Nitric Oxide Inhibits Contraction of Isolated Pig Ureteral Smooth Muscle

0022-5347/96/1552-0763$03,00/0 THE JOLWNAL OF UROLOGY Copyright 0 1996 by AMERICAN UROLOCICAL ASSOCIATION.INC.

Val. 155,763-767, February 1996 Printed in U S A

NITRIC OXIDE INHIBITS CONTRACTION OF ISOLATED PIG URETERAL SMOOTH MUSCLE C. E. ISELIN,* P. ALM, N. C. SCHAAD, B. LARSSON, P. GRABER

AND

K.-E. ANDERSON

From the Urology Clinic, Department of Surgery, and Department of Psychiatry, Geneua University Hospital, Geneua, Switzerland, and the Departments of Pathology and CZinical Pharmacology, Lund Uniuersity Hospital, Lund,Sweden

ABSTRACT

Purpose: To investigate the L-arginindnitric oxide (NO) pathway in the pig isolated ureter. Materials and Methods: Functional inhibitory effects mediated by NO were assessed and correlated with cyclic nucleotide levels. Nitric oxide synthase (NOS) activity was measured by monitoring the conversion of [3H]-arginine to [3H]-citrulline. Immunohistochemical studies were performed. Results: The NO-donor SIN-1 reduced in a concentration-dependent manner the frequency of contractions, whereas NO completely interrupted the contractile activity. In precontracted strips exposed to SIN-1 or NO, there were 6- and 12-fold increases of the cyclic GMP levels in comparison with control preparations. Activity of NOS was moderate. Overall innervation of the ureter was sparse, and there were few NOS-immunoreactive nerves. Conclusion: Although few NOS-containing nerves were found, pathways regulating the cyclic GMP levels of pig ureteral smooth muscle were demonstrated. Such pathways may be important targets for drugs producing relaxation of the mammalian ureter. KEYWORDS: muscle, smooth; nitric oxide; muscle contraction Functional and biochemical studies. Preliminary studies showed that the tissue was viable as long as 30 hours after harvesting. However, the majority of the experiments were conducted within 12 hours. Longitudinal ureteral strips of approximately 6 x 2 x 1 mm. were prepared. Silk threads were attached to both ends. Preparations were then transferred to small perfusion chambers (internal volume 0.18 ml.) superfused with Krebs solution maintained at 37C by a thermoregulated water circuit. The Krebs solution was bubbled with a mixture of 95% 0, and 5% CO,, maintaining pH at 7.4. The strips were connected by one of the silk ligatures to Grass Instrument F'T03C force-displacement transducers (Grass Instruments, Quincy, Massachusetts) for registration of isometric tension. The transducer output was recorded on a Gould Polygraph model 2400 (Gould Instruments, Oxnard, California) or a Grass Polygraph model 7H. The preparations were initially stretched to a tension of 10 mN and then left for an equilibration period of at least 45 minutes. When agents with a very short half-life, such as NO, were tested, the preparations were placed in 5-ml. organ baths to allow direct application of the drug. To study inhibitory effects, the preparations were precontracted with 5-HT (serotonin). Due to the development of a pronounced tachyphylaxis of the tissue to this agonist, 2 consecutive concentration-response curves could not be conMATERIALS A N D METHODS structed in the same preparation. However, the frequency of Ureters were taken from young pigs (3 to 6 months old) at the contractions induced by a single concentration of 5-HT or 3 X M.) was reproducible for as many as 8 a local slaughterhouse. Urinary bladders with attached ureters were removed within a few minutes after death and exposures (variability limits: see analysis of data). 5-HT was placed in cold (4C) aerated (5% CO, in 0,) Krebs solution. superfused for 1 minute and then the preparation was Specimens from the lower third of ureter (at least 3 cm. washed for 29 minutes. In organ baths, 5-HT was present for proximal to the bladder) were dissected, and care was taken about 3 minutes; there was an interval of 90 minutes beto remove adjacent connective and fatty tissues. tween the applications of 5-HT. The NO-donor SIN-1 (3morpholino-sydnonimin hydrochloridell ) and the cyclic GMP Accepted for publication August 25, 1995. * Re uests for reprints: Urology Clinic, Department of Surgery, specific phosphodiesterase inhibitor zaprinast were added 3 and 20 minutes before the 5-HT-induced contraction. To com%pita? Cantonal Universitaire, 1211 Geneva 14,Switzerland. This work was supported by the Swiss National Science Founda- pletely recover the 5-HT response after incubation with tion (Grant 34-40751.94), by the De ,Fteuter Foundation, Geneva, SIN-1, the washing period of the superfusion chambers was Switzerland, and by the Swedlsh Medlcd Research Council (grants extended to 1 hour. When evaluating its threshold concen6837 and 11205). 763

There has been considerable debate whether ureteral peristalsis is a myogenic or neurogenic phenomenon. Ureteral smooth muscle has no discrete neuromuscular junctions; it is considered as a syncytium in which excitation spreads electronically from one muscle cell to another.',, However, ureteral nerves have been demonstrated in many mammals, including man3.4 and pig," and the common trend is at present to consider that ureteral innervation is at least modulatory.5 Calcitonin-gene related peptide (CGRP)6-S and substance p6 have been considered likely candidates for nonadrenergic noncholinergic (NANC) inhibitory neurotransmission in mammalian upper urinary tract smooth muscle. However, NO (nitric oxide) seems to be a n important mediator of NANC neurotransmission in various smooth muscles, including the lower urinary tract.9 So far, the functional importance of NO in the upper urinary tract has not been well described although NO synthase (NOS) positive nerves have been identified in the human3 and mouse ureter.l0 The aim of this study was to assess the possible functional effects of NO on the pig ureter, and their correlation with cyclic nucleotide levels. In addition, the immunohistochemical occurrence of NOS-immunoreactive nerves and of NOS enzyme activity was studied.

764

NO INHIBITS CONTRACTION OF PIG URETERAL SMOOTH MUSCLE

tration, NO was added in the organ baths 30 to 60 seconds mtibodies. As cross-reactions with antigens sharing similar after the beginning of the 5-HT induced phasic contraction. xmino acid sequences cannot be excluded, the structures When quantifying the NO concentration-response effect, all lemonstrated are referred to as NOS- or PGP-IR (immunothe concentrations were added 30 seconds after the begin- reactive). Drugs and solutions. The following drugs were used: seroning of the phasic contraction. Electrical field stimulation was performed by means of a tonin hydrochloride, prazosin hydrochloride, zaprinast, tetroGrass S8 or S88 stimulator (Grass Instruments) connected to dotoxin, N"-nitro-Garginine (Sigma); ketanserin tartrate 2 platinum electrodes. These were circular (3 mm. diameter) (Janssen Pharmaceutica, Beerse, Belgium). SIN-1 (3-morand fixed at each end of the superfusion chambers (16 mm. pholino-sydnonimin hydrochloride) was kindly supplied by apart). In the organ baths, 3 mm. long electrodes were used, Dr. R. Henning (Cassella AG, Frankfurt, Germany); it was mounted parallel to the preparations 4 mm. apart. Square kept in dark vessels to minimize light-induced degradation. wave pulses of 0.2 to 0.8 msec. duration were delivered at a Nitric oxide was prepared by flushing 99.5% NO gas (AGA frequency of 0.5 to 80 Hz.The voltage was supramaximal, gas AB, Stockholm, Sweden) through a sealed bottle for 1 hour. Isotonic saline was deoxygenated by flushing it with and the train duration was 1to 10 seconds. After registration of mechanical activity in the organ 0,-free helium for 1hour through a sealed bottle. The NO gas baths, the preparations were rapidly frozen in liquid nitro- was aspirated with a syringe and injected into the deoxygengen, and the concentration of cGMP (guanosine 3'5' cyclic- ated Krebs solution.17 A saturated solution of NO (3 X lo-' monophosphate) and CAMP(adenosine 3'5' cyclic-monophos- M.) was obtained by adding 5 ml. NO gas to 20 ml. deoxyp h t e ) were determined as detailed elsewhere.12 The content genated isotonic saline solution. The Krebs solution used had the following composition of cyclic nucleotides in the strips was measured 1)during the 5-HT-induced contractile activity, 2) at the moment of com- (mM.):NaCl 119, NaHCO, 15, KC1 4.6, NaH,P04 1.2, MgC1, plete abolition of this activity (to baseline level) by NO (2 x 1.2, CaCl, 1.5 and glucose 11. Analysis of data. The average contraction frequency of the M.)and 3) while induced contractile activity was decreased by SIN-1 (3 x M.).The samples were harvested controls before and after testing of the inhibitory agent was 30 to 45 seconds aRer the beginning of the contraction, when calculated. If the frequencies of both control contractions were within 5% of this average, the inhibitory effect was the relaxant effect of NO or SIN-1 was maximal. Nitric oxide synthase assay. Ureteral strips of approxi- validated. Preparations with a higher variation of frequency mately 20 x 5 