Human native Cav1 channels in chromaffin cells: contribution to exocytosis and firing of spontaneous action potentials.

The present study was performed to evaluate the Cav1 channel subtypes expressed in human chromaffin cells and the role that these channels play in exocytosis and cell excitability. Here we show that human chromaffin cells obtained from organ donors express Cav1.2 and Cav1.3 subtypes using molecular and pharmacological techniques. Immunocytochemical data demonstrated the presence of Cav1.2 and Cav1.3 subtypes, but not Cav1.1 or Cav1.4. Electrophysiological experiments were conducted to investigate the contribution of Cav1 channels to the exocytotic process and cell excitability. Cav1 channels contribute to the exocytosis of secretory vesicles, evidenced by the block of 3µM nifedipine (36.5±2%) of membrane capacitance increment elicited by 200ms depolarizing pulses. These channels show a minor contribution to the initiation of spontaneous action potential firing, as shown by the 2.5 pA of current at the threshold potential (-34mV), which elicits 10.4mV of potential increment. In addition, we found that only 8% of human chromaffin cells exhibit spontaneous action potentials. These data offer novel information regarding human chromaffin cells and the role of human native Cav1 channels in exocytosis and cell excitability.

Therapeutic concentrations of varenicline in the presence of nicotine increase action potential firing in human adrenal chromaffin cells.

Varenicline is a nicotinic acetylcholine receptor (nAChR) agonist used to treat nicotine addiction, but a live debate persists concerning its mechanism of action in reducing nicotine consumption. Although initially reported as α4ß2 selective, varenicline was subsequently shown to activate other nAChR subtypes implicated in nicotine addiction including α3ß4. However, it remains unclear whether activation of α3ß4 nAChRs by therapeutically relevant concentrations of varenicline is sufficient to affect the behavior of cells that express this subtype. We used patch-clamp electrophysiology to assess the effects of varenicline on native α3ß4* nAChRs (asterisk denotes the possible presence of other subunits) expressed in human adrenal chromaffin cells and compared its effects to those of nicotine. Varenicline and nicotine activated α3ß4* nAChRs with EC50 values of 1.8 (1.2-2.7) µM and 19.4 (11.1-33.9) µM, respectively. Stimulation of adrenal chromaffin cells with 10 ms pulses of 300 µM acetylcholine (ACh) in current-clamp mode evoked sodium channel-dependent action potentials (APs). Under these conditions, perfusion of 50 or 100 nM varenicline showed very little effect on AP firing compared to control conditions (ACh stimulation alone), but at higher concentrations (250 nM) varenicline increased the number of APs fired up to 436 ± 150%. These results demonstrate that therapeutic concentrations of varenicline are unlikely to alter AP firing in chromaffin cells. In contrast, nicotine showed no effect on AP firing at any of the concentrations tested (50, 100, 250, and 500 nM). However, perfusion of 50 nM nicotine simultaneously with 100 nM varenicline increased AP firing by 290 ± 104% indicating that exposure to varenicline and nicotine concurrently may alter cellular behavior such as excitability and neurotransmitter release.

α-Conotoxins Identify the α3ß4* Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells.

Ligands that selectively inhibit human α3ß2 and α6ß2 nicotinic acetylcholine receptor (nAChRs) and not the closely related α3ß4 and α6ß4 subtypes are lacking. Current α-conotoxins (α-Ctxs) that discriminate among these nAChR subtypes in rat fail to discriminate among the human receptor homologs. In this study, we describe the development of α-Ctx LvIA(N9R,V10A) that is 3000-fold more potent on oocyte-expressed human α3ß2 than α3ß4 and 165-fold more potent on human α6/α3ß2ß3 than α6/α3ß4 nAChRs. This analog was used in conjuction with three other α-Ctx analogs and patch-clamp electrophysiology to characterize the nAChR subtypes expressed by human adrenal chromaffin cells. LvIA(N9R,V10A) showed little effect on the acetylcholine-evoked currents in these cells at concentrations expected to inhibit nAChRs with ß2 ligand-binding sites. In contrast, the ß4-selective α-Ctx BuIA(T5A,P6O) inhibited >98% of the acetylcholine-evoked current, indicating that most of the heteromeric receptors contained ß4 ligand-binding sites. Additional studies using the α6-selective α-Ctx PeIA(A7V,S9H,V10A,N11R,E14A) indicated that the predominant heteromeric nAChR expressed by human adrenal chromaffin cells is the α3ß4* subtype (asterisk indicates the possible presence of additional subunits). This conclusion was supported by polymerase chain reaction experiments of human adrenal medulla gland and of cultured human adrenal chromaffin cells that demonstrated prominent expression of RNAs for α3, α5, α7, ß2, and ß4 subunits and a low abundance of RNAs for α2, α4, α6, and α10 subunits.

Donation after cardiac death: results of the SUMMA 112 - Hospital 12 de Octubre Program.

BACKGROUND: