Effects of carbonyl cyanide p-trichloromethoxyphenylhydrazone (CCCP) and of ruthenium red (RR) on capsaicin-evoked neuropeptide release from peripheral terminals of primary afferent neurones

Summary In the superfused isolated rat urinary bladder, capsaicin as well as electrical field stimulation evoked the release of calcitonin gene-related peptide-like immunoreactivity (CGRP-IR). Carbonyl cyanide p-trichloromethoxyphenylhydrazone (CCCP, threshold 2 M) reduced both, the capsaicin- and the electrical field stimulation-evoked release of CGRP-IR while a low concentration of Ruthenium Red (RR, 0.6 M and 2 M) selectively attenuated the capsaicin-evoked release of CGRP-IR but did not influence the effect of electrical field stimulation. 20 M RR nearly abolished the capsaicin-evoked release, but also attenuated the effect of electrical field stimulation.In the isolated guinea-pig bronchus, electrical field stimulation and capsaicin induced non-cholinergic contractions which are known to be caused by tachykinin release from afferent nerve terminals. CCCP (0.6 M) only reduced the response to field stimulation; a ten-fold higher concentration of CCCP attenuated field stimulation as well as capsaicin-induced contractions. This is in contrast to the reported selective inhibition of capsaic-ininduced contractions by RR.The present data demonstrate that CCCP generally inhibits evoked neuropeptide release, regardless of the kind of stimulation used while low concentrations of RR preferentially inhibit capsaicin-evoked neuropeptide release.Send offprint requests to: R. Amann at the above address     

Effect of Bay K 8644 and ryanodine on the refractory period,action potential and mechanical response of the guinea-pig ureter to electrical stimulation

We have investigated the effect of the dihydropyridine calcium channel agonist, Bay K 8644, and of the plant alkaloid blocker of calcium-induced calcium release (CICR) from the sarcoplasmic reticulum, ryanodine, on the refractory period, action potential and mechanical response of the guinea-pig isolated ureter to electrical stimulation. All experiments were performed in ureters pre-exposed to 10 M capsaicin to eliminate the inhibitory influence exerted by local release of sensory neuropeptides on ureteral excitability and contraction. In organ bath experiments, electrical field stimulation with parameters which produce direct excitation of ureteral smooth muscle (train of pulses at 10 Hz, 5 ms pulse width, 60 V for 1 s) produced tetrodotoxin- (1 M) resistant phasic contractions. The response to EFS was abolished by nifedipine (1 nM-3 M) and was enhanced by Bay K 8644 (1 nM-3 M). In the presence of Bay K 8644 (1 M), nifedipine (30 M) abolished the evoked contractions. Ryanodine (10–100 M) had no significant effect on the amplitude of evoked contraction. The response of the guinea-pig ureter to direct electrical stimulation of smooth muscle is characterized by a refractory period: at least 40 s interstimulus interval was required to produce a second response in all preparations tested. Bay K 8644 (1 M) markedly reduced the refractory period of the ureter and a similar effect was observed with ryanodine (100 M). To further analyze the effect of Bay K 8644 and ryanodine on the refractory period, the response of the ureter was investigated over a 10 s period of stimulation (other parameters as above). In control ureters, continuous stimulation for 10 s produced only one phasic contraction just after the beginning of the train of stimuli. In the presence of Bay K 8644 or ryanodine, more than one phasic contraction developed during a 10 s stimulation, i.e. the refractory period became shorter than the train duration. When both Bay K 8644 and ryanodine were tested on the same preparations, an additive excitatory effect was observed on the mechanical response to electrical stimulation. A slight elevation of KCI concentration (5–10 mM) reduced the refractory period of the ureter as observed with ryanodine or Bay K 8644. Application of KCI (80 mM) produced a biphasic contractile response of the ureter: a series of phasic contractions occurred first, which were then replaced by a slowly developing tonic response. Bay K 8644 (1 M) enhanced both components of the response to KCI. Ryanodine (10 and 100 M) markedly prolonged the duration of phasic contractions evoked by KCI and, at 100 M, slightly (about 25%) reduced the amplitude of tonic contraction.In sucrose gap experiments, electrical stimulation (single pulse, 40–130 V, 1–3 ms pulse duration) evoked an action potential and accompanying phasic contraction which were abolished by 1 M, nifedipine. Bay K 8644 (1 M) produced a marked prolongation of action potential duration, increased the number of spikes and enhanced contraction amplitude and duration. Ryanodine (100 M) depolarized the membrane, reduced the delay between stimulus application and onset of the action potential, shortened the action potential at 50% of repolarization and increased afterhyperpolarization, without producing marked effects on the accompanying mechanical response. KCI (5 mM) likewise produced a slight membrane depolarization and decreased latency between stimulus application and onset of the action potential but did not affect action potential duration. The combined administration of ryanodine and Bay K 8644 produced additive effects on action potential and contractions: furthermore, the contractile phase of the overall contraction-relaxation cycle was significantly prolonged by the combined administration of the two agents, an effect not observed with either drug alone. In the presence of both Bay K 8644 and ryanodine, multiple action potentials and contractions were observed during a train of pulses delivered at a frequency of 1 Hz for 12 s: when a second action potential was triggered before relaxation of the preceding contraction, a summation of the contractile response was observed. These findings demonstrate that availability of voltage-dependent L-type calcium channels is a major mechanism in determining the refractory period of the guinea-pig ureter and, consequently, can be considered as a limiting step in regulating the maximal frequency of ureteral peristalsis. Furthermore, a ryanodine-sensitive mechanism regulates the excitability and contraction-relaxation cycle of ureteral smooth muscle. The increased electrical excitability of the ureter observed in the presence of ryanodine may involve blockade of transient outward currents triggered by spontaneous calcium release from the store and consequent membrane depolarization.     

Effect of ω-conotoxin on cholinergic and tachykininergic excitatory neurotransmission to the circular muscle of the guinea-pig colon

The aim of this study was to compare the stimulus-response characteristics of the cholinergic and tachykininergic excitatory transmission to the circular muscle of the guinea-pig proximal colon and their susceptibility to inhibition by the N-type calcium channel blocker -conotoxin (CTX). All experiments were performed in the presence of guanethidine (3 M), indomethacin (10 M), L-nitroarginine (L-NOARG, 30 M) and apamin (0.1 M). In the presence of the tachykinin receptor antagonists, FK 888 (10 M) and GR 94800 (3 M), to block NK₁ and NK₂ receptors, respectively, electrical field stimulation (EFS) produced frequency-dependent atropine- (1 M) sensitive contractions. In the presence of atropine (1 M), EFS produced tachykininergic contractions which were abolished by the combined administration of FK 888 (10 M) and GR 94 800 (3 M). The maximal responses produced by cholinergic and tachykininergic neurotransmission ranged between 80 and 100% of the maximal contractile response to 80 mM KCI. The frequency of stimulation, pulse width and voltage required to produce 50% of the maximal cholinergic and tachykininergic contraction were not different from each other, although cholinergic transmission appeared more efficient in producing twitch contractions in response to single pulse EFS. Furthermore, cholinergic transmission was more efficient than tachykininergic transmission in producing contraction in response to short periods of EFS.CTX (0.1 M for 30 min) produced a large and comparable rightward shift of the cholinergic and tachykininergic frequency-response curve (19 and 17 fold increase in the frequency of stimulation producing 50% of the maximal response, respectively) and markedly depressed (51 and 43% inhibition, respectively) the maximal concentrations response. CTX failed to affect the contraction of the colon produced by submaximally effective concentrations of the muscarinic receptor agonist, methacholine (0.1–0.3 M) and those produced by the tachykinin NK₁ and NK₂ receptor selective agonists [Sar⁹] substance P sulfone and [\Ala⁸] neurokinin A (4–10) (1–3 nM).The present findings demonstrate that the cholinergic and tachykininergic components of the excitatory transmission to the circular muscle of the guinea-pig colon are activated at comparable intensities of nerve stimulation and are both inhibited, in a qualitatively and quantitatively comparable manner, by CTX at the prejunctional level. These findings are consistent with the idea that acetylcholine and tachykinins are co-released from the same population of enteric motoneurones which innervate the circular muscle of the colon. Correspondence to: C. A. Maggi at the above address     

Tachykinin receptors mediate atropine-resistant rat duodenal reflex contractions in vivo

The study aimed to establish the possible role of tachykinins as mediators of atropine-resistant reflex contractions evoked by balloon distension in the proximal duodenum of urethane-anesthetized, guanethidine (34 mol/kg s.c.)-pretreated rats. Distension of the balloon with a small amount (0.2–0.3 ml) of saline induced the appearance of phasic rhythmic contractions (about 11 mmHg in amplitude) which were promptly suppressed by either atropine (3 mol/kg i.v.) or hexamethonium (28 mol/kg i.v.). Despite the continuous i.v. infusion of atropine (2 mol/h), low-amplitude rhythmic phasic contractions recovered, which were promptly suppressed by hexamethonium, to indicate the involvement of an atropine-resistant excitatory reflex. The amplitude of these atropine-resistant contractions was increased to about 4–5 mmHg by further distension of the balloon (0.4–0.6 ml) : under these conditions, the atropine-resistant contractions undergo a progressive fading. The fading was prevented by i.v. administration of the nitric oxide (NO) synthase inhibitor, L-nitroarginine methyl ester (L-NAME, 55 mol/h), to provide a suitable baseline (amplitude of contractions was 7–8 mmHg) for studying the effect of tachykinin receptor antagonists.I.v. administration of the selective tachykinin NK₂ receptor antagonists, MEN 10,627 (10–100 nmol/kg) and SR 48968 (100–300 nmol/kg) or of the selective NK₁ antagonist SR 140333 (100 nmol/kg), at doses which do not affect the duodenal contractions induced by acetylcholine (5.5 µmol/kg i.v.), produced a prompt and long lasting suppression of the atropine-resistant reflex duodenal contractions produced by balloon distension in urethane-anesthetized rats, whilst SR 48965 (300 nmol/kg), the enantiomer of SR 48968 devoid of NK₂ receptor blocking activity, was without effect.I.v. administration of the selective NK_i receptor agonists [Sar⁹] substance P sulfone and septide or of the NK₂ receptor selective agonist, Ala⁸] neurokinin A(4–10) produced dose-dependent contractions of the duodenum. SR 140333 (100 nmol/kg i.v.) selectively antagonized the duodenal contractions produced by [Sar⁹] substance P sulfone and septide without affecting those produced by [Ala⁸] neurokinin A(4–10). On the other hand, MEN 10,627 (30–100 nmol/kg i.v.) and SR 48968 (100–300 nmol/kg i.v.) but not SR 48965 (300 nmol/kg i.v.) antagonized, at a comparable extent, duodenal contractions induced by both the selective NK₂ and NK₁ receptor agonists.We conclude that endogenous tachykinins are involved in mediating atropine-resistant reflex contractions evoked by distension of the rat duodenum in vivo: both NK₁ and NK₂ receptors are activated by endogenous ligands to produce NANC contractions of rat duodenum in vivo. However, the contractile response to i.v. administered NK₁ receptor agonists, [Sar⁹] substance P sulfone and septide, may involve the release of mediators producing smooth muscle contraction via NK₂ receptors.     

Vascular endothelial growth factor and p53 expressions in liver and abdominal metastases from colon cancer.

OBJECTIVE: To determine the relationship between p53 overexpression and vascular endothelial growth factor (VEGF) upregulation in liver and abdominal metastases from colon cancer. The analysis in the two metastatic sites was carried out to evaluate the potential role of microenvironment in the molecular regulation of VEGF. METHODS: Bioptic specimens of liver and abdominal metastases from colon carcinomas were examined by immunohistochemistry for p53 and VEGF expressions. Consecutive cases with assessable tumor tissue were selected. RESULTS: The study population consisted of 24 cases having liver metastases and 34 cases having abdominal metastases. Abdominal metastases showed a higher number of VEGF-positive cases and a higher intensity of VEGF immunoreactivity than liver metastases did (p = 0.01). The combined analysis of p53 and VEGF showed a strong association between the two markers in the 24 liver metastases; 9 cases were VEGF positive/p53 positive and 15 cases were VEGF negative/p53 negative. This relationship was not found in the 34 abdominal metastases, which showed concordance between the two markers in 9 VEGF-positive/p53-positive cases only. CONCLUSIONS: Microenvironment factors like hypoxia may have a predominant role in inducing VEGF expression and they can override the molecular control of p53 on VEGF.     

