY rats suggesting hyperinnervation of NGF-dependent sympathetic nerves. These results support the findings of Tong et al. w33x demonstrating elevated concentrations of sympathetic neurotransmitters in synaptosomal preparations from bladders of SHRs compared to WKYs. Moreover, neuronal hypertrophy occurs in both the postganglionic efferent limb Žmajor pelvic ganglion. and sensory afferent limb Ždorsal root ganglion. of the micturition reflex pathway of SHRs when compared to WKYs, suggesting that both sympathetic and sensory changes in neuronal innervation of the bladder occur in SHRs w4x. Finally, adult SHRs void at a higher frequency than WKYs, but void the same total volume, suggesting enhanced reflexes are the cause rather than increased urine production. The data presented do not isolate the changeŽs. responsible for increased voiding frequency, whether sensory, sympathetic, visceral motor andror central circuits associated with one or more of these. Experiments designed to distinguish among these possibilities could yield valuable information on the determinants of bladder circuit function. The observed changes in voiding frequency do not appear to result solely from hypertension because similar differences are observed in 4-week old SHR and WKY rats, prior to the development of high blood pressure w16x. Increased voiding frequency does not derive from the fact that SHRs have smaller bladders as adult SHRs void more frequently than 4-week old WKYs with smaller bladders and the ratio of bladder to body weight is not different between strains. These findings are consistent with the hypothesis that overexpression of NGF by bladder smooth muscle leads to hyperinnervation of the bladder, resulting in hyperactive voiding behavior in affected rats. If hypertensive states in some humans have causes similar to those in SHRs, one would predict that humans suffering from hypertension may also exhibit alterations in the physiology of bladder filling and emptying. Indeed, patients with hypertension are also more likely to suffer symptoms of prostatism and thus voiding dysfunction w1,25,26x. The
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SHR strain may represent the first genetic model to study neurally mediated hyperactive voiding.
Acknowledgements We would like to thank Pamela Neff for her assistance with RT–PCR, Cheryl Talley for her help in maintenance of the rat colony and blood pressure measurements, and Disheng Men for her help with norepinephrine measurements. This work was supported by the generosity of the taxpayers of the United States of America through the National Institutes of Health Grants DK 45179 and DK 49431 and a grant from the American Heart Association, Virginia Affiliate.
References w1x P. Boyle, P. Napalkov, The epidemiology of benign prostatic hyperplasia and observations on concomitant hypertension, Scand. J. Urol. Nephrol. 168 Ž1995. 7–12, Supplement. w2x M. Carman-Krzan, B.C. Wise, Arachidonic acid lipoxygenation may mediate interleukin-1 stimulation of nerve growth factor secretion in astroglial cultures, J. Neurosci. Res. 34 Ž1993. 225–232. w3x D.B. Clemow, W.D. Steers, J.B. Tuttle, Stretch-activated nerve growth factor secretion from bladder smooth muscle, Am. Soc. Cell Biol. Abstr. 7-S Ž1996. 530a. w4x D.C. Clemow, R. McCarty, W.D. Steers, J.B. Tuttle, Efferent and afferent neuronal hypertrophy associated with micturition pathways in spontaneously hypertensive rats, Neurol. Urodynamics 16 Ž1997. 293–303. w5x D. Creedon, J.B. Tuttle, Nerve growth factor synthesis in vascular smooth muscle, Hypertension 18 Ž1991. 730–741. w6x W.C. de Groat, Mechanisms underlying the recovery of lower urinary tract function following spinal cord injury, Paraplegia 33 Ž1995. 493–505. w7x S.J. Donohue, R.J. Head, R.E. Stitzel, Elevated nerve growth factor levels in young spontaneously hypertensive rats, Hypertension 14 Ž1989. 421–426. w8x A.P. Fields, G. Tyler, A.S. Kraft, W.S. May, Role of nuclear protein kinase C in the mitogenic response to platelet-derived growth factor, J. Cell Sci. 96 Ž1990. 107–714. w9x B. Folkow, Early structural changes in hypertension: pathophysiology and clinical consequences, J. Cardiovasc. Pharmacol. 22 Ž1993. S1–S6. w10x R.J. Head, Hypernoradrenergic innervation: its relationship to functional and hyperplastic changes in the vasculature of the spontaneously hypertensive rat, Blood Vessels 26 Ž1989. 1–20. w11x R.J. Head, L.A. Cassis, R.L. Robinson, D.P. Westfall, R.E. Stitzel, Altered catecholamine contents in vascular and nonvascular tissues in genetically hypertensive rats, Blood Vessels 22 Ž1985. 196–204. w12x E.D. Hendley, M.A. Cierpial, R. McCarty, Sympathetic-adrenal medullary response to stress in hyperactive and hypertensive rats, Physiol. Behav. 44 Ž1988. 47–51. w13x E.D. Hendley, W.G. Ohlsson, Two new inbred rat strains derived from SHR: WKHA, hyperactive, and WKHT, hypertensive, rats, Am. J. Physiol. 261 Ž1991. H583–H589. w14x C. Holmes, G. Eisenhofer, D.S. Goldstein, Improved assay for plasma dihydroxyphenylacetic acid and other catechols using highperformance liquid chromatography with electrochemical detection, J. Chromatogr. B Biomed. Appl. 653 Ž1994. 131–138.
