Interleukin-1beta in synergism gabapentin with tramadol in murine model of diabetic neuropathy.

Neuropathic pain is a complication of cancer and diabetes mellitus and the most commonly used drugs in the treatment of the diabetic neuropathic pain have only limited efficacy. The aim of this study was to evaluate the role of the biomarker interleukin-1beta (IL-1ß) in the pharmacological interaction of gabapentin with tramadol in a model of diabetic neuropathic pain. CF-1 male mice, pretreated with 200 mg/kg i.p. of streptozocin (STZ), were used and at day 3 and 7 were evaluated by the hot plate test and the spinal cord level of IL-1ß was determined. Antinociceptive interaction of the coadministration i.p. of gabapentin with tramadol, in basic of the fixed the ratio 1:1 of their ED50 values alone, was ascertained by isobolographic analysis. Tramadol was 1.13 times more potent than gabapentin in saline control mice, 1.40 times in STZ mice at 3 days and 1.28 times in STZ at 7 days. The interaction between gabapentin and tramadol was synergic, with an interaction index of 0.30 and 0.22 for mice pretreated with STZ at 3 and 7 days. The combination of gabapentin with tramadol reversed the increased concentration of IL-1ß induced by STZ in diabetic neuropathic mice. These findings could help clarify the mechanism of diabetic neuropathy.

Utility of retrograde ureterocelogram in management of complex ureterocele.

INTRODUCTION: Symptomatic pediatric ureterocele has diverse manifestations, making evidence-based management impractical. Thus, detailed visualization of ureterocele anatomy prior to first surgical incision is invaluable. Retrograde ureterocelogram (RUC) is a simple, underutilized radiologic technique that can be performed during cystoscopy. This study sought to determine whether RUC changes surgical management by more accurately depicting the complex ureteral and ureterocele anatomy, compared with renal ultrasound (US) and voiding cystourethrography (VCUG). METHODS: Patients who underwent surgical management of ureterocele from 2003 to 2015 were identified; those who received concomitant fluoroscopic RUC were selected for the case series. Data collected included: demographics, pre-operative evaluation, surgical interventions, and outcomes. The RUC images were individually examined, and the anatomic impression compared with previous renal US and VCUG. Novel RUC findings not previously appreciated by the pre-operative evaluation were noted. The RUC was performed by cystoscopically inserting a needle into the ureterocele and injecting contrast retrograde. If indicated, simultaneous PIC (Positioning the Instillation of Contrast) cystography was performed. RESULTS: Of the 43 patients that underwent surgery for suspected ureterocele, 28 underwent cystoscopy + RUC (10 M: 18 F) at a median age of 4.6 months and median follow-up of 37.0 months. All patients had prior US, 25 had prior VCUG, and 20 had prior radionuclide studies. Ureteroceles were either duplex system (n = 21) or single system (n = 7); 17 were ectopic into the bladder neck or urethra; seven were intravesical; and four were pseudoureteroceles. Fourteen patients underwent concomitant transurethral incision of the ureterocele (TUIU); two were deferred for surgery; and 11 received concomitant definitive surgery (e.g., nephrectomy). The RUC illuminated novel aspects of the anatomy in 20 of the 28 patients. No adverse events occurred. Notably, in nine of the 28 children, significant observations from RUC prompted change to the pre-operative surgical plan. DISCUSSION: Retrograde ureterocelogram clearly revealed ureterocele ectopy, pseudoureterocele, ureterocele disproportion, and unsuspected duplex systems, making it a useful adjunct to standard US and VCUG studies. Retrograde ureterocelogram can also be used to fluoroscopically verify decompression of the ureterocele post incision, document severity of ureteral dilation, and teach residents about the great damage generated by ureterocele variations. Limitations of RUC included increasing radiation dose and overall cost. The study design was limited by its small size, retrospective approach, selection bias, and availability of RUC images. CONCLUSIONS: While not indicated in routine ureterocele management, intraoperative RUC further defined ureterocele anatomy in nearly all cases and yielded changes to the original surgical plan frequently enough to merit greater use in complex patients.