A Clinical and Histologic Comparison of Percutaneous Dilational Versus Conventional Surgical Tracheostomy

To directly compare percutaneous dilational tracheostomy (PDT) with conventional surgical tracheostomy, a prospective study was performed in 83 patients requiring tracheostomy for prolonged mechanical ventilation in the intensive care unit or after surgery for a large tumor in the upper respirodigestive tract. Median follow-up was 355 days after PDT and 338 days after conventional tracheostomy. The overall morbidity rate was significantly lower with PDT than with conventional tracheostomy (6.4% vs 36.1%; P < 0.001). Compared with conventional tracheostomy, PDT was also associated with a significantly lower incidence of postoperative bleeding (2.1% vs 13.9%; P < 0.05) and postoperative wound infection (0% vs 22.2%; P < 0.001). There were no clinical signs of laryngotracheal stenosis in either group. In conclusion, PDT is a simple, fast, safe bedside procedure that is associated with significantly lower morbidity than standard surgical tracheostomy.     

Surgical scar endometriosis after Cesarean section: a case report.

BACKGROUND: Cutaneous endometriosis is a rare condition. CASE REPORT: A 37-year-old woman came to our observation 3 years after Cesarean section for a nodule under the scar that became spontaneously painful during menstrual bleeding. Transabdominal ultrasound examination, serum CA125 determination and histopathological analysis of the nodule were performed. Ultrasound revealed the presence of an oval-shaped hypoechogenic neoformation, while the serum CA125 level was slightly increased, and a diagnosis of endometriosis was confirmed by the histopathological analysis of a surgical specimen. CONCLUSION: This is an interesting case of surgical scar endometriosis, and the etiopathogenetic mechanism of this location may be explained by a dissemination of endometrial tissue during the Cesarean section.     

The 'QUAD SHOT'--a phase II study of palliative radiotherapy for incurable head and neck cancer.

BACKGROUND AND PURPOSE: The primary objective of this study was to estimate the rate of tumour response to a cyclical hypofractionated palliative radiotherapy regimen (QUAD SHOT) in previously untreated patients with incurable squamous cell carcinoma of the head and neck. Secondary objectives were to prospectively evaluate toxicity, quality of life (QoL) and survival in these patients. PATIENTS AND METHODS: The QUAD SHOT consisted of 14 Gy in four fractions, given twice a day and at least 6h apart, for 2 consecutive days. This regimen was repeated at 4 weekly intervals for a further two courses if there was no tumour progression. The QoL tool used was an abbreviation of the EORTC QLQ-C30. RESULTS: Thirty eligible patients (29 Stage IV, 20 performance status 2-3) had at least one treatment and 16 patients completed all three cycles. Sixteen patients (53%) had an objective response (2CR, 14PR) and a further seven had stable disease. Median overall survival was 5.7 months, median progression free survival was 3.1 months. The treatment was very well tolerated, with improved QoL in 11 of 25 evaluable patients (44%). CONCLUSION: The QUAD SHOT regimen is an effective palliative treatment with minimal toxicity and a good response rate, which impacts positively on patients' QoL.     

Optimal treatment of poor-risk renal cell carcinoma patients with mTOR inhibitors and anti-VEGFR agents

Poor-risk metastatic renal cell carcinoma (RCC) includes a subgroup of patients with unfavorable prognosis, according to both the Motzer and Heng criteria. Overall, owing to the poor prognosis of these patients, the approach is still a challenge for the first and subsequent lines of treatment, particularly for rare histologies other than clear cell renal cell carcinoma. In this review, we investigated the present treatment option of poor-risk metastatic RCC. Areas covered are data with first and further line of therapy with mTOR inhibitors and other agents but without cytoreductive nephrectomy or rare histologies. The current data on systemic therapy in poor-risk metastatic RCC maintain temsirolimus as the preferred first-line therapy. New agents targeting immune checkpoints are being developed in clinical trials.