x 1mm. were prepared and homogenized (4C) were discarded. The inhibitory effects of NO and SIN-1 on the with a polytron (10 to 15 seconds) in a buffer (1 to 2 ml.) 5-HT-induced contractile activity were expressed as percent containing N-[2-hydroxyethyllpiperazine-N'-[2-ethanesul- of the average calculated above. Results are given as mean values 5 the standard error of fonic acid] (HEPES; 50 d.), pH 7.4 at 25C and ethylenediamine tetraacetic acid (EDTA; 1 mM.). Ion-exchange resin the mean; n denotes the number of ureters. Probability val(AG5OWX-8, sodium form, 100 mg./g. of tissue) was then ues were determined by a two-tailed Student's t test considadded. After 5 minutes, the suspension was centrifuged ered significant if lower than 0.05.In the case of multiple (12,000 g, 30 minutes, 4C) to remove tissue debris and resin. comparisons between groups, corrections according to the Nitric oxide synthase activity was essayed in triplicate in a Bonferroni method were performed. 10 ml. homogenate by measuring the conversion of [,HIarginine to [3H]-citrulline in the presence of Ca2' (3.75 mM.), RESULTS EDTA (1mM.), flavin adenine dinucleotide (FAD; 10 mM.), Functional and biochemical studies. Seventy-nine percent flavin mononucleotide (FMN,10 mM.), 5,6,7,8-tetrahydro- of 177 strips from 166 ureters responded to 5-HT. Of these, biopterin (100 mhf.), calmodulin (10 mgJml.), arginine (25 73% displayed a stable contractile activity with a contraction mM.), dithiothreitol(1 mM.) and nicotinamide adenine dinu- frequency of over 10 per minute (figs. 1 and 2, left tracings) cleotide phosphate (NADPH; 1mM.), according to our modi- and were used for the experiments. No tachyphylaxis was fication13 of the method of Bredt and Snyder.14 observed. The 5-HT, receptor blocker ketanserin (lo-' M.) h t e i n quantification was determined by a dye-binding completely inhibited the 5-HT-induced contractions (n = 6; method with bovine serum albumin as a standard. results not shown). This inhibition was totally reversible Zmmunohistochemical studies. Tissue specimens were im- after washing of the preparations for 1 hour. Since ketanmersion fixed, rinsed and frozen, whereupon cryostat sec- serin may p a r t i d y antagonize smooth muscle a,-adrenoceptions were cut and further processed as previously de- tors, we tested the effect of the a,-adrenoceptor antagonist acribed.16 In brief, the sections were first incubated in prazosin on the 5-HT-induced contractions. Prazosin (2 X phosphate buffered saline (PBS) with 0.2% Triton X-100 for 2 M. to M.) did not produce any inhibition of this hours at Mom temperature and then incubated overnight at 4C with a rabbit antiserum to PGP (protein gene product 9.5; 1:2000, Ultraclone, Wellow, Isle of Wight, United Kingdom), 5HT w or a rabbit antiserum to NO synthase (1:1280).15 After rins5HT w ing in PBS, the sections were incubated for 90 minutes with t t t t F'ITC (fluorescein is0thiocyanat.e)-conjugatedgoat anti-rabbit immunoglobulins (1:80; Sigma Chemical Co., St. Louis, Missouri), rinsed and mounted. The immunoreactive nerve structures were subjectively evaluated with respect to type (nerve trunks and nerve terminals) and number. In the evaluation of the latter parameter, immunoreactivity for PGP was used as an internal standard for total innervation. Protein gene product is a 4cytoplasmic protein that can be used as a marker of all types control 3 mln SN-1 (3x101 M) of nerve fibers.16 The sections were examined in an Olympus 3 x 50 system FIG. 1. Original tracing showin effect of SIN-1on isolated pig M. ring 60 seconds) induces phasic microscope, equipped with epi-illumination. In control exper- ureter. On left, 5-HT(3 X contractile activit (control). On right, contraction frequency ie iments, no immunoreactivities could be detected in sections markedlx diminided after preincubation (3 minutes) with SIN-1 incubated with primary antisera absorbed with excess of the (3X 10- M.). In this preparation, 5-HTinduced contractile r e s p o m respective antigens (100 mg./ml.) or with only the secondary with some delay.

765

NO INHIBITS CONTRACTION OF PIG URETERAL SMOOTH MUSCLE wn

t

W

5-M

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t

W

Responses of preparations of the prerontracted isolated pig ureter to different nitrir ox& (NO) concentrations.

NO concentration

M.