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w15x J.C. Juskevich, D.S. Robinson, D. Whitchorn, Effect of hypothalamic stimulation in spontaneously hypertensive rats, Eur. J. Pharmacol. 51 Ž1978. 429–439. w16x L.T. Lais, L.L. Rios, S. Boutelle, G.F. DiBona, M.J. Brody, Arterial pressure development in neonatal and young spontaneously hypertensive rats, Blood Vessels 14 Ž1977. 277–284. w17x A. Levitzki, From epinephrine to cAMP, Science 241 Ž1988. 800– 806. w18x G.R. Lewin, A.M. Ritter, L.M. Mendell, Nerve growth factor-induced hyperalgesia in the neonatal and adult rat, J. Neurosci. 13 Ž1993. 2136–2148. w19x H. Metzgar, E. Linder, The positive-ionotropic-acting forskolin, a potent adenylate cyclase activator, Arzneimittel-Forschung 30 Ž1981. 1248–1250. w20x S.F. Morrison, D. Whitchorn, Enhanced preganglionic sympathetic nerve responsiveness in spontaneously hypertensive rats, Brain Res. 296 Ž1984. 152–155. w21x Okamoto, K. Acki, Development of a strain of spontaneously hypertensive rats, Jpn. Circ. J. 27 Ž1963. 282–293. w22x G.K. Owens, P.S. Rabinovich, S.M. Schwartz, Smooth muscle cell hypertrophy versus hyperplasia in hypertension, Proc. Natl. Acad. Sci. U.S.A. 78 Ž1981. 7759–7763. w23x K. Persson, J.J. Sando, J.B. Tuttle, W.D. Steers, Protein kinase C in cyclic stretch-induced nerve growth factor production by urinary tract smooth muscle cells, Am. J. Physiol. 269 Ž1995. C1018–C1024. w24x K. Persson, W.D. Steers, W.D. Tuttle, Regulation of nerve growth factor secretion in smooth muscle cells cultured from rat bladder body, base and urethra, J. Urol. 157 Ž1997. 2000–2006. w25x J.L. Pool, Role of the sympathetic nervous system in hypertension and benign prostatic hyperplasia, Br. J. Clin. Pract. 74 Ž1994. 13–17, Symposium Supplement. w26x J.L. Pool, Doxazosin: a new approach to hypertension and benign prostatic hyperplasia, Br. J. Clin. Pract. 50 Ž1996. 154–163. w27x J.M. Spitsbergen, J.S. Stewart, J.B. Tuttle, Altered regulation of nerve growth factor secretion by cultured VSMCs from hypertensive rats, Am. J. Physiol. 269 Ž1995. H621–H628. w28x W.D. Steers, J. Ciambotti, S. Erdman, W.C. de Groat, Morphological plasticity in efferent pathways to the urinary bladder of the rat following urethral obstruction, J. Neurosci. 10 Ž1990. 1943–1951. w29x W.D. Steers, J. Ciambotti, B. Etzel, S. Erdman, W.C. de Groat, Alterations in afferent pathways from the urinary bladder of the rat in response to partial urethral obstruction, J. Comp. Neurol. 310 Ž1991. 401–410. w30x W.D. Steers, D.J. Creedon, J.B. Tuttle, Immunity to nerve growth factor prevents afferent plasticity following urinary bladder hypertrophy, J. Urol. 155 Ž1996. 379–385. w31x W.D. Steers, W.C. de Groat, Effect of bladder outlet obstruction on micturition reflex pathways in the rat, J. Urol. 140 Ž1988. 864–871. w32x W.D. Steers, S. Kolbeck, D. Creedon, J.B. Tuttle, Nerve growth factor in the urinary bladder of the adult regulates neuronal form and function, J. Clin. Invest. 88 Ž1991. 1709–1715. w33x Y.C. Tong, Y.C. Hung, S.N. Lin, J.T. Cheng, Comparisons of neurotransmitter concentrations in the synaptosomal preparation of the normotensive and hypertensive rat urinary bladder, Neurosci. Lett. 204 Ž1996. 141–143. w34x J.B. Tuttle, J.M. Spitsbergen, J.S. Stewart, R.M. McCarty, W.D. Steers, Altered signalling in vascular smooth muscle from spontaneously hypertensive rats may link medial hypertrophy, vessel hyperinnervation and elevated nerve growth factor, Clin. Exp. Pharmacol. Physiol. 22 Ž1995. S117–S119. w35x C. Zettler, D.C.M. Bridges, X.-F. Zhou, R.A. Rush, Detection of increased tissue concentrations of nerve growth factor with an improved extraction procedure, J. Neurosci. Res. 46 Ž1996. 581–594. w36x C. Zettler, R.A. Rush, Elevated concentrations of nerve growth factor in heart and mesenteric arteries of spontaneously hypertensive rats, Brain Res. 614 Ž1993. 15–20.
Research report
Neurally mediated hyperactive voiding in spontaneously hypertensive rats John M. Spitsbergen a b
c,1
, David B. Clemow a,1, Richard McCarty b , William D. Steers c , Jeremy B. Tuttle a,c,)
Department of Neuroscience, Box 230, UniÕersity of Virginia Health Sciences Center, CharlottesÕille, VA 22908, USA Department of Psychology, Box 230, UniÕersity of Virginia, Health Sciences Center, CharlottesÕille, VA 22908, USA c Department of Urology, Box 230, UniÕersity of Virginia, Health Sciences Center, CharlottesÕille, VA 22908, USA Accepted 13 January 1998
Abstract The development of hypertension in spontaneously hypertensive rats ŽSHR. and hyperactive voiding in rats with urethral obstruction are characterized by abnormal smooth muscle growth, increased tissue levels of nerve growth factor ŽNGF. and altered patterns of innervation. The present study was undertaken to determine if bladder smooth muscle from SHRs contains and secretes elevated levels of NGF, and if so, whether the augmented NGF contributes to changes in bladder innervation and function without tissue hypertrophy. Voiding behavior was monitored using specially designed metabolic cages. NGF levels in tissue homogenates and conditioned cell culture media were measured by ELISA. NGF mRNA in cultured bladder smooth muscle cells ŽBSMCs. was quantified using reverse transcriptase PCR. Noradrenergic innervation was assessed by staining with glyoxylic acid and assaying norepinephrine ŽNE. content in bladders with high performance liquid chromatography. SHRs voided more frequently than WKY rats. NGF content was higher in bladders from adult SHRs when compared to Wistar–Kyoto normotensive rats ŽWKYs.. No significant difference in NGF mRNA content was observed between SHR and WKY BSMCs. However, SHR BSMCs secreted NGF at a higher rate and amount per unit mRNA than did WKY BSMCs. SHR bladders contained more NE and were more densely stained for catecholaminergic fibers than bladders from WKY rats. The results support the hypothesis that elevated NGF secretion by bladder smooth muscle is associated with hyperinnervation of bladder and hyperactive voiding in SHRs. Thus, the SHR strain may represent a genetic model to study changes in bladder function resulting from altered patterns of innervation. q 1998 Elsevier Science B.V. Keywords: Nerve growth factor; Sympathetic; Micturition; Autonomic; Bladder smooth muscle; Hypertension
1. Introduction Urethral obstruction causes smooth muscle growth, changes in morphology and physiology of afferent w29x and efferent w28x innervation, augmented nerve growth factor content w32x as well as altered bladder filling and emptying w31x. Afferent and efferent neurons that are dependent on nerve growth factor ŽNGF. and innervate the bladder, respond to elevated NGF levels with an increased cell body size, enlarged dendritic arborization and terminal sprouting w6,28,29,32x. Autoimmunity to NGF prevents the morphological and functional changes of the nervous system and urinary frequency associated with obstruction w29,30,32x. These data support a role for elevated bladder ) Corresponding author. Box 230, Health Sciences Center, University of Virginia, Charlottesville, VA 22908, USA. Fax: q1-804-982-4159. 1 First two authors share equal credit.
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 0 6 1 - 4
NGF in the quantifiable neural changes linked to voiding pathology. The spontaneously hypertensive rat ŽSHR. and its genetically normotensive control strain, the Wistar–Kyoto ŽWKY. rat, have been used to study structural and functional changes associated with hypertension. SHRs develop progressive hypertension by 10 weeks of age, while agematched WKY rats do not w16,21x. Smooth muscle hypertrophy and hyperplasia w22x, enhanced responsiveness of the sympathetic nervous system w12,15,20x and increased noradrenergic innervation of vascular tissues w11x have all been observed in SHRs. Vascular beds receiving dense innervation in adult SHRs express higher levels of NGF during development than the same vascular beds in WKYs w7x. One source of NGF for neurons innervating blood vessels is vascular smooth muscle. Cultured vascular smooth muscle cells ŽVSMCs. from SHRs have a disturbance in NGF regulation that causes heightened NGF
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output when compared to VSMCs from WKYs w27x. Elevated target NGF is thought to play a role in the vascular hyperinnervation that leads to vascular remodeling and ultimately high blood pressure in SHRs w9x. Similarities exist between the progression of changes following bladder outlet obstruction and the development of hypertension in SHRs, both of which are characterized by hyperinnervation. For example, in both cases, elevated tissue levels of NGF are observed prior to changes in innervation w10,32x. The purpose of the present study was fourfold. First, do bladder smooth muscle cells ŽBSMCs. from SHRs contain the same defect observed in VSMCs leading to elevated secretion of NGF? Second, does elevated secretion of NGF by SHR BSMCs parallel increased tissue content? Third, do SHR bladders contain increased noradrenergic innervation as is found in the vasculature? Fourth, if there are neural differences in SHRs when compared to WKYs, are the differences reflected in voiding behavior?