Results of distal hypospadias repair after pediatric urology fellowship training: A comparison of junior surgeons with their mentor.

BACKGROUND: Teaching and learning hypospadias repair is a major component of pediatric urology fellowship training. Educators must transfer skills to fellows, without increasing patient complications. Nevertheless, few studies report results of surgeons during their first years of independent practice. PURPOSE: To review outcomes of distal hypospadias repairs performed during the same 2-year period by consecutive, recently matriculated, surgeons in independent practice, and to compare them to results by their mentor (with >20 years of experience). MATERIALS: Exposure to hypospadias surgery during fellowship was determined from case logs of five consecutive fellows completing training from 2007-2011. TIP was the only technique used to repair distal hypospadias. No fellow operated independently or performed complete repairs under supervision. Instead, the first 3 months were spent assisting their mentor, observing surgical methodology and decision-making. Then, each performed selected portions under direct supervision, including: degloving, penile straightening, developing glans wings, incising and tubularizing the urethral plate, creating a barrier layer, sewing the glansplasty, and skin closure. Overall fellow participation in each case was <50%. In 2011-2012, urethroplasty complications (fistula, glans dehiscence, meatal stenosis, urethral stricture, diverticulum) were recorded for consecutive patients undergoing primary distal repair by these recent graduates in their independent practices. The fellow graduating in 2011 provided 1 year of data. All patients undergoing repair during the study period were included in the analysis, except those lost to follow-up after catheter removal. Composite urethroplasty complications were compared between junior surgeons, and between junior surgeons and their mentor, with Fisher's exact contingency test. RESULTS: Training logs indicated fellow participation ranged from 76-134 hypospadias repairs, including distal, proximal and reoperative surgeries. Post-graduation case volumes ranged from 25-68 by junior surgeons versus 136 by the mentor. With similar mean follow-up, urethroplasty complication rates were statistically the same between the former fellows, and between them versus the mentor, ranging from 5-13%. Nearly all were fistulas or glans dehiscence. Junior surgeons reported they performed TIP as learned during fellowship, with one exception who used 7-0 polydioxanone rather than polyglactin for urethroplasty. DISCUSSION: This is the first study directly comparing hypospadias surgical outcomes by recently graduated fellows in independent practice with those of their mentor. We found junior surgeons achieved similar results for distal TIP hypospadias repair. Although their participation during training largely comprised observation and surgical assistance, with discrete performance of key steps, skills sufficient to duplicate the mentor's results were transferred. These data suggest there should be no learning curve for distal hypospadias after training. This report raises several considerations for surgical educators. First, mentors should review their own results, to be certain that they are correctly performing and teaching procedures. Second, programs need to determine key steps for procedures they teach, and then emphasize their optimal performance. Finally, mentors should expect former fellows to report back their initial results of hypospadias repair to be certain lessons taught were learned. Otherwise, preventable complications resulting from technical errors will be multiplied in the children operated by their trainees as they enter independent practice.

Evaluation of TSH Levels in the Program of Congenital Hypothyroidism Newborn Screening in a Pilot Study of Preterm Newborns in Bogotá, Colombia

Abstract Introduction: Preterm infants (<37 weeks of gestation) have low levels of thyroid hormones due to multiple factors. Objective: To evaluate levels of thyroid-stimulation hormone (TSH) in the program congenital hypothyroidism (CH) newborn screening in a sample of preterm infants in the city of Bogotá, Colombia. Methods: The Secretaría de Salud Distrital screening protocol for CH (blood sample is collected from the umbilical cord in all the newborns) remeasured the serum TSH and heel TSH when preterm infants completed 37 weeks of gestation. Results: A total of 59 preterm neonates were rescreened, of which 2 neonates had elevated levels of TSH and 1 neonate had transient hypothyroxinemia. The Kolmogorov-Smirnov 2-sample/bilateral statistical test was used to compare the neonatal TSH levels of preterm and full-term newborns, which do not follow the same distribution. Conclusion: In our pilot study, 2 of the rescreened infants presented high levels of TSH and 1 had transient hyperthyrotropinemia, suggesting the need for rescreening of preterm infants. Additionally, a larger study should be performed to determine the screening cutoff values for preterm newborns.

[Hip fracture, antiplatelet drugs treatment and postoperative complications]. / Fractura de cadera, antiagregantes plaquetarios y complicaciones postoperatorias.

OBJECTIVES: To assess the incidence of postoperative complications, blood transfusions and survival at one month, in the old patients operated for hip fracture undergoing chronic treatment with antiplatelet drugs. MATERIAL AND METHODS: Two hundred twenty three patients operated for hip fracture were studied retrospectively, separated into 3 groups: patients who received acetylsalicylic acid (group I), patients who were given 100mg/day of acetylsalicylic acid or 300mg/day of triflusal (group II) and patients receiving>100mg/day of acetylsalicylic acid, or>300mg/day of triflusal or thienopyridines (group III). Surgery was delayed for 4 days in patients in group III. Demographic, biological, clinical and treatment characteristics, postoperative complications and survival at one month were recorded. RESULTS: Patients in group III were older and sustain worse general health status. Patients with a higher transfusion requirement were those of group II (73.8%) (P=0.192), who also showed a higher percentage of anaemia on admission. Severe cardiovascular complications were experienced by 5.4% of group III patients, 4.8% of group II patients and 2.1% of group I patients. Patients from group III presented a significant amount of respiratory complications (P=0.007). CONCLUSIONS: Our results suggest that delaying surgery for 4 days in patients treated with clopidogrel can be associated to an increase in postoperative respiratory complications and severe adverse cardiovascular events, without increasing the tranfusional index, hospital stay, mortality, and without complications related to neuraxial anaesthesia.

Eight years experience with liver transplantation in Rio Hortega hospital.

OBJECTIVES: We sought to evaluate our transplant series in light of the parameters outlined in the quality criteria established by the Spanish Hepatic Transplant Society (Sociedad Española de Trasplante Hepático [SETH]). METHODS: We retrospectively analyzed 240 hepatic transplantations performed in 223 patients from November 2001 to December 2009. RESULTS: Among the series, 57% were in Child class C, 50% had cirrhosis without hepatocellular carcinoma, and 32% had this neoplasm. The most common cause for the illness was alcohol, followed by a virus, namely hepatitis C virus in 76% of cases. The average waiting list time was 45.14 days. The total graft ischemia averaged 460 minutes (range, 265-937). The 4.1% (n = 10), incidence of an urgent retransplantation was mainly due to primary graft failure or arterial thrombosis. During the perioperative period the mortality rate was 2.5% (n = 6) and the 1-month mortality rate was 6.6% (n = 16). The raw survival rates at 1, 3, and 5 years after the operation are 85%, 78%, and 72%, respectively. CONCLUSION: Our perioperative as well as the long-term results fall within the quality standards established by SETH.

Estradiol inhibits the estrone sulfatase activity in normal and cancerous human breast tissues.

It is well accepted that estradiol (E2) plays an important role in the genesis and evolution of breast cancer. Quantitative evaluation indicates that in human breast tumor, estrone sulfate (E1S) 'via sulfatase' is a much more likely precursor for E2 than is androstenedione 'via aromatase'. In previous studies, it was demonstrated that in isolated MCF-7 and T-47D breast cancer cell lines, estradiol can block estrone sulfatase activity. In the present study, the effect of E2 was explored using total normal and cancerous breast tissues. This study was carried out with post-menopausal patients with breast cancer. None of the patients had a history of endocrine, metabolic or hepatic diseases or had received treatment in the previous 2 months. Each patient received local anaesthetic (lidocaine 1%) and two regions of the mammary tissue were selected: (A) the tumoral tissue and (B) the distant zone (glandular tissue) which was considered as normal. Samples were placed in liquid nitrogen and stored at -80 degrees C until enzyme activity analysis. Breast cancer histotypes were ductal and post-menopausal stages were T2. Homogenates of tumoral or normal breast tissues (45-75 mg) were incubated in 20 mM Tris-HCl, pH 7.2 with physiological concentrations of [3H]-E1S (5 x 10(-9)M) alone or in the presence of E2 (5 x 10(-5) to 5 x 10(-7) M) during 30 min or 3 h. E1S, E1 and E2 were characterized by thin layer chromatography and quantified using the corresponding standard. The sulfatase activity is significantly more intense with the breast cancer tissue than normal tissue, since the concentration of E1 was 3.20 +/- 0.15 and 0.42 +/- 0.07 pmol/mg protein, respectively after 30 min incubation. The values were 27.8 +/- 1.8 and 3.5 +/- 0.21 pmol/mg protein, respectively after 3 h incubation. Estradiol at the concentration of 5 x 10(-7) M inhibits this conversion by 33% and 31% in cancerous and normal breast tissues, respectively and by 53% and 88% at the concentration of 5 x 10(-5) M after 30 min incubation. The values were 24% and 18% for 5 x 10(-7) M and 49% and 42% for 5 x 10(-5) M, respectively after 3h incubation. It was observed that [3H]-E1S is only converted to [3H]-E1 and not to [3H]-E2 in normal or cancerous breast tissues, which suggests a low or no 17beta-hydroxysteroid dehydrogenase (17beta-HSD) Type 1 reductive activity in these experimental conditions. In conclusion, estradiol is a strong anti-sulfatase agent in cancerous and normal breast tissues. This data can open attractive perspectives in clinical trials using this hormone.

Transperitoneal and retroperitoneal laparoscopic heminephrectomy--what approach for which patient?

PURPOSE: We report our experience with laparoscopic heminephrectomy using the transperitoneal and retroperitoneal approaches in 48 pediatric patients. MATERIALS AND METHODS: A total of 48 laparoscopic heminephrectomies were performed in 35 girls and 13 boys 45 days to 17 years old (mean 4.08 years) between September 1998 and March 2005. The procedures consisted of 44 upper pole heminephrectomies with partial or total ureterectomies and 4 lower pole heminephroureterectomies. Surgeries were performed using a transperitoneal approach in 32 patients (67%) and a retroperitoneal approach in 16 (33%). RESULTS: Followup ranged from 0.75 to 7.25 years (mean 3.53). In the retroperitoneal group 2 procedures required conversion, 1 to open heminephrectomy and 1 to a transperitoneal laparoscopic approach. Complications were seen in 5 of 48 patients (10%). Complications in the retroperitoneal group were seen in 2 patients. One patient had a postoperative urinary leak that resolved spontaneously. Another patient had development of a urinoma that was treated conservatively. Complications in the transperitoneal group were seen in 3 patients. One patient required an intraoperative chest tube due to pneumothorax, 1 had recurrent urinary tract infection that required excision of a short ureteral remnant and 1, 6-month-old boy had development of postoperative hypertension. Four of the 5 complications (80%) were seen in patients younger than 1 year. CONCLUSIONS: Transperitoneal and retroperitoneal laparoscopic heminephrectomy can be performed for benign disease in children with minimal morbidity, improved cosmesis and short hospital stay. Complication rate does not depend on the surgical approach, but rather on the age of the patient.