Abolition of contraction (n)

p

~

~

No;kct ~ ~

_ _ ..-.... ..

0

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M. 4 X 10.' M. 8 X lO-'M. 2 x 10-4 M.

4 9 1 4 7 2 1 10 7 0 0 7 17 2 0 19 (N denotes the number of strips). The preparations responded in an all or none type of way, which made it impossible to quantify a concentrationresponse relationship beyond the threshold concentrations. 2 X

CCiltml

FIG. 2. Original tracing showing effect of NO on isolated preconM.during tracted preparation of pig ureter. On left, 5-HT(3 x 2 minutes 15 seconds) induces phasic contractile activity (control). M.) interrupts this activity. On right, NO (2 X

contraction ( n = 5; results not shown), nor did tetrodotoxin M.; n = 7; results not shown). The NO donor SIN-1 (3 X lo-" to 3 X M.) reduced in a concentration-dependent fashion the frequency of contractions induced by 5-HT (figs. 1 and 3). This inhibition was totally reversible a t each concentration tested, The control contraction frequency was reduced to about half its maxiM. mum by SIN-1 3 x Nitric oxide had distinct inhibitory effects on the ureteral contractions induced by 5-HT (fig. 2). It completely interrupted the contractions for a concentration-dependent (4 X lo-' M. and 2 x M.) time interval ranging between 10 and 30 seconds (n = 10). I t acted always within 10 seconds of its administration and its effect was reversible. When contractions reappeared, the frequency was initially diminished in comparison t o the control. The threshold concentration of NO for complete abolition of the 5-HT-induced contractions was 4 X lop5 M. (table). The cGMP specific phosphodiesterase inhibitor zaprinast inhibited the 5-HT-induced contraction with a threshold concentration of lo-' M. (fig. 4). The inhibitory effect of SIN-1 (lo-" M.; figs. 3 and 4) was enhanced 2.5-fold (p <0.001) in the presence of the threshold concentration of zaprinast (fig.

M.)and SIN-1 (lo-' M.) sepaFIG. 4. Effects of zaprinast rately and together on phasic contractions of isolated pig ureter induced by 5-HT (3 x 10-6 M.), bsul~ are expressed as percent of average contraction frequen of control and given as mean 2 standard error of mean (verticalyam). n = 8 to 9. *** = p
(19.11 -t 3.04 pMJmg. protein and 39.59 5 3.79 pM./mg. protein) in comparison to the control strips (p <0.001;fig. 5). Cyclic AMP levels of strips exposed to SIN-1 and to NO were not significantly different in comparison to control ( n = 7-8). Nitric oxide synthase assay. Activity of NO synthase was determined in 6 ureters. I t averaged 63 z 13 pmol./mg.

501

-6

-5

4

ttt 7

-3

Concentration SIN-1 ( log [MI )

FIG. 3. Concentration-dependent inhibitory effect of SIN-1 (3 X M.) on phasic contractions of isolated p1g ureter M. to 3 X induced by 5-HT (3 x lo-' M.). Results are expressed as percent of average contraction frequency of control before and after treatment, and given as mean 2 standard ermr of mean (vertical bars). n = 7 to 10.

Control

SIN-1

NO

FIG. 5. Effects of SIN-1(3 X 10 -' M.) and nitric oxide (NO 2 x M.) on cyclic GMP concentrations of isolated precontraded strips of pig ureter. Concentrations of cGMP are e reseed in pMI mg. protein, and given as means ? standard ermr 3 m e a n (vertical bars). n = 7 to 8 for each column. *** = p <0.001 (versus control).