2. Materials and methods 2.1. Bladder smooth muscle cell (BSMC) cultures and conditions SHR and WKY breeders were maintained from breeding stock originally purchased from Taconic Farms. For all of the studies, SHR Ž300–350 g. and WKY Ž400–450 g. adult rats between 14 and 16 weeks of age were screened for resting systolic BP with tail cuff plethysmography w13x. In this procedure, rats were restrained in clear Plexiglas tubes, and ambient temperature was maintained at less than 278C. Each rat was in a restraint tube for at least 10 min before BP was recorded. Precautions were taken to ensure that rats were calm when BP recordings were made. A tail cuff with a photosensitive cell was placed around the base of each rat’s tail. Changes in pressure applied to the cuff were transduced and amplified ŽModel 29, IITC. and the output traced on a chart recorder. Systolic BP values for each rat were based on an average of three to five tracings spaced at least 2 min apart. This is an age after the development of hypertension but before the age of peak BP. An average BP of at least 150 mmHg was required for all SHR and one of not more than 130 mmHg for all WKY. Bladders were dissected from rats following CO 2 euthanasia and placed in filtered Ca2q–Mg 2q-free tyrodes buffer with antibioticsrantimycotics. Bladders were cleaned of large blood vessels, mucosa and connective tissue and cut into small pieces Žapprox. 1 mm.. Minced bladders were then incubated Ž1–2 h. in saline containing trypsin Ž1 mgrml. and collagenase ŽSigma: 1 mgrml.. Tissue was periodically triturated to break up remaining clumps. Following enzymatic digestion, cells were centrifuged, culture medium containing 10% FBS was added,
and the cells were resuspended and plated in culture dishes. For routine culture BSMCs were plated at a density of 5 = 10 3 cellsrcm2 and maintained in growth medium consisting of Dulbecco’s Modified Eagle Medium ŽDMEM; Gibco. supplemented with 10% FBS, streptomycin Ž100 m grml., penicillin Ž100 unitsrml. and amphotericin B Ž250 ngrml; AntibioticrAntimycotic; Sigma.. All cultures were maintained in a humidified chamber containing 5.0% CO 2 at 378C. Cultures were made from six different primary cell lines Ž3 WKY and 3 SHR., each derived from four bladders Ž2 male:2 female rats.. Each set of rats represented the offspring of different breeders. There were no apparent differences between cell lines derived from a particular strain. Only cultures between passages 3 and 6 were used. To confirm the smooth muscle character of the cultured cells, immunohistochemical staining with anti-smooth muscle a-actin specific antibody ŽSigma. was used as previously described w5x. To study the secretion of NGF by smooth muscle, BSMCs were plated into 12-well tissue culture plates at a density of 5 = 10 3 cellsrcm2 and maintained in serum containing medium until confluence Žentire plate covered by cells such that no part of plate was visible with cultures being checked daily.. Once confluent, the culture medium was changed to serum free medium ŽSFM. consisting of growth medium without FBS, supplemented with insulin Ž1 Urml., transferrin Ž5 m grml., and ascorbic acid Ž200 m molrl; Sigma.. Culture medium was changed 48 and 72 h after the initial change to SFM upon which fresh SFM or SFM containing 100 m M forskolin, 100 nM phorbol–12myristate–13-acetate ŽPMA: Sigma., or 1 nM platelet-derived growth factor ŽPDGF: Upstate Biotechnology. was added Žconcentrations based upon literature search.. Each culture well was then sequentially sampled Ž250 m l each time point from the same medium. at 4, 6 and 24 h after treatment. Each experimental paradigm Ž n s 8. was run in triplicate. Following 24 h of treatment, cultures were rinsed with 0.1 molrl phosphate buffered saline ŽPBS., fixed for 20 min with acetic acid:H 2 O:ethanol Ž1:1:20., rinsed with PBS and stored in 70% alcohol prior to staining with bisbenzimide ŽSigma. for determination of cell numbers. For studies in which NGF mRNA levels were examined, BSMCs were plated in 100 mm Ž78.5 cm2 . dishes and grown in serum containing medium for 2 days. Prior to reaching confluence, the medium was changed to SFM and cultures were maintained for an additional 48 h. After 48 h in SFM, fresh SFM was added to the cultures. Culture medium was sampled and cells harvested after 24 h. For each experiment, parallel cultures were established; one culture was fixed for determination of cell number, the other was used for RNA collection. Conditioned medium was collected from both cultures to be assayed for NGF protein. Values are based on five experiments Ž n s 5: mRNA; n s 10: protein.. All experiments were performed on cultures between passages 2 and 5.
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2.2. Conditioned medium NGF determination For the quantification of NGF, cell culture conditioned medium samples, stored at y408C until assayed, were applied directly to ELISA plates. A two-site ELISA for NGF was used as described previously w27x. Briefly, 96well plates ŽNunc. were incubated with a monoclonal antibody raised against NGF ŽBoehringer Mannheim.. Remaining sites were blocked with 1.0% BSA in 50 mmolrl NaCO 3 buffer. After several rinses with wash buffer, 50 m l of conditioned medium or NGF standard Žpurified murine b-NGF kindly provided by E.M. Johnson, Jr., Washington University., freshly diluted in sample buffer immediately prior to each assay, was added to each well. The following day, wells were washed and incubated with an anti-NGF antibody conjugated to b-galactosidase ŽBoehringer Mannheim.. Three 45-min washes followed, after which a fluorescent substrate solution with 4-methylumbelliferyl-8-D-galactopyranoside Ž1 mgr10 ml; Sigma. was added to the wells and allowed to incubate overnight. Plates were read on a Titertek Fluoroscan II ŽFlow Laboratories. plate reader. For each assay, a standard curve was calculated from the known NGF standard concentrations. For cultured smooth muscle cells, the rate of NGF secretion for each sample period Ž0–4, 4–6, 6–24 h. was calculated by subtracting the total amount of NGF at the start of each time period Žconcentration of NGF in conditioned media multiplied by the volume of media. from that present at the end of each sample period and divided by the time between samples. The assay was sensitive to 1.0 pgrml of NGF. Single time point samples from each well were assayed in quadruplicate.