Trombosis en el infarto agudo del miocardio en hospitales chilenos: la experiencia del grupo GEMI / Thrombolysis in the acute myocardial infarction in chilean hospitals: the gemi group experience

Antecedentes: La trombolisis es uno de los métodos de reperfusión coronaria que permite reducir la mortalidad del infarto agudo del miocardio (IAM). Lamentablemente no todos los pacientes con indicación de trombolítico reciben el tratamiento. Objetivo: Evaluar los cambios en el empleo de trombolíticos a través del tiempo en pacientes con IAM y supradesnivel ST y analizar las variaciones en mortalidad según el período de registro. Método: Se compara la información de tres registros efectuados los años 93-95 (R1), 97-98 (R2), y los pacientes incluidos en el año 2001 del registro GEMI actualmente en curso (R3), en el que participan 23 hospitales de Santiago y regiones. Se recolecto información sobre latencia en administración del trombolítico, motivo de la no utilización y evolución intrahospitalaria de los pacientes que ingresaron con el diagnóstico de IAM Q o con supradesnivel ST (SDST). Resultados: En R1 se recolectaron 2.155 pacientes con IAM y SDST, en R2: 1.436 pacientes y en R3: 789 pacientes. El porcentaje que recibió trombolíticos fue de 37,8 por ciento, 41,4 por ciento y 45,1 por ciento, respectivamente. La mortalidad global en cada uno de los registros fue de R1: 11,2 por ciento, R2: 9,9 por ciento y R3: 8,9 por ciento ( p para tendencias: NS). Cuando se analiza según sexo, la mortalidad en hombres fue de 8,1 por ciento 7,4 por ciento y 7,1 por ciento (p para tendencias: NS). En mujeres estas proporciones fueron 23,6 por ciento 19,2 por ciento y 14,8 por ciento, respectivamente (p para tendencias: <0,05). El motivo de no uso trombolítico, dato consignado en R2 y R3, se debió a: ingreso tardío (45 por ciento y 38 por ciento respectivamente), contraindicación (9,5 y 12,5), no disponibilidad de él (1,5 por ciento y 0,23 por ciento). En el resto de los pacientes se consignó como “otro” el motivo de no uso. (De este análisis se excluyeron los pacientes sometidos a angioplastia primaria). Se observa un aumento en la proporción de pacientes sometidos a trombosis, asociado a una reducción en la mortalidad global en ellos. Conclusión: La reducción de la mortalidad en mujeres es determinante en la mejoría del pronóstico intrahospitalario en la población de trombolisados. Estos hallazgos pueden reflejar una mejor indicación y oportunidad del empleo de trombolíticos, así como de los fármacos de eficacia demostrada para el tratamiento de IAM con SDST...

[Cardiovascular risk factors in middle-age women with acute coronary syndromes]. / Impacto de los factores de riesgo coronario en mujeres de edad media.

BACKGROUND: In Chile, 40% of deaths due to acute myocardial infarction occur in women. AIM: To assess the presence of cardiovascular risk factors in women with acute coronary syndromes. PATIENTS AND METHODS: Thirty four women aged 46 to 55 years old, admitted to the hospital due to an acute coronary syndrome and 102 age matched healthy women were studied. A clinical history was obtained, blood pressure and fasting serum lipids were measured. RESULTS: A history of high blood pressure was present in 65 and 16% of patients and healthy controls respectively. Seventy seven percent of patients and 36% of controls smoked, 76% of patients and 48% of controls were postmenopausal, 21% of patients an 5% of controls were diabetic. In patients and controls respectively serum total cholesterol was 230.1 +/- 36.2 and 211.2 +/- 34.8 mg/dl, serum triacylglycerol was 213.4 +/- 109.4 and 143.2 +/- 76.9 mg/dl and serum HDL cholesterol was 44.1 +/- 10.8 and 49.8 +/- 13.3 mg/dl (p < 0.001 or less). In univariate analysis, the risk of acute coronary syndrome increased with high blood pressure (OR: 9.3, CI: 2.5-18.6), menopause (OR: 8.3, CI: 2.2-31:5), smoking (OR: 6.9, CI: 2.5-18.6), diabetes mellitus (OR: 5.0, CI: 1.4-17.5), a high total cholesterol/HDL cholesterol ratio (OR: 6.6, CI: 1.8-12.5) and hypertriglyceridemia (OR: 3.6, CI: 1.5-8.5). Logistic regression analysis showed that hypertension and menopause had the higher predictive values for acute coronary syndrome. CONCLUSIONS: In this group of women with acute coronary syndromes, the main coronary risk factors were high blood pressure and menopause.

