Characterization of kinin receptors in human cultured detrusor smooth muscle cells.

BACKGROUND AND PURPOSE: Kinins have an important role in inflammatory cystitis and in animal pathophysiological models, by acting on epithelium, fibroblasts, sensory innervation and smooth muscle. The aim of this study was to characterize the receptors responsible for direct motor responses induced by kinins on human detrusor. EXPERIMENTAL APPROACH: Human detrusor cells from biopsies were isolated and maintained in culture. B(1) and B(2) kinin receptors were characterized by means of radioligand and functional experiments (PI accumulation and PGE(2) release). KEY RESULTS: [(3)H]-[desArg(9)]-Lys-BK and [(3)H]-BK saturation studies indicated receptor density (B(max)) and K (d) values of 19 or 113 fmol mg(-1), and 0.16 or 0.11 nM for the B(1) or B(2) receptors, respectively. Inhibition binding studies indicated the selectivity of the B(1) receptor antagonist [desArg(9)Leu(8)]-Lys-BK and of the B(2) receptor antagonists Icatibant and MEN16132. [DesArg(9)]-Lys-BK and BK induced PI accumulation with an EC(50) of 1.6 and 1.4 nM and different maximal responses (E(max) of [desArg(9)]-Lys-BK was 10% of BK). BK also induced prostaglandin E(2) release (EC(50) 2.3 nM), whereas no response was detected with the B(1) receptor agonist. The incubation of detrusor smooth muscle cells with interleukin 1beta (IL-1beta) or tumour necrosis factor-alpha (TNF-alpha) (10 ng ml(-1)) induced a time-dependent increase in radioligand-specific binding, which was greater for the B(1) than for the B(2) receptor. CONCLUSIONS AND IMPLICATIONS: Human detrusor smooth muscle cells in culture retain kinin receptors, and represent a suitable model to investigate the mechanisms and changes that occur under chronic inflammatory conditions.

Penile carcinoma in patients with genital lichen sclerosus: a multicenter survey.

PURPOSE: In this observational descriptive study we reviewed the histology and the clinical records of 130 patients with LS involving the male genitalia to determine the presence of premalignant or malignant lesions. MATERIALS AND METHODS: A total of 130 male patients (from 1991 to 2001) with genital LS were treated at our centers. Mean patient age at diagnosis was 42.5 years. In all patients with a clinical diagnosis of LS, the histology was reexamined to look for evidence of LS, applying strict histological criteria. All cases of histologically proven epithelial malignancy, namely SCC, VC and EQ, were reviewed to confirm the presence of neoplastic changes and ascertain the degree of SCC differentiation. RESULTS: Of 130 men 11 (8.4%) with genital LS showed premalignant or malignant histopathological features including 7 (64%) with SCC, 2 (18%) with VC, 1 (9%) with EQ and 1 (9%) with SCC associated with VC. In 6 of 11 patients (55%) the histological study showed the presence of epithelial dysplasia. CONCLUSIONS: Survival of patients with penile carcinoma depends on early diagnosis and treatment, and all patients with genital LS should be observed closely to detect the development of neoplastic or preneoplastic lesions as early as possible.

Interim outcomes of dorsal skin graft bulbar urethroplasty.

PURPOSE: We update our interim results of bulbar urethroplasty using a skin graft placed on the dorsal urethral surface. MATERIALS AND METHODS: A total of 45 patients with an average age of 45 years underwent dorsal onlay skin graft urethroplasty between January 1994 and December 2000. Of the patients 23 had undergone an average of 2.6 prior endoscopic procedures (range 1 to 14). Preoperative evaluation include clinical history, physical examination, retrograde and voiding urethrography, and ultrasonography. In all patients the bulbar urethra was opened along its dorsal surface, the graft was sutured, splayed and quilted to the corpora cavernosa, and the urethra was rotated to cover the graft. In all patients was used penile skin as substitution material. Mean graft length was 4.7 cm (range 2.5 to 11). Three weeks after surgery voiding cystourethrography was performed. RESULTS: Average followup was 71 months (range 41 to 110). Clinical outcome was considered a failure when postoperative instrumentation was needed, including dilation. Of 45 cases 33 (73%) were classified as successful and 12 (27%) were failures. The 12 failures were treated with internal urethrotomy (1), end-to-end-anastomosis (1), skin graft urethroplasty (2) and 2-stage urethroplasty (6). Six of the 12 initial failures had a satisfactory final outcome. The remaining 6 patients refused further surgical procedures and received a definitive perineal urethrostomy. CONCLUSIONS: Penile skin grafts used as a dorsal onlay for bulbar urethral reconstruction in a homogeneous series of patients showed a tendency to deteriorate with time. Longer followup is required to compare penile skin with buccal mucosa as substitute materials for bulbar urethral reconstruction.

Intravesical infusion of resiniferatoxin by a temporary in situ drug delivery system to treat interstitial cystitis: a pilot study.

PURPOSE: Interstitial cystitis (IC), a syndrome characterized by motor and sensory dysfunction of the lower urinary tract, represents a diagnostic and therapeutic challenge even to highly skilled physicians. We investigated the technical feasibility and the clinical efficacy of a prolonged intravesical instillation of RTX by in situ drug delivery system in patients with IC. MATERIAL AND METHODS: 5 female patients (mean age 48.7 years) received a prolonged infusion of a saline solution containing 10nM of resiniferatoxin at the flow rate 25microl/h by the MiniMed 407C Infusion Pump (MiniMed Sylmar, CA, USA), connected to sovrapubic 5Fr mono Pigtail catheter, for 10 days. All patients reported frequency, nocturia and urgency, and symptoms of pelvic pain for at least six months. They showed the absence of urinary tract infection within the last three months, the absence of functional disorders of lower urinary tract and no other vesical or urethral pathology. The pre-treatment (PT) frequency/volume (FV) chart and a pain score (VAS score) were recorded. Patients were evaluated after 30 days from the end of infusion (primary end point, PEP) and after three months (secondary end point, SEP). RESULTS: At PEP frequency reduced from 11.3+/-1.39 to 7.4+/-1.51 (p<0.01) and nocturia from 3.6+/-0.54 to 1.2+/-0.44 (p<0.01). A highly significant reduction of pain score was observed at PEP: it decreased to 2.4+/-0.54 from 6.7+/-0.83 (p<0.01). The pain score remained significantly lower at SEP (3.2+/-0.44 p<0.05). Nocturia was also statistically reduced at SEP (1.9+/-0.74) as well as frequency (8.7+/-1.76). No side effects were reported during the infusion as well as after the removal of the catheter. CONCLUSION: The present study demonstrates that the prolonged intravesical instillation of a drug by in situ drug delivery system is a feasible procedure and seems to support the efficacy of RTX in the treatment of IC patients. However further studies are necessary and mandatory to confirm our results and to define the exact action mechanism of prolonged infusion of RTX, the dosage and the treatment schedule.

Urodynamic and clinical evidence of acute inhibitory effects of intravesical nociceptin/orphanin FQ on detrusor overactivity in humans: a pilot study.

PURPOSE: Management of neurogenic incontinence is complex and available treatments are not satisfactory. Nociceptin/orphanin FQ, a recently discovered neuropeptide, has been reported to inhibit the voiding reflex in the rat. These experimental results prompted us to investigate the urodynamic and clinical effects of intravesical instillation of nociceptin/orphanin FQ in humans. MATERIAL AND METHODS: Our study involved 5 normal subjects (group 1) with a mean age of 40.4 years (range 21 to 54) and 9 patients (group 2) 40.4 years (24 to 54). All patients in group 2 presented with detrusor hyperreflexia refractory to standard therapy. They were invited to undergo a filling cystometrogram with saline solution and after 30 minutes, a new one with a solution containing 1 microM. nociceptin/orphanin FQ. The urodynamic parameters that were recorded included bladder capacity, volume threshold for the appearance of detrusor hyperreflexia and maximum bladder pressure. Clinical and urodynamic followup was performed after 15 days. The data were statistically analyzed with 1-way analysis of variance followed by the Dunnett test for multiple comparison considered statistically significant with p <0.05. RESULTS: Intravesical instillation of 1 microM. nociceptin/orphanin FQ in group 1 did not produce significant functional changes. This infusion in group 2 produced a statistically significant increase in mean bladder capacity and volume threshold for the appearance of detrusor hyperreflexia from 164 plus or minus standard deviation (SD) 84 to 301 +/- 118 and 93 plus or minus SD 41 to 231 +/- 104 ml. (p <0.05, respectively). Mean maximum bladder pressure decreased from 79 plus or minus SD 25 to 54 +/- 44 cm. water but was not statistically significant (p = 0.19). After 15 days an absence of clinical improvement was noticed in group 2, and the urodynamic control did not show any significant changes compared to the values before nociceptin/orphanin FQ treatment. No severe symptomatic reactions were observed during infusion of 1 microM. nociceptin/orphanin FQ. CONCLUSIONS: Our results demonstrate that nociceptin/orphanin FQ is able to elicit a robust inhibitory effect on voiding reflex in group 2 but not 1. The ideal dosage, route of administration of nociceptin/orphanin FQ and treatment interval are not yet established.

Long-term outcome of urethroplasty after failed urethrotomy versus primary repair.

PURPOSE: A urethral stricture recurring after repeat urethrotomy challenges even a skilled urologist. To address the question of whether to repeat urethrotomy or perform open reconstructive surgery, we retrospectively review a series of 93 patients comparing those who underwent primary repair versus those who had undergone urethrotomy and underwent secondary treatment. MATERIALS AND METHODS: From 1975 to 1998, 93 males between age 13 and 78 years (mean 39) underwent surgical treatment for bulbar urethral stricture. In 46 (49%) of the patients urethroplasty was performed as primary repair, and in 47 (51%) after previously failed urethrotomy. The strictures were localized in the bulbous urethra without involvement of penile or membranous tracts. The etiology was ischemic in 37 patients, traumatic in 23, unknown in 17 and inflammatory in 16. To simplify evaluation of the results, the clinical outcome was considered either a success or a failure at the time any postoperative procedure was needed, including dilation. RESULTS: In our 93 patients primary urethroplasty had a final success rate of 85%, and after failed urethrotomy 87%. Previously failed urethrotomy did not influence the long-term outcome of urethroplasty. The long-term results of different urethroplasty techniques had a final success rate ranging from 77% to 96%. CONCLUSIONS: We conclude that failed urethrotomy does not condition the long-term result of surgical repair. With extended followup, the success rate of urethroplasty decreases with time but it is in fact still higher than that of urethrotomy.

Urodynamic assessment during intravesical infusion of capsaicin for the treatment of refractory detrusor hyperreflexia.

PURPOSE: Parameters to predict outcome and the urodynamic effects during infusion of capsaicin, seem not to have been assessed in patients with chronic cord injury. We monitored bladder activity urodynamically during infusion of high dosage of capsaicin. MATERIAL AND METHODS: Thirty patients, 18 women and 12 men (average age 29 years, range 20-59 years), suffering from chronic spinal myelopathy, who presented a refractory detrusor hyperreflexia, were studied. They received saline solution containing 10(-3) M capsaicin at a flow rate of 2 ml min(-1) for 15 min (total volume 30 c.c.). The detrusor activity was monitored by a real-time cystometrogram during infusion and 15 min after the end of the infusion itself. New filling cystometrograms were recorded after 30 days and after 6 months. RESULTS: We obtained a clinical and significant urodynamic improvement in 15 of the 30 patients (50%), confirming that intravesical capsaicin may represent a therapeutic option for a selected group of patients suffering from refractory detrusor hyperreflexia due to chronic spinal upper motor neuron lesion. Best results were observed in patients who showed, during the infusion of capsaicin, early uninhibited bladder contractions which disappeared within 10-12 min from the beginning of the infusion (desensitisation). The patients of this group presented a significant increase of mean cystomanometric capacity after 6 months (from 190.7 to 396.7 ml). No significant clinical or urodynamic improvement was observed in the group of patients in whom uninhibited activity of detrusor was recorded for all the time of infusion. CONCLUSION: Our results support the idea of a major complexity of spinal reflex in paraplegic patients and may offer a clue to explain the failure of therapy with capsaicin. The present results support a new approach in the treatment of detrusor hyperreflexia. The ideal dosage and treatment interval are not at present established and further studies are needed to explain substantial differences in the outcome according to different urodynamic responses.

Intravesical resiniferatoxin for the treatment of detrusor hyperreflexia refractory to capsaicin in patients with chronic spinal cord diseases.