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Triton w X-100, 0.02% sodium azide, 0.1 m grml pepstatin A, 5 m grml aprotinin, 0.5 m grml antipain, 167 m grml benzamidine, 5.2 m grml phenylmethyl sulfonyl fluoride using 6-ml polypropylene tubes. Samples were then basetreated to pH 11 using 4 M NaOH. After centrifugation at 13,000 = g Ž48C, 15 min., the supernatant was acid-treated to pH 3 using glacial acetic acid followed by centrifugation at 13,000 = g Ž48C, 30 min.. The supernatant was then neutralized to pH 7 using 10 M NaOH. After a final centrifugation at 13,000 = g Ž48C, 15 min. the supernatant was assayed using the previously described ELISA for NGF. The samples were vortexed and left undisturbed for 5 min before each centrifugation. 2.4. Determination of cell number Fixed cultures that had been stored in alcohol were rinsed 1 = 5 min with distilled water, 1 = 5 min in PBS followed by incubation in PBS containing 0.5 m grml bisbenzimide ŽHoechst 33258 Dye. for 15 min. Stained nuclei were visualized with a 20 = objective under ultraviolet illumination. The cell number value for each 3.8 cm2 culture well of a 12-well plate consisted of the mean number from nine separate 250,000 m m2 areas each separately framed and counted using NIH Image software. The cell number value for each 100 mm Ž78.5 cm2 . dish consisted of the mean number from 24 separate areas. Each of the areas were the same for every wellrplate. Due to the possible effects of cell number on NGF secretion in a given experiment, rates of NGF secretion were expressed as fg NGF hy1 100 cellsy1 . 2.5. NGF mRNA determination
2.3. Bladder tissue NGF determination Bladders were dissected out of male rats Ž n s 8 WKY and 8 SHR. and frozen at y808C. Bladders were prepared based upon a protocol developed by Zettler et al. w35x. Frozen bladders were finely pulverized in liquid N2 and prepared for 1 min by Polytron w tissue disruption at 1:40 Žwrv. in a high saltrhigh detergent buffer: 100 mM Tris–HCl, 1 M NaCl, 2% BSA, 4 mM EDTA, 2%
Competitive, quantitative reverse transcriptase polymerase chain reaction ŽcqRT–PCR. was used to analyze tissue culture mRNA levels as previously described w23x. In brief, total RNA was extracted using TRIzole ŽGibco., according to manufacturer’s instructions. The amount of NGF mRNA in the isolate was determined with cqRT–PCR utilizing an internal, competitive cRNA standard w23x. First-strand cDNA synthesis was accomplished using Mal-
Table 1 Bladder NGF and norepinephrine content
Body weight Bladder weight Ratio of bladder to body weight NGFrbladder NGFrmg bladder weight NErmg bladder weight NErm g bladder protein
WKY
SHR
430 " 30 g 134 " 8 mg 0.032% 1.8 " 0.08 pgrbladder 0.014 " 0.001 pgrmg 35.7 " 2.2 pgrmg 2.2 " 0.11 pgrm g
328 " 20 g) 100 " 5 mg) 0.031% 3.4 " 0.22 pgrbladder) 0.034 " 0.002 pgrmg) 58.6 " 2.9 pgrmg) 5.30 " 0.32 pgrm g)
Bladder nerve growth factor ŽNGF. content was determined using an acidrbase-based protocol to extract NGF protein into a supernatant which was then analyzed with an ELISA. Norepinephrine ŽNE. content was determined with high-performance liquid chromatography with electrochemical detection. The asterisks Ž). denote significant differences between strains.
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Table 2 NGF mRNA and protein in BSMCs
Total RNA Žpg RNAr100 cells. NGF mRNArtotal RNA Žpg mRNArm g total RNA. NGF mRNA Žpg mRNAr100 cells. NGF secretion Žfg hy1 100 cellsy1 . NGF proteinrmRNA Žfg NGF hy1 pgy1 mRNA.
WKY BSMCs
SHR BSMCs
4.7 = 10 3 " 1.6 = 10 3 2.6 " 1.0 1.8 = 10y2 " 0.7 = 10y2 4.0 " 0.4 223 " 22
7.0 = 10 3 " 1.5 = 10 3 2.2 " 1.1 2.2 = 10y2 " 1.4 = 10y2 9.5 " 1.0) 431 " 46)
Cells were plated in 100 mm tissue culture dishes at a density of 5 = 103 cellsrcm2 . Cultures were maintained in normal growth medium for 48 h, then serum free medium ŽSFM. for 48 h, followed by SFM for 24 h after which the medium was sampled for NGF. Preconfluent, parallel cultures were either fixed for cell counting, or RNA was harvested using TRIzol reagent. The asterisks Ž). denote significant differences between strains.
oney-murine leukemia virus RT ŽGibco. in a mixture of internal standard Ž1 of 4 different bracketing amounts., native mRNA, and the downstream NGF mRNA primer resulting in four separate competitive RT–PCRs. The RT reaction products were used for PCR amplification. Amplified cRNA was analyzed with gel electrophoresis on a 1.3% agaroser0.5 = Tris–borate gel stained with ethidium bromide Ž10 mgrml.. Band intensities were measured from digitized images using an EagleEye system ŽStratagene. and NIH Image software. The ratio of native cDNA to the standard cDNA was plotted Žlogrlog. against the amount of cRNA standard added Ž50, 10, 5, 1 pgrml. at the beginning of the cqRT–PCR. The amount of native NGF mRNA was calculated using linear regression w23x. 2.6. Glyoxylic acid staining Bladders were removed from male SHR Ž n s 5. and WKY Ž n s 5. rats, frozen in cryomolds in a cryostat
Žy308C. and sectioned lengthwise from dome to base at 20 m M. The entire bladder was serially sectioned and kept in order from the first to last slice. As each slide was filled with bladder sections, it was dipped in buffered saline Ž3 = 1 s. containing 200 mM sucrose, 236 mM KH 2 PO4 and 1% glyoxylic acid ŽpH 7.4.. Slides were air dried for 10 to 15 min and placed in an 808C oven for 5 min. After heating, slides were coverslipped with mineral oil, heated on a hot-plate for 90 s to remove bubbles and viewed under ultraviolet illumination on a microphot TMD microscope ŽNikon.. To determine if innervation differed in bladders from SHR and WKY rats, two methods of quantification were used. First, we calculated the percentage of sections containing stained fibers Ž200 = magnification., as well as the percentage of sections containing fibers in the bladder base, bladder body and bladder dome. The entire area of every section of every bladder was examined. It is important to note that perivascular staining was clearly evident and not counted as stained fibers. Second,
Fig. 1. NGF secretion by bladder smooth muscle cells (BSMCs): The effect of 1 nM PDGF, 100 nM PMA or 100 m M forskolin on BSMCs under confluent, serum free conditions quantified from conditioned samples taken 4, 6, and 24 h from the start of each experiment. Cultures were derived from bladders of adult WKYs ŽA. and SHRs ŽB.. Nerve growth factor content of culture medium was determined using an ELISA. The asterisks Ž). denote significant differences from control. The solid circles Žv . represent significant differences between strain control values.