Neuroendocrine differentiation of the LNCaP prostate cancer cell line maintains the expression and function of VIP and PACAP receptors.

The molecular mechanisms involved in differentiation of prostate cancer cells to a neuroendocrine (NE) cell phenotype are not well understood. Here we used the androgen-dependent human prostate cancer cell line LNCaP to perform a systematic and broad analysis of the expression, pharmacology, and functionality of vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (PACAP) receptors. Reverse transcription polymerase chain reaction experiments, together with pharmacological approaches with a set of specific agonists and antagonists, demonstrated the presence of the three VIP/PACAP receptor subtypes (PAC1, VPAC1, and VPAC2 with a major role for VPAC1, acting through adenylate cyclase (AC) stimulation. An essentially similar pattern was observed by NE differentiated cells (4 days after serum deprivation) in spite of the important morphological changes observed. However, the expression of the prostate-specific antigen (PSA) decreased in NE cells (and increased again by dihydrotestosterone, DHT, treatment). The present demonstration of the induction of NE transdifferentiation in LNCaP cells by increasing concentrations of VIP adds value to previous observations on the role of cAMP in this process, an interesting topic in the comprehension of the molecular changes that are involved in the progression of prostate cancer to androgen independence.

Low expression of Galpha protein subunits in human prostate cancer.

PURPOSE: Because of increasing interest in G protein regulation of cell growth, differentiation and oncogenesis, we studied the functionality and expression of different G protein subunits in human prostate adenocarcinoma. MATERIALS AND METHODS: Surgical prostate specimens from control patients with bladder cancer and patients with prostate cancer were used. The functionality of alphas and alphai G protein subunits was evaluated by studying somatostatin or guanyl-5'-yl-imidotriphosphate regulation of forskolin stimulated adenylyl cyclase activity. The expression of alphas, alphai and beta subunits was studied by reverse transcriptase-polymerase chain reaction and immunoblot analysis. RESULTS: Adenylyl cyclase sensitivity to somatostatin inhibition decreased in prostate cancer. Low guanyl-5'-yl-imidotriphosphate doses inhibited forskolin stimulated adenylyl cyclase, whereas the opposite was true at high concentrations, evidencing the functionality of alphai and alphas, respectively, in normal and cancer tissue samples. Reverse transcriptase-polymerase chain reaction revealed RNA encoding for alphas and alphai1,2,3 subclasses in normal and pathological conditions. However, immunoblot analysis showed that the level of beta subunits was maintained, whereas that of alphas and alphai subunits decreased 30% to 40% after neoplastic transformation. The levels of alphas and alphai1,2 subunits correlated inversely with serum prostate specific antigen in patients with prostate cancer. CONCLUSIONS: The functionality and expression of G protein subunits are selectively modified in human prostate adenocarcinoma. Low alphas and alphai levels in prostate cancer suggest an important regulatory role of G proteins for cell proliferation and neoplastic transformation in the human prostate and they may have prognostic value.

Vasoactive intestinal peptide (VIP) stimulates rat prostatic epithelial cell proliferation.