OBJECTIVE: Resiniferatoxin (RTX), a substance isolated from some species of Euphorbia, a cactus-like plant, shows pharmacological effects similar to those of capsaicin. We have studied the possibility of treating detrusor hyperreflexia refractory to intravesical capsaicin in patients with chronic spinal cord injuries, thereby providing insight into the mechanism of action of RTX on sensory neurons and its possible future pharmacological and clinical use. MATERIALS AND METHODS: RTX saline solution (30 ml at a concentration of 10(-5) M) was instilled into the bladder of 7 patients with detrusor hyperreflexia, refractory to intravesical capsaicin therapy, and left in place for 30 min. Effects on bladder function were monitored during the treatment and at follow-up (15 days and 4 weeks later). RESULTS: Fifteen days after RTX, the mean cystomanometric capacity increased significantly from 190 ml +/- 20 ml to 407.14 ml +/- 121.06 (p < 0.01), and it remained high four weeks later (421.66 +/- 74.40 p < 0.01). After 15 days, four patients had a pharmacologically induced detrusor areflexia. They emptied their bladders by clean intermittent catheterization. After four weeks, only two patients still had a pharmacologically induced detrusor areflexia. Clinically, three patients remained dry, and the other three reported a significant improvement in their incontinence and symptoms (frequency, urgency and nocturia). CONCLUSIONS: By interfering with sensory unmyelinated fibers, intravesical RTX seems to be a promising treatment option for selected cases of detrusor hyperreflexia. The ideal dosage and treatment interval have not yet been established, and further studies are necessary to confirm our preliminary results.

Iatrogenic external iliac artery disruption during open pelvic lymph node dissection: successful repair with hypogastric artery transposition.

We report the first case of a wide, iatrogenic, proximal disruption of the right external iliac artery, occurring during staging open lymph node dissection for prostate cancer, which was repaired by hypogastric artery transposition. The hypogastric artery was mobilized and rotated anteriorly, and sutured to the distal segment of the external iliac artery. This is a feasible, innovative and safe technique which permits, by a single anastomosis, the secure reconstruction of a vascular axis to the leg when other procedures are not accessible.

Vesical dysfunction in systemic sclerosis (scleroderma)

There have been only a few reports on the involvement of the urinary tract in patients with systemic sclerosis, a disease of the connective tissue characterized by thickening and fibrosis of the skin, abnormality of the small arteries, and involvement of the gastrointestinal tract, heart, lung and kidney. We report the urodynamic assessment and histological examination of 9 women with scleroderma. Three patients voided less than 100 ml. with a significant residual volume and 4 presented with detrusor areflexia during a filling cystometrogram. Histopathological examination in all patients with detrusor areflexia demonstrated the presence of arterial lesions and derangement of the capillary bed of the detrusor musculature. Our data provide evidence for the functional and histological involvement of the bladder in patients with systemic sclerosis.

Characterization of the tachykinin neurokinin-2 receptor in the human urinary bladder by means of selective receptor antagonists and peptidase inhibitors.

The tachykinin (NK2) receptor-mediating contraction of the human isolated bladder to NKA was investigated by studying the affinities of eight structurally different receptor-selective antagonists (linear peptides, cyclic peptides and pseudopeptides, nonpeptide NK2 receptor antagonists). The affinities of the antagonists were compared to those measured for the same ligands at the NK2 receptors previously characterized in the rabbit pulmonary artery and hamster trachea. In the presence of a cocktail of peptidase inhibitors (bestatin captopril and thiorphan, 1 microM each) no significant correlation was found between pA2 values measured in the human bladder vs. those measured in the other two NK2 receptor-bearing preparation. In the presence of the aminopeptidase inhibitor amastatin, however, pA2 values of linear antagonists bearing an N-terminal Asp residue MEN 10,207 and MEN 10,376 were significantly enhanced and these pA2 values were used for analysis; a significant correlation was found between pA2 values measured in the human urinary bladder and rabbit pulmonary artery. The pseudopeptide analog of NKA (4-10), MDL 28,564 which also bears a N-terminal Asp residue behaved as an agonist and its action was enhanced by amastatin. We conclude that the NK2 receptor-mediating contraction of the human urinary bladder smooth muscle is similar to that previously characterized in the rabbit pulmonary artery (NK2A receptor category); in the human bladder smooth muscle an amastatin-sensitive peptidase (possibly aminopeptidase A) limits biological activity of linear peptide derivatives of NKA bearing a N-terminal Asp residue.

Relief of pain following intravesical capsaicin in patients with hypersensitive disorders of the lower urinary tract.

We have extended our earlier observations on pain relief produced by intravesical instillation of capsaicin (10 microM in saline) in patients with hypersensitive disorders of the lower urinary tract. Patients in group A (n = 15) received intravesical capsaicin on days 0, 14 and 28: on each occasion the drug produced a warm or burning sensation, reduction in bladder capacity and a delayed, transient improvement or disappearance of symptoms. Patients in group B (n = 5) received intravesical capsaicin (10 microM at cystometry) 3 times on day 0. The initial sensation of warmth was experienced on each occasion, indicating that no significant desensitisation has been produced by the first instillation. Clinical improvement similar to that found in group A was observed. Three patients (group C) received warm saline (42 degrees C) at cystometry. This produced a pricking sensation, no change at cystometry and no subjective clinical improvement. Apart from the initial sensation of warmth, no patient in group A or B experienced side effects, either local or systemic. These findings confirm that intravesical instillation of capsaicin has a beneficial effect on patients with hypersensitive bladder disorders. Counter-irritation rather than desensitisation of primary afferents could be a possible mechanism of action. Further studies are needed to establish whether the intravesical administration of capsaicin or capsaicin-like agents represents a new form of treatment for relief of bladder pain.

NK2 tachykinin receptors and contraction of circular muscle of the human colon: characterization of the NK2 receptor subtype.

The contractile effect of substance P, neurokinin A, receptor selective agonists for tachykinin receptors and NK2 tachykinin receptor antagonists was investigated in mucosa-free circular strips of the human isolated colon. Neurokinin A and substance P produced concentration-dependent contractions which approached 80-90% of the maximal response to carbachol. Neurokinin A was about 370 times more potent than substance P. The action of neurokinin A and substance P was not modified by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). The NK2 receptor selective agonist, [beta-Ala8]neurokinin A-(4-10) closely mimicked the response to neurokinin A while NK1 and NK3 receptor selective agonists were active only at microM concentrations. The pseudopeptide, MDL 28,564, which is one of the most selective NK2 ligands available, behaved as a full agonist. Responses to [beta-Ala8]neurokinin A were antagonized by NK2 receptor selective antagonists, with the rank order of potency MEN 10,376 greater than L 659,877 much greater than R 396. These data indicate that NK2 tachykinin receptors play a dominant role in determining the contraction of the circular muscle of the human colon to peptides of this family. The NK2 receptor subtype responsible for this effect belongs to the same subtype (NK2A) previously identified in the rabbit pulmonary artery and guinea-pig bronchi.

Ruthenium red as a selective capsaicin antagonist of the motor response to capsaicin in the human isolated ileum.

We have investigated the effect of ruthenium red, omega-conotoxin fraction GVIA (CTX) and tetrodotoxin (TTX) on the relaxation produced in the circular muscle of the human isolated ileum by capsaicin, electrical field stimulation (EFS) or vasoactive intestinal polypeptide (VIP). Ruthenium red (10 microM) selectively blocked the capsaicin-evoked relaxation while leaving the response to EFS or VIP unaffected. CTX had no significant effect on the various stimuli. TTX blocked the relaxation due to EFS but not that due to capsaicin or VIP. It is concluded that capsaicin excitation of primary afferents in the human ileum, leading to VIP release and muscle relaxation, occurs with mechanisms similar to those operating in animal tissues and that ruthenium red acts as a selective capsaicin antagonist in the human ileum.

Direct evidence for the involvement of vasoactive intestinal polypeptide in the motor response of the human isolated ileum to capsaicin.

Capsaicin (1 microM) produced complex motor responses in longitudinal and circular muscle strips from the human isolated small intestine (jejunum and ileum). In the longitudinal muscle, inhibition of the nerve-mediated contractions (electrical field stimulation) was the dominant response, while capsaicin had a weak and inconsistent effect on tone and spontaneous activity. In contrast, relaxation and decreased spontaneous activity were the responses of the circular muscle to capsaicin. These effects of capsaicin were not reproduced by a second application of capsaicin, indicating desensitization, a feature of the specific action of this drug on sensory nerves. All the effects of capsaicin in the longitudinal and circular muscle were closely mimicked by exogenous vasoactive intestinal polypeptide (VIP). Further, the inhibitory motor effect of capsaicin in both muscle layers was blocked by an anti VIP serum. In the longitudinal muscle, VIP, like capsaicin, inhibited the electrically evoked nerve-mediated contractions but not the tetrodotoxin-resistant myogenic contractions, suggesting a prejunctional site of action. The inhibitory effect of both capsaicin and VIP in the circular muscle was tetrodotoxin-resistant suggesting direct inhibition of muscle cells. Capsaicin (1 microM) evoked a tetrodotoxin-resistant release of VIP-like immunoreactivity from the human small intestine. On high pressure liquid chromatography, a major peak of the immunoreactive material released by capsaicin co-eluted with authentic VIP and a minor, unidentified peak eluted shortly afterward. We conclude that authentic VIP is involved in the local motor response to capsaicin in the human small intestine. These findings raise the possibility that VIP might be present in sensory nerves of the human gut from which it is released by capsaicin.

Human isolated ileum: motor responses of the circular muscle to electrical field stimulation and exogenous neuropeptides.

(1) Circularly-oriented muscle strips from the human ileum responded to electrical field stimulation (1-50 Hz) with frequency-related primary relaxation at low frequency and primary contractions at high frequencies of stimulation. Both responses were abolished or markedly reduced by tetrodotoxin (1 microM). (2) Atropine (3 microM) or omega conotoxin (0.1 microM) reduced but dit not abolish contraction to electrical field stimulation and enhanced the relaxation. Omega conotoxin (0.1 microM) did not affect carbachol-induced contraction nor isoprenaline-induced relaxation. (3) Neurokinin A and substance P (1 nM-1 microM) produced a concentration-dependent contraction. The NK-1 receptor selective agonist, [Pro9]SP sulfone and the NK-2 receptor selective agonist [beta Ala8]NKA(4-10) produced a contraction superimposable to that of substance P and neurokinin A, respectively. On the other hand, [MePhe7]-neurokinin B, an NK-3 receptor selective agonist was ineffective up to 1 microM. The response to substance P or neurokinin A was unaffected by atropine (3 microM). (4) Galanin, up to 0.1 microM, produced a weak and inconsistent contraction. (5) Vasoactive intestinal polypeptide (10 nM-1 microM) produced a concentration-dependent relaxation while human alpha calcitonin gene-related peptide exerted a weak and inconsistent relaxant effect. (6) These findings indicate that both cholinergic excitatory and non-cholinergic inhibitory nerves affect the motility of the circular muscle of the human small intestine. Transmitter release from excitatory nerves seems largely mediated by activation of omega conotoxin-sensitive (N-type) calcium channels. Tachykinins exert a potent contractile effect, independently of cholinergic nerves, via NK-1 and NK-2 receptors.

Motor response of the human isolated colon to capsaicin and its relationship to release of vasoactive intestinal polypeptide.

The aim of this study was to obtain indirect evidence of the presence of capsaicin-sensitive afferents in the human colon by studying the motor response to capsaicin of longitudinal strips from the human isolated taenia coli in parallel to the ability of capsaicin or KCl to induce peptide release from the human superfused colon. Capsaicin (1 microM) evoked a relaxation of the taenia, approaching 60-80% of the response to isoprenaline. Tachykinins evoked contractions of the taenia, while calcitonin gene-related peptide induced a relaxation. Neither tachyphylaxis to calcitonin gene-related peptide nor preincubation with an anti-calcitonin gene-related peptide serum did block the response to capsaicin which was also unaffected by tetrodotoxin, apamin, naloxone or an anti-galanin serum. Vasoactive intestinal polypeptide produced a concentration-dependent tetrodotoxin-resistant relaxation which was shifted rightward in the presence of anti-vasoactive intestinal polypeptide serum. The anti-vasoactive intestinal polypeptide serum reduced the response to capsaicin and application of capsaicin prevented the ability of anti-vasoactive intestinal polypeptide serum to block exogenous vasoactive intestinal polypeptide. Capsaicin (1 microM) evoked a significant release of vasoactive intestinal polypeptide-like immunoreactivity from the superfused muscle but not mucosa of the human colon. A significant vasoactive intestinal polypeptide-like immunoreactivity release was also observed in response to KCl (80 mM). KCl but not capsaicin evoked a significant release of neurokinin A-like immunoreactivity from colonic muscle and mucosa. No significant release of either substance P-, neuropeptide Y-, galanin- or calcitonin gene-related peptide-like immunoreactivity was detected in response to capsaicin or KCl although detectable levels of each peptide were evident in tissue extracts.(ABSTRACT TRUNCATED AT 250 WORDS)

Contractile responses of the human urinary bladder, renal pelvis and renal artery to endothelins and sarafotoxin S6b.

1. Endothelin-1 (ET-1), endothelin-3 (ET-3) and sarafotoxin S6b (SRFTX) produced a concentration-dependent tonic contraction of the human isolated urinary bladder, renal pelvis and renal artery with threshold at nM concentration. 2. In the bladder, the following order of potency was found: ET-1 greater than SRFTX greater than ET-3. In the renal pelvis, all peptides displayed similar affinity but, at high concentrations the maximal response was highest for SRFTX followed by ET-1 and ET-3. In the renal artery ET-1 and SRFTX were about equipotent and equieffective while ET-3 produced only a slight and inconsistent (2 out of 5 cases) vasoconstrictor response. 3. As shown previously for the human bladder muscle, the response to ET-1 in the renal pelvis was nifedipine (1 microM)-resistant while a consistent fraction of the response was blocked by nifedipine in the human renal artery. 4. These findings indicate that peptides of the endothelin family exert a potent contractile effect on various human smooth muscles. Participation of dihydropyridine- and voltage-sensitive calcium channels in the contractile response produced by these peptides may vary from one organ to another.

Multiple sources of calcium for contraction of the human urinary bladder muscle.

1. KCl, carbachol, neurokinin A and endothelin produced concentration-dependent contractions of mucosa-free muscle strips from the dome of the human urinary bladder. The maximal response to carbachol or neurokinin A exceeded that to KCl, while the maximal response to endothelin approached that to KCl. 2. Nifedipine (1 microM) abolished the response to KCl, reduced the response to carbachol or neurokinin A but had no effect on the response to endothelin. Bay K 8644 (1 microM) markedly potentiated the response to KCl but had little or no effect on the response produced by the other stimulants. 3. Superfusion of the strips with a nominally calcium (Ca)-free medium containing EDTA (1 mM) for 30 min markedly reduced the response to carbachol, neurokinin A and endothelin, although a small response was still evident at high concentrations. Likewise, after a prolonged (60 min) superfusion of the strips with a high K (80 mM) Ca-free medium plus EDTA (1 mM) these three agonists still produced a small contractile response. 4. The nifedipine (1 microM) resistant response to carbachol, neurokinin A or endothelin was markedly depressed by LaCl3 (1 mM). In contrast, the nifedipine-(1 microM) resistant response to carbachol was not modified by NiCl2 (0.1 mM) or omega-conotoxin (0.1 microM). 5. Caffeine produced divergent effects depending upon the temperature of incubation: a relaxation at 37 degrees C and a concentration-dependent (2.5-20 mM) contraction at 25 degrees C. The latter was markedly inhibited by procaine (3 mM) but unaffected by nifedipine (1 microM). 6. After a prolonged (60 min) superfusion with a high K, Ca-free medium containing EDTA the response to carbachol (100 microM) was abolished by previous exposure to procaine (3 mM). Conversely, the response to endothelin (1 microM) was unaffected by procaine. The response to endothelin in these experimental conditions was also resistant to LaCl3 (1 mM). 7. These findings indicate that multiple sources of Ca are mobilized for contraction of the human bladder muscle by different stimulants. Dihydropyridine- and voltage-sensitive Ca channels provide the major if not the sole source of Ca for the response to KCl, play some role in the response to muscarinic (carbachol) or NK-2 tachykinin receptor stimulation but are not involved in the response to endothelin. Carbachol, neurokinin A and endothelin all mobilize a Ca pool (either extracellular or located at membrane level) which is LaCl3-sensitive but nifedipine-resistant. Neither T- nor N-type channels appear to be involved in the response to carbachol. In addition, these agents mobilize a tightly bound Ca pool independently from membrane depolarization. This latter pool is probably a procaine-sensitive intracellular source of activator Ca mobilized by caffeine and carbachol. The failure of procaine to prevent the response to endothelin in high K, Ca-free medium raises the possibility that this peptide mobilizes an intracellular source of activator Ca, distinct from the caffeine- and carbachol-sensitive pool.