Marcia A. Wheeler

BACTERIAL INFECTION INDUCES NITRIC OXIDE SYNTHASE IN HUMAN NEUTROPHILS

The identification of human inflammatory cells that express inducible nitric oxide synthase and the clarification of the role of inducible nitric oxide synthase in human infectious or inflammatory processes have been elusive. In neutrophil-enriched fractions from urine, we demonstrate a 43-fold increase in nitric oxide synthase activity in patients with urinary tract infections compared with that in neutrophil-enriched fractions from noninfected controls. Partially purified inducible nitric oxide synthase is primarily membrane associated, calcium independent, and inhibited by arginine analogues with a rank order consistent with that of purified human inducible nitric oxide synthase. Molecular, biochemical, and immunocytochemical evidence unequivocally identifies inducible nitric oxide synthase as the major nitric oxide synthase isoform found in neutrophils isolated from urine during urinary tract infections. Elevated inducible nitric oxide synthase activity and elevated nitric oxide synthase protein measured in patients with urinary tract infections and treated with antibiotics does not decrease until 6-10 d of antibiotic treatment. The extended elevation of neutrophil inducible nitric oxide synthase during urinary tract infections may have both antimicrobial and proinflammatory functions.

A comparison of multiple urine markers for interstitial cystitis.

PURPOSE We measured several urine markers in 24-hour specimens from patients with interstitial cystitis and healthy controls. For each marker we determined whether the urine level was significantly different in interstitial cystitis and control cases, and whether the marker level correlated with the symptom score. MATERIALS AND METHODS Study participants included 36 female patients with interstitial cystitis and 36 age matched female volunteers. Multiple urine aliquots were obtained to measure the various markers. RESULTS Certain markers were significantly increased in interstitial cystitis, including anti-proliferative factor, epidermal growth factor, insulin-like growth factor (IGF) binding protein-3 and interleukin (IL)-6. Markers significantly decreased in interstitial cystitis were heparin-binding epidermal growth factor-like growth factor, cyclic guanosine monophosphate and methylhistamine. Other markers were not significantly different in the interstitial cystitis and control groups, including total glycosaminoglycans, epitectin, hyaluronic acid, IL-8, IL-1 and nitrates plus nitrites. IGF-1 was undetectable in 24-hour urine samples but spot voided samples from the same interstitial cystitis population had IGF-1 levels similar to previously reported levels. The only significant association of marker with symptom score was a positive correlation of IL-6 with nocturia. For all markers the conclusions were the same whether the marker was normalized to creatinine or to 24 hours. CONCLUSIONS This study confirmed several previously reported urine alterations in interstitial cystitis, including increased anti-proliferative factor, epidermal growth factor, IGF binding protein-3 and IL-6, and decreased heparin-binding epidermal growth factor-like growth factor and cyclic guanosine monophosphate. Of all markers studied anti-proliferative factor had the least overlap in the interstitial cystitis and control groups, and so it is the most likely candidate to become a diagnostic test.

NG-nitro-L-arginine inhibits non-adrenergic, non-cholinergic relaxation in rabbit urethral smooth muscle

Electrical field stimulation induced a relaxation response in female rabbit urethral smooth muscle strips precontracted with phenylephrine. The relaxation response was inhibited by tetrodotoxin, but not by atropine, propranolol, or hexamethonium. The relaxation response thus results from stimulation of inhibitory non-adrenergic, non-cholinergic nerves. The electrically induced relaxation response was inhibited by an inhibitor of nitric oxide biosynthesis, NG-nitro-L-arginine. This inhibition was overcome by addition of a precursor of nitric oxide, L-arginine. An inhibitor of soluble guanylate cyclase, methylene blue, reduced the relaxation response, and a selective cyclic GMP phosphodiesterase inhibitor, M & B 22948, potentiated the relaxation response. These data indicate that agents which affect the biosynthesis of nitric oxide are associated with the urethral relaxation response evoked by electrical field stimulation, and that cyclic GMP may mediate the relaxation response.

INDUCIBLE NITRIC OXIDE SYNTHASE WITH TRANSITIONAL CELL CARCINOMA OF THE BLADDER

PURPOSE Nitric oxide (NO) plays a critical role as both a cell signaling molecule and as a cytotoxic/cytostatic mediator. Nitric oxide synthase (NOS) present in macrophages and neutrophils produces NO in response to immune stimulation. We evaluated NO production in both bladder tissue and urine from patients with transitional cell carcinoma (TCC) of the bladder. MATERIALS AND METHODS Inducible NOS (iNOS) RNA and protein were evaluated in bladder tissue from patients with and without TCC. Human iNOS-RNA products were identified with the reverse transcriptase-polymerase chain reaction (RT-PCR). Western blot analysis using a polyclonal antibody directed against iNOS recognized immunoreactive iNOS protein. Using the same iNOS antibody, the distribution of iNOS was examined in formalin-fixed, paraffin embedded samples of various grades of TCC. NOS activity was measured in the urine particulate fraction from patients with TCC and from controls by the conversion of [14C]-L-arginine to [14C]-L-citrulline. RESULTS Inducible NOS-RNA products and iNOS specific proteins were found in bladder tissue that contained TCC but not in control bladder tissue. Inducible NOS was uniformly localized in inflammatory cells within the carcinomas. Scattered tumor cells expressed iNOS in 8 of 12 specimens. There was no clear relationship between tumor immunoreactivity and tumor grade. NOS activity in urine from patients with TCC was not significantly elevated or decreased in comparison with control urine. CONCLUSIONS Inducible NOS is expressed by cells comprising and surrounding human bladder tumors. It is primarily localized to inflammatory cells, but also is demonstrated within individual tumor cells.

Urinary Nitric Oxide Synthase Activity and Cyclic GMP Levels are Decreased with Interstitial Cystitis and Increased with Urinary Tract Infections

PURPOSE Since urinary nitric oxide synthase (NOS) activity correlates with certain disease process affecting the urinary tract and since nitric oxide increases cyclic GMP levels by activating guanylyl cyclase, urinary particulate NOS activity and cyclic GMP levels are evaluated in female patients with interstitial cystitis (IC) and compared with those from female controls and female patients with urinary tract infections (UTIs). MATERIALS AND METHODS Urinary NOS activity is measured as the formation of [(14)C]-L-citrulline from [(14)C]-L-arginine, and urinary cyclic GMP levels are measured by an [(125)I]-radioimmunoassay. RESULTS Female patients with IC have significantly less NOS activity in their urine pellet particulate fractions than female control females UTIs, 2.3 +/- 1.0, 14 +/- 3.0, and 120 +/- 10 pmol. citrulline formed/min./mg. protein. Urinary cyclic GMP levels are significantly lower in IC patients than in female controls or females with UTIs: 0.50 +/- 0.06, 0.82 +/- 0.14. and 3.72 +/- 0.81 micromol. cyclic GMP/g. creatinine. CONCLUSIONS Regulation of urinary NOS activity with subsequent changes in nitric oxide and cyclic GMP may be an important determinant of symptoms and immunologic responses to UTIs and IC.

A Randomized Double-blind Trial Of Oral L-arginine For Treatment Of Interstitial Cystitis

PURPOSE Nitric oxide synthase activity is decreased in the urine of patients with interstitial cystitis compared to the urine of controls. In a preliminary trial oral L-arginine, the substrate for nitric oxide synthase, increased urinary nitric oxide synthase activity and improved interstitial cystitis symptoms. This randomized, double-blind, placebo controlled study further investigates the efficacy of L-arginine treatment for interstitial cystitis. MATERIALS AND METHODS A total of 53 interstitial cystitis patients were assigned to receive daily 1,500 mg. L-arginine or placebo orally for 3 months. Interstitial cystitis symptoms were assessed by interviews at 2 weeks, and 1, 2 and 3 months. RESULTS The trial was completed by 21 of 27 patients in the L-arginine group and 25 of 26 in the placebo group. Using per protocol analysis 29% (6 of 21 patients) in the L-arginine group and 8% (2 of 25) in the placebo group were clinically improved by the end of the trial (p = 0.07). A Likert scale showed greater global improvement in the L-arginine group (48%, 10 of 21) compared to the placebo group (24%, 6 of 25) at 3 months (p = 0.05) with a decrease in pain intensity (p = 0.04), and tendency toward improvement in urgency (p = 0.06) and frequency of pain (p = 0.09). Using an intention to treat approach to analysis there were no differences between groups. CONCLUSIONS Oral L-arginine (1,500 mg. daily) may decrease pain and urgency in a subset of interstitial cystitis patients.

Improvement in Interstitial Cystitis Symptom Scores During Treatment With Oral L-Arginine

PURPOSE Urinary nitric oxide synthase activity is decreased in patients with interstitial cystitis. Since nitric oxide may be an important determinant of the symptoms and immunological responses associated with interstitial cystitis, patients with this disease were treated with oral L-arginine, the substrate for nitric oxide synthase. MATERIALS AND METHODS Ten patients took 1.5 gm. L-arginine orally daily for 6 months. Interstitial cystitis symptoms were surveyed before and during the 6-month trial. RESULTS Oral L-arginine treatment resulted in a significant decrease in urinary voiding discomfort, lower abdominal pain and vaginal/urethral pain. Urinary frequency during the day and night also significantly decreased. CONCLUSIONS This self-controlled study provides evidence that long-term oral L-arginine improves interstitial cystitis related symptoms.

Urine Markers Do Not Predict Biopsy Findings or Presence of Bladder Ulcers in Interstitial Cystitis/Painful Bladder Syndrome

PURPOSE We tested for associations between urine markers, bladder biopsy features and bladder ulcers in interstitial cystitis/painful bladder syndrome. MATERIALS AND METHODS Subjects were 72 patients with interstitial cystitis/painful bladder syndrome undergoing bladder distention and biopsy. Urine was collected before the procedure. Urine marker levels were correlated with biopsy and cystoscopic findings. Patients with no previous interstitial cystitis/painful bladder syndrome treatments (47) were analyzed separately from previously treated patients (25). RESULTS For untreated patients urine interleukin-6 and cyclic guanosine monophosphate were associated with urothelial epidermal growth factor receptor staining (for interleukin-6 r = 0.29; 95% CI 0.07, 0.51; p = 0.01 and for cyclic guanosine monophosphate r = 0.34; 95% CI 0.13, 0.55; p = 0.002). Urine interleukin-8 was negatively associated with urothelial heparin-binding epidermal growth factor-like growth factor staining (r = -0.34; 95% CI -0.55, -0.12; p = 0.002) and positively associated with lamina propria mast cell count (r = 0.29; 95% CI 0.06, 0.52; p = 0.01). The latter association also was seen in treated patients (r = 0.46; 95% CI 0.20, 0.73; p <0.001). None of the urine markers was significantly different for ulcer vs nonulcer groups. All of the patients with ulcer had extensive inflammation on bladder biopsy including severe mononuclear cell infiltration, moderate or strong interleukin-6 staining in the urothelium and lamina propria, and leukocyte common antigen staining in more than 10% of the lamina propria. However, these features also were seen in 24% to 76% of the patients without ulcer. CONCLUSIONS Overall urine markers did not associate robustly with biopsy findings. The strongest association was a positive association between urine interleukin-8 levels and bladder mast cell count. Patients with ulcer consistently had bladder inflammation but the cystoscopic finding of ulcers was not a sensitive indicator of inflammation on bladder biopsy.

Involvement of nitric oxide and cyclic GMP in rabbit urethral relaxation

Soluble and particulate fractions from rabbit urethra converted [14C]arginine to [14C]citrulline, indicating the presence of nitric oxide synthase activity in these fractions. Both soluble and particulate nitric oxide synthase activities were NADPH dependent, and the soluble activity was Ca2+ dependent. Three nitric oxide synthase (NOS) inhibitors affected transmural nerve stimulation induced relaxation responses in the rabbit urethra and the activity of soluble nitric oxide synthase with the same rank order of potency, i.e., NG-nitro-L-arginine (NNA) > NG-methyl-L-arginine (NMA) > canavanine (CAN). The rank order of potency with respect to particulate NOS activity was CAN > NMA = NNA. The relaxation responses to electrical stimulation were accompanied by increases in cyclic GMP. These results suggest that NOS activity found in the soluble fraction of urethral homogenates produces nitric oxide that in turn increases cyclic GMP levels which mediates the relaxation responses induced by transmural nerve stimulation in the rabbit urethra.

Age-dependent Alterations in beta-adrenergic Responsiveness of Rat Detrusor Smooth Muscle

The relaxant effects of norepinephrine (NE, 10(-7) to 10(-4) M.) and isoproterenol (ISO, 10(-9) to 10(-4) M.) on maximal KCl-induced tonic contractions and the relaxant effects of ISO on contractions induced by electrical field stimulation (EFS) were measured in detrusor muscle strips obtained from 22-25 day, 90-95 day and 22-month-old male Fischer 344 rats. The maximum relaxant response to NE and ISO on KCl-induced tonic contractions decreased significantly with increasing age. The ED50 values for ISO, but not NE, increased with age. The maximum relaxant response to ISO on EFS-induced contractions also was reduced significantly in the old bladders. The relaxation effects of forskolin (10(-6) to 3 x 10(-5) M.), dibutyryl cyclic AMP (DBcAMP, 10(-4) to 3 x 10(-3) M.) and cholera toxin (10 micrograms/ml.) were examined on maximal KCl-induced contractions of the muscle strips obtained from the three age groups. The relaxant responses to forskolin decreased significantly with increasing age, whereas DBcAMP relaxed the muscle strips from the three age groups equally. Cholera toxin (10 micrograms) attenuated KCl-induced phasic contractions, and this effect was impaired in the aged rat detrusor. The density of beta-adrenergic receptors, as determined by radioligand binding with [125I]iodopindolol ([125I]-PIN) decreased with increasing age. These data demonstrate an age-related decrease in the responsiveness of the bladder detrusor to beta-adrenergic stimulation that may be related to the decreased density of beta-adrenergic receptors and decreased cyclic AMP (cAMP) production.