BACKGROUND: Androgens play a major role in supporting normal growth and functional maintenance in the prostate. However, this gland contains an array of neuroendocrine peptides that can play a regulatory role in its physiopathology. Among these peptides, one of the best studied is vasoactive intestinal peptide (VIP), which is abundant in autonomic nerves surrounding both human and rat prostatic acini. This neuropeptide may act through interaction with two types of high-affinity receptors, named VPAC(1) and VPAC(2) receptors. Another regulatory peptide, the pituitary adenylate cyclase-activating peptide (PACAP), interacts with these receptors with the same affinity as VIP, but binds with higher affinity to PAC(1) receptors. Human prostate tumors and rat prostate show a major presence of VPAC(1) receptors, whereas various findings suggest a role for VIP in prostatic development. Here we studied the effects of VIP on the proliferation of rat prostatic epithelial cells in culture. METHODS: We studied the [(3)H]-thymidine uptake by rat prostatic epithelial cells in culture, characterized previously by using biomarkers such as cytokeratin and vimentin. In these cells we tested the effect of VIP and PACAP-27 on two different signaling pathways, the cyclic AMP (cAMP) and the inositol phosphate (IPs). RESULTS: The rat prostatic cells in culture were cytokeratin (5,6,8) and vimentin positive, indicating that the culture was predominantly epithelial. The proliferation curves showed that the cells followed different states of growth: a quiescent, an exponential proliferative, and a steady state. Cyclic AMP production, but not inositol phosphate production, was increased in the presence of VIP and PACAP-27, which suggests the expression of VPAC(1) and/or VPAC(2) receptors primarily. VIP significantly increased prostatic cell proliferation in a bimodal manner, as shown for dibutyryl cyclic AMP (dbcAMP), which suggests that the effect of VIP upon prostatic proliferation is cAMP-dependent. CONCLUSIONS: Here, we demonstrate that VIP increased [(3)H]thymidine uptake by rat prostatic epithelial cells in culture, conceivably by the activation of the adenylate cyclase.

Effects of the luteinising hormone-releasing hormone (LH-RH) agonist leuprolide on adenylyl cyclase regulation through G-protein coupled receptors in rat ventral prostate.

Luteinising hormone-releasing hormone (LH-RH) agonists are widely used for the therapy of advanced prostate cancer through the suppression of testosterone secretion. Furthermore, recent studies indicate the existence of prostate LH-RH receptors coupled to signalling pathways resulting in direct antiproliferative effects. In order to shed light on the mechanisms through which these compounds inhibit prostate cell growth, we investigated the effects of leuprolide (a LH-RH agonist) treatment of rats compared with the effects of surgical castration on the behaviour of G-protein coupled receptors acting through adenylyl cyclase in the ventral prostate. Important decreases of both plasma testosterone levels and ventral prostate weight were observed 5 weeks after subcutaneous (s.c.) injection of a leuprolide-depot preparation (1.5 mg/kg body weight (b.w.)) or 5 days after bilateral gonadectomy. However, leuprolide treatment increased the number of vasoactive intestinal peptide (VIP) receptors and the ability of this neuropeptide to stimulate adenylyl cyclase activity in prostate membranes, whereas surgical castration decreased both parameters. Moreover, leuprolide resulted in significant increases of prostate alpha(s) and alpha(i1-3) (but not alpha(i1) and beta) G-protein levels, while the four G-protein subunits were overexpressed after gonadectomy. The estimation of alpha(s) and alpha(i) activity by experiments with Gpp[NH]p and forskolin indicated a potentiation of the two arms of adenylyl cyclase regulation in leuprolide-treated rats. Present observations suggest that leuprolide treatment leads to an antimitogenic response by acting mainly through the activation of Gi proteins negatively coupled to adenylyl cyclase.

Expression of vasoactive intestinal peptide (VIP) receptors in human uterus.

We show the existence of functional vasoactive intestinal peptide (VIP) receptors in normal human female genital tract (endometrium, myometrium, ovary and Fallopian tube) as well as in leiomyoma (a frequent uterine pathology). The correlation between VIP binding and stimulation of adenylyl cyclase activity for all studied tissues was linear (r = 0.86) suggesting the expression of VIP receptors throughout the human female genital tract. Immunodetection of VIP receptor subtypes gave different molecular weights for VPAC(1) (47 kDa primarily) and VPAC(2) (65 kDa), which may be due to different glycosylation extents. In conclusion, this study demonstrates the expression of both subtypes of VIP receptors and their functionality in human female genital tract, suggesting that this neuropeptide could play an important physiological and pathophysiological role at this level.