l

766

NO INHIBITS CONTRACTION OF PIG URETERAL SMOOTH MUSCLE

of the cGMP phosphodiesterase selective inhibitor zaprinast, given in a threshold concentration for relaxation, the relaxant effect of SIN-1was significantly increased. Comparable results have been obtained in lower urinary tract smooth muscle with a clearly defined NO pathway.9 Our experimental work had been completed when we became aware of a preliminary study reporting inhibition of pig ureteral contraction by NO, associated with an increase of cGMP (Bfold) in the presence of an inhibitor of its degradation.lg These results strengthen the present evidence of a NO pathway in the pig ureter. The present study confirmed previous morphologic findings on the small amount of overall innervation of the pig ureter.4 Moreover, it revealed that, as in the human ureter? there are NOS-IR nerves in the pig ureter; however, these were few in comparison to the overall innervation. To further evaluate the importance of ureteral NOS,measurement of its DISCUSSION activity was performed by monitoring the conversion of L3H]The present study demonstrated that exogenous NO tem- arginine to [SH]-citrulLine. This investigation showed that porarily abolished 5-IiT-induced contractions of pig ureteral NOS exerted catalytic activity in the pig ureter. This NOS smooth muscle. However SIN-1,at the maximum concentra- activity was constitutive, since it was Ca2+-dependent. It M.),did not inhibit more than 50% of the cannot be excluded that the Ca2+-independentactivity detion used (3 x response. to 5-€lT.This may be &bed to the fact that, as in tected in 3 preparations may be ascribed to subclinical infecvascular smooth muscle,*l SIN-1may produce contracting tion. Under the same experimental conditions, the activity in factors such as eupemxide anions in the pig ureter. the pig urethra, which has a prominent NO pathway,g was It is well eetablished that NO relaxes smooth muscle by found to be 7-fold higher than in the ureter (unpublished activating guanylate cyclase, which increases the intracellu- observations). lar cGMP concentration. This was found also in the present These immunohistochemical and biochemical findings sugstudy, which showed that NO and SIN-1markedly increased gest the presence of NOS in pig ureteral nerves. However, 2 (respectively 12-and Sfold) the cGMP levels. In the presence observations in this study question the neural origin of NO. First, the scarce nitrergic innervation observed contrasts with the marked functional and biochemical effects that were found after exposure to exogenous NO. The disproportion between the small amount of NOS-IR nerves and NOS activity may be due to methodological limitations of the immunohistochemistry. Although scarce, this does not exclude that the nitrergic innervation of the upper urinary tract may be physiologically relevant. The ureter is a syncytial smooth muscle that spreads its excitation electronically from cell to ceU,1.2 and coordinated motility may not need an extensive neural network. Therefore, a physiologically relevant NO pathway may not require a high density of NOS-IR nerves in the pig ureter. Second, the electrically-induced relaxations were not inhibited by tetrodotoxin, which blocks the Na'channels that initiate neurotransmitter release. These relaxations were also resistant to the NOS inhibitor Nu-nitro& arginine. However, electrical stimulation may directly influence the smooth muscle of the ureter, interfering with the contractile effect of 5-HT. Whereas the physiological relevance of NO in the ureter remains to be proven, its clear-cut inhibitory effects on 5-HTinduced contractions support the view that drugs acting through an increase in cGMP, such as nitroglycerin and specific cGMP phosphodiesterase inhibitors, may have a clinical application in the treatment of renal colic and ureteral spasm."O Because the systemic route is unlikely to be u s e l l for NO-donors, such agents will probably require a local (intraureteral) administration. thereby limiting their indications to ureteral spasm relief during ureteral endoscopic surgery. In conclusion, this study shows that NO effectively inhibits the contraction of isolated pig ureter. This effect is associated with an increase in the cGMP levels in the smooth muscle cells. The sparse presence of NOS-IR nerves and the low levels of NOS activity in the ureter raise questions about the physiological relevance of the ureteral LarpinineNO pathha. 6. Pig ureter, smooth m d a t u r e . A. few PGP-IR nerve way. However guanylate cyclase stimulation and inhibition fibers (arrows). B, single gracile NOSIR varicose terminals (arrows); of cGMP PhosPhodiestermes seem to be key mechanisms for putative drugs inhibiting ureteral contraction. FlTC fluorescence micrqlraphs. x 190 and 3715.

protein per hour. The Ca"-dependency of this activity was assessed in 12 preparations. In the absence of Ca2+, NOSactivity averaged 8.6 % 5.8% of the total activity (Ca2+independent activity was detected in 3 preparations). Zrnrnunohistochernicalstudies. In the ureter, the number of FGP-IR nerves was sparse (fig. 6, A) compared with the pig urinary bladder and urethra.'8 Few varicose nerve terminals were observed in relation to smooth muscle bundles, and single coarse nerve trunks ran in the surrounding connective tissue. Vessels, mostly arteries, in all locations were often encircled by varicose nerve terminals. No PGP-IR nerve structure could be detected in relation to the urothelium. In comparison to PGP-IR nerves, NOS-IR nerves were s m , with single varicose terminals following smooth muscle bundles (fig. 6, B)and vessels. mostly arteries. No coarse NOSIR nerve trunks were seen.

NO INHIBITS CONTRACTION OF PIG URETERAL SMOOTH MUSCLE

Acknowledgernents. We thank Brita Sunden for performing the cyclic nucleotide assays. Many thanks also to J d l e Manot, Bent Holmskov, Dominique Mastrangelo, Lars Ny and Petter Hedlund for helpful technical assistance. REFERENCES

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