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to examine fiber density and fiber branching, we took photographs Ž20 = objective. of stained fibers. We then projected these images onto a screen, superimposed a grid onto these and counted the number of times fibers crossed the grid lines, giving an estimate of the extent of fiber length and branching in the sections. For quantification, one picture was taken from each of 15 sections from each bladder. Because of the size differences between SHR Žmean s 50 sectionsrbladder. and WKY Žmean s 63 sectionsrbladder. bladders, sections used for pictures were approximately selected in 6.7% serial increments through
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the bladder, i.e., for a bladder with 50 sections the first section used for pictures was the 3rd section while the 4th section was used for a bladder with 63 sections. Because of the high amount of variability in staining density in a specifically defined area of a given section, the picture taken for a section was of the most densely stained area for that section. 2.7. Norepinephrine (NE) analysis For measurement of NE in tissue, male bladders Ž n s 6 WKY and n s 6 SHR. were removed from rats following
Fig. 2. Sympathetic fiber staining in bladder: Bladders were removed from adult SHRs and WKYs. Sections were cut at 20 m M and stained for catecholamine containing fibers using glyoxylic acid. Two methods were used to quantify bladder staining. First, the percent of sections containing positively stained fibers were calculated, as well as the percent of sections containing fibers in the bladder base, bladder body and bladder dome ŽA: the asterisks Ž). denote significant differences between strains.. Second, to examine fiber density and fiber branching, we took photographs Ž20 = . of particular regions containing fibers and superimposed a grid onto these. We then calculated the number of times fibers crossed the grid lines for a given measured area and compared the frequency distributions for WKY and SHR bladders ŽB: the asterisk Ž). denotes that the frequency distributions are significantly different..
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CO 2 euthanasia and snap frozen on dry ice. Frozen samples were weighed and homogenized in 4 = volume of cold 0.2 N perchloric acid. Fifty microliters of homogenate were removed for determination of protein according to manufacturer’s instructions: Bio-Rad protein assay kit. The remaining homogenate was centrifuged at 15,000 rpm for 30 min at 48C. The resulting supernatant was removed and NE and other catechols measured by batch alumina extraction followed by high-performance liquid chromatography with electrochemical detection w14x. 2.8. Voiding studies Rats used for voiding studies were housed in individual cages with free access to food and water Ž30 adult rats: 11 F, 18 M and 10 4-week old rats: 5 F, 5 M for both WKY and SHR.. Animals were kept on a 12-h lightrdark cycle starting at 7:00 AM. Voiding studies were run in mid-light cycle Ž6-h time period: 10:00 AM–4:00 PM.. For the measurement of voiding frequency, SHR and WKY rats were placed in individual metabolic cages ŽNalge. with unrestricted food and water. A graduated cup was attached to an FT03 force transducer ŽGrass. and positioned to collect all urine during the sample period. Void events were amplified using a model 7D amplifier and traced on a polygraph ŽGrass.. Previous testing allowed comparisons between volume of individual voids and the magnitude of graph deflections. 2.9. Statistical analyses Comparisons between two variables in different strains were made using Student’s t-test for independent samples, while comparisons within the same strain were made using Student’s paired t-test. Analysis of NGF mRNA levels and glyoxylic acid staining were made using the Mann–Whitney U-test. Analysis of nerve fiber frequency distributions within bladder sections was performed using the Kolmogorov–Smirnov test. Values shown represent the mean " S.E.M. For all comparisons significance was set to p - 0.05.
3.2. NGF mRNA and protein in cultured BSMCs NGF mRNA levels and the rate of NGF secretion were examined in preconfluent cultures. Although all of the cultures were plated at the same density, SHR BSMCs Ž10 = 10 3 cellsrcm2 . proliferated more rapidly resulting in a greater preconfluent cell number than WKY BSMCs Ž6 = 10 3 cellsrcm2 .. There was no difference between strains in the amount of total RNA or NGF mRNA, but the per cell rate of NGF secretion was significantly higher in SHR than WKY preconfluent cultures ŽTable 2.. Furthermore, SHR BSMCs secreted a significantly greater amount of NGF per unit NGF mRNA ŽTable 2. suggesting that SHR BSMCs are more efficient at translating their mRNA into protein. 3.3. Pharmacology of NGF secretion by BSMCs SHR BSMCs Ž6.3 = 10 4 cellsrcm2 . grew to a greater packing density than did WKY BSMCs Ž3.2 = 10 4 cellsrcm2 . when allowed to grow to confluence. Preconfluent cultures secreted NGF at a higher rate than did confluent cultures. Confluent cultures of SHR BSMCs Ž2.3 " 0.3; 3.5 " 0.6; 2.3 " 0.2. secreted NGF at a higher rate than did WKY BSMCs Ž1.2 " 0.2; 1.6 " 0.3; 0.9 " 0.2 fg hy1 100 cellsy1 . between 0–4, 4–6 and 6–24 h, respectively ŽFig. 1.. Treatment of confluent cultures with PDGF caused a significant increase in NGF secretion between 0–4 h in SHR BSMCs, while increasing NGF secretion between 4–6 h in WKY BSMCs. Direct activation of protein kinase C ŽPKC. signaling pathways with the phorbol ester PMA had no effect on the rate of NGF secretion for either SHR or WKY BSMCs. Stimulation of cAMP-dependent pathways with forskolin decreased the rate of NGF secretion between 4–6 and 6–24 h for both SHR and WKY BSMCs ŽFig. 1.. 3.4. Noradrenergic staining of the bladder Analysis of glyoxylic acid-stained bladder sections revealed that a greater percentage of SHR bladder sections
3. Results
Table 3 Sympathetic nerve fiber density
3.1. NGF and norepinephrine (NE) content of SHR and WKY bladders
Mean grid crossings per quantified area
WKY
SHR
Total Base Body Dome
19.4"1.2 27.2"2.7 16.3"1.3 11.5"2.6
26.7"0.8) 35.6"2.9) 21.4"1.6) 16.6"3.8
Total body and bladder weights were less in SHRs when compared to WKYs. However, the ratio of bladder weight to body weight was identical between the two strains ŽTable 1.. The concentration of NGF calculated as per whole bladder or per mg of bladder wet weight was higher in SHRs ŽTable 1.. The amount of NE determined as either per mg of bladder weight or per m g bladder protein was higher in SHRs ŽTable 1..
Bladders were serially sectioned lengthwise from dome to base at 20 m M and stained with glyoxylic acid, thereby marking catecholamine containing nerves. An estimate of fiber density and branching was examined Ž20=. by counting the nerve fiber crossings of a superimposed grid. For quantification, one viewing area was quantified from each of 15 sections from each bladder. Areas were counted in the bladder dome, body and base.
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contained positively-stained fibers in the dome, body and base than did WKY sections ŽFig. 2A.. Nerve fiber grid crossings of a superimposed grid revealed that regions exhibiting positively stained nerve fibers contained a higher density of fibers in SHR bladders when compared to WKY bladders. Not only were grid crossings higher for SHRs in
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measured areas of the bladder dome, body and base ŽTable 3. but the whole bladder frequency distribution of grid crossings per measured area was significantly different between SHRs and WKYs ŽFig. 2B.. 3.5. Voiding frequency and Õolume in SHR and WKY rats Voiding frequency and volume were examined in 4week old and adult rats. Both 4-week old and adult SHR rats voided significantly more frequently ŽFig. 3A. but at a reduced volume per void ŽFig. 3B. than did age-matched WKYs. No difference between strains was observed in the total volume voided during the 6-h experimental time period ŽFig. 3C.. Due to the design of the experimental apparatus, only approximate measurements of water intake were possible. No differences were observed between strains.
4. Discussion
Fig. 3. Void frequency and Õolume: SHRs and WKYs were placed in individual metabolic cages for a period of 6 h with unrestricted food and water. A graduated cup was attached to a force transducer and positioned to collect all urine during the sample period. Void events were amplified and traced on a polygraph. Previous testing allowed comparisons between volume of individual voids and the magnitude of graph deflections. Mean voiding frequency ŽA., void volume ŽB., and total volume ŽC. are shown. The asterisks Ž). denote significant differences between strains.
Our results show that BSMCs from SHRs contain a similar defect to that found in VSMCs such that cultured SHR BSMCs secrete an elevated level of NGF compared to normotensive WKY BSMCs. SHR bladder tissue NGF content is also elevated when compared to WKY bladders. SHR bladders contain higher concentrations of NE and NGF-dependent catecholamine nerve fibers, suggesting they are hyperinnervated. Disturbances in SHR bladder NGF production and neural innervation are concomitant with SHRs hyperactive voiding behavior and may be an underlying causal factor. Under both preconfluent and confluent conditions, SHR BSMCs secrete more NGF than WKY BSMCs. This finding mirrors results from studies in cultured VSMCs, in which SHR VSMCs secrete NGF at a greater rate than WKY VSMCs w27x. Although the rate of NGF secretion by BSMCs is more than 20 times that of the rate observed for VSMCs, the ratio of NGF secretion by BSMCs from SHRs and WKYs is almost identical to that of VSMCs. It is tempting to suggest that the differences in NGF production between strains by BSMCs and VSMCs in culture reflect differences in innervation in vivo. The effect of PDGF, PMA or forskolin on NGF secretion from VSMCs is markedly different in cultures from SHRs and WKYs w34x. We tested these treatments in the present study to determine if the regulation of NGF secretion is similar in bladder and vascular smooth muscle and to determine if the alterations in regulation found in SHR VSMCs exist in BSMCs. Direct activation of PKC signaling pathways with PMA causes a dramatic increase in the rate of NGF secretion in VSMCs from both SHR and WKY rats that returns to control levels between 8 and 24 h in WKY VSMCs but remains significantly elevated in SHR VSMCs w34x. In contrast, the present study shows
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that treatment of cultures with PMA has no effect on the rate of NGF secretion in either SHR or WKY BSMCs. The cytokine PDGF causes a dramatic increase in the rate of NGF secretion in both SHR Ž3200% of control. and WKY VSMCs Ž1300% control.. This effect is blocked by PKC down-regulation w34x. In BSMCs, PDGF causes only a doubling of the rate of NGF output and is equally effective in cells from SHRs and WKYs. The only difference between strains is that elevated NGF output occurs at an earlier time point in SHR BSMC cultures ŽFig. 1.. The limited effect of PDGF, which acts in part through activation of PKC signaling pathways w8x, and the lack of effect of PMA on NGF secretion by BSMCs may indicate that PKC signaling pathways play less of a significant role in the regulation of NGF production in BSMCs as compared to VSMCs. However, this may not always be the case. PDGF and PMA increase NGF output from Wistar BSMCs 200–400% in the presence of 5% horse serum w24x. Furthermore, control of BSMC NGF secretion by PKC signaling pathways is strongly suggested in culture conditions where mechanical load is applied w3,23x. Moreover, an unknown defectŽs. in mechanical signal transduction leads to elevated NGF output in stretched SHR BSMC Ž1400% control. cultures when compared to WKY BSMC Ž165% control. cultures w3x suggesting there may be multiple irregularities in the regulation of NGF output in SHR smooth muscle. The effect of serum suggests culture conditions and cellular phenotype have an influence on the regulation of NGF secretion. b-Adrenoceptor agonists generally inhibit while aadrenoceptor agonists activate NGF secretion from smooth muscle. Forskolin mimics b-adrenergic stimulation by triggering cAMP elevations by directly activating adenylate cyclase w19x and thus protein kinase A ŽPKA. signaling pathways w17x. Forskolin causes a normal decrease in the rate of NGF secretion by WKY VSMCs, but actually increases the rate of NGF secretion by SHR VSMCs w34x. Forskolin potently inhibits NGF secretion in both SHR and WKY BSMCs suggesting that the defects in adrenergic regulation of SHR VSMC NGF production are not found in BSMCs. The observation that there is little difference in the response of SHR and WKY BSMCs to treatment with PDGF and PMA or following treatment with forskolin may indicate that the abnormal NGF secretion measured in cultured SHR BSMCs is not due to alterations in PKC or PKA signaling pathways. Other mechanisms, such as phospholipase-A2 linked signaling pathways w2x, associated with NGF output may be relevant to altered NGF secretion in SHR BSMCs. Hypertension in SHRs is characterized by an elevation in vascular bed NGF levels associated with noradrenergic hyperinnervation as the disease progresses w7,36x. A similar phenomenon occurs following bladder outlet obstruction. NGF content of obstructed bladders increases dramatically w32x. In a time-dependent manner, elevated NGF is followed by morphological changes in innervation that
include hypertrophy of afferent and efferent neurons supplying the bladder w28,29,32x. Bladder decentralization does not prevent the neuronal size increases induced by obstruction w28x. Enhanced spinal reflex discharges appear after obstruction which are linked to an increase in voiding frequency w31x. Afferent thresholds are lowered by exogenous NGF w18x. Perhaps elevated secretion of NGF by SHR bladder smooth muscle results in bladder hyperinnervation, similar to that of SHR vascular smooth muscle, and bladder hyperinnervation in the SHR is associated with voiding dysfunction resembling that observed with obstructed, hyperinnervated bladders. The present findings demonstrate that this may indeed be the case. Cultured BSMCs from SHRs secrete more NGF than WKY BSMCs, and there is an elevated level of NGF in SHR bladder tissue. SHR bladders exhibit a higher concentration of NE and a greater density of catecholaminergic fibers than bladders from WKY rats suggesting hyperinnervation of NGF-dependent sympathetic nerves. These results support the findings of Tong et al. w33x demonstrating elevated concentrations of sympathetic neurotransmitters in synaptosomal preparations from bladders of SHRs compared to WKYs. Moreover, neuronal hypertrophy occurs in both the postganglionic efferent limb Žmajor pelvic ganglion. and sensory afferent limb Ždorsal root ganglion. of the micturition reflex pathway of SHRs when compared to WKYs, suggesting that both sympathetic and sensory changes in neuronal innervation of the bladder occur in SHRs w4x. Finally, adult SHRs void at a higher frequency than WKYs, but void the same total volume, suggesting enhanced reflexes are the cause rather than increased urine production. The data presented do not isolate the changeŽs. responsible for increased voiding frequency, whether sensory, sympathetic, visceral motor andror central circuits associated with one or more of these. Experiments designed to distinguish among these possibilities could yield valuable information on the determinants of bladder circuit function. The observed changes in voiding frequency do not appear to result solely from hypertension because similar differences are observed in 4-week old SHR and WKY rats, prior to the development of high blood pressure w16x. Increased voiding frequency does not derive from the fact that SHRs have smaller bladders as adult SHRs void more frequently than 4-week old WKYs with smaller bladders and the ratio of bladder to body weight is not different between strains. These findings are consistent with the hypothesis that overexpression of NGF by bladder smooth muscle leads to hyperinnervation of the bladder, resulting in hyperactive voiding behavior in affected rats. If hypertensive states in some humans have causes similar to those in SHRs, one would predict that humans suffering from hypertension may also exhibit alterations in the physiology of bladder filling and emptying. Indeed, patients with hypertension are also more likely to suffer symptoms of prostatism and thus voiding dysfunction w1,25,26x. The
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SHR strain may represent the first genetic model to study neurally mediated hyperactive voiding.
Acknowledgements We would like to thank Pamela Neff for her assistance with RT–PCR, Cheryl Talley for her help in maintenance of the rat colony and blood pressure measurements, and Disheng Men for her help with norepinephrine measurements. This work was supported by the generosity of the taxpayers of the United States of America through the National Institutes of Health Grants DK 45179 and DK 49431 and a grant from the American Heart Association, Virginia Affiliate.
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