Effect of flutamide-induced androgen-receptor blockade on adenylate cyclase activation through G-protein coupled receptors in rat prostate.

The effect of the antiandrogen flutamide on the prostatic vasoactive intestinal peptide (VIP) receptor/effector system was studied in rats. Rats were s.c. injected with a daily dose of flutamide (15 mg/kg B.W.) or vehicle for 14 days. Drug treatment resulted in histological evidence of gland involution and increased plasma membrane fluidity as estimated by fluorescence spectroscopy. The number of VIP receptors and the stimulatory effect of VIP on adenylate cyclase activity in prostatic membranes decreased in flutamide-treated rats. However, the pattern of forskolin stimulation of the enzyme activity was not modified by this drug. Androgen-receptor blockade by flutamide also decreased the prostatic levels of alpha(s,) alpha(i1/2), and alpha(i3/0) G-protein subunits, as estimated by an immunological procedure. Whereas apoptotic DNA fragmentation was evidenced in prostate from 3-day castrated animals, a heterogeneous electrophoretic pattern was observed after flutamide treatment. Thus, androgen-receptor blockade by flutamide results in an important impairment of the components of the VIP receptor/effector system in rat prostate as well as in a modification of their coupling extent, which is presumably due to differences observed in plasma membrane fluidity. These results represent a crosstalk in the prostate between two mechanisms of signal transduction involved in cell proliferation.

Immunohistochemical localization and distribution of VIP/PACAP receptors in human lung.

VIP and PACAP are distributed in nerve fibers throughout the respiratory tract acting as potent bronchodilators and secretory agents. By using RT-PCR and immunoblotting techniques, we have previously shown the expression of common VIP/PACAP (VPAC(1) and VPAC(2)) and specific PACAP (PAC(1)) receptors in human lung. Here we extend our aims to investigate by immunohistochemistry their localization and distribution at this level. A clear immunopositive reaction was obtained in human lung sections by using either anti-VPAC(1) or -VPAC(2) receptor antibodies but not with anti-PAC(1) receptor antibody. However, PAC(1) receptor (and VPAC(1) and VPAC(2) receptors) could be identified in lung membranes by immunoblotting which supports that the PAC(1) receptor is expressed at a low density. Both VPAC(1) and VPAC(2) receptors showed similar immunohistochemical patterns appearing in smooth muscle cells in the wall of blood vessels and in white blood cells (mainly in areas with inflammatory responses). The results agree with previous evidence on the importance of both peptides in the immune system and support their anti-inflammatory and protective roles in lung.

Impairment of adenylate cyclase activity and G-proteins in human uterine leiomyoma.

The mechanisms responsible for the growth of uterine leiomyoma (a frequent cause of infertility in women) are largely unknown. Some data supports that cAMP plays a role in the growth of uterine cells but there are no reports on the status of the cAMP producing system in this human benign neoplasia. In this study, biopsies from leiomyoma and the adjacent myometrium were taken from menstruating women subjected to total hysterectomy for leiomyoma. Adenylate cyclase activity was determined by a protein-binding method, and the expression of alpha(s), alphai1/2, alphai3 and alphai0) G-protein subunits was analysed by immunoblot. The leiomyoma samples exhibited a decreased expression of as and ai1/2 with respect to the adjacent myometrial tissue. No differences were observed in alphai3 and alphaio protein expression. The basal adenylate cyclase activity as well as the efficacy (as assessed by the maximal stimulation levels) of either forskolin or, to a lesser extent, Gpp[NH]p on stimulation the enzyme activity was significantly lower in leiomyoma than in myometrium, whereas the potency (as assessed by the ED50 values) of these two agents did not vary. Present data indicate that the human leiomyoma is associated with low levels of cAMP. It is conceivable that the loss of sensitivity of adenylate cyclase to endogenous regulatory molecules could be related to the pathogenesis of human leiomyomas given that cAMP inhibits the MAP-kinase cascade in uterine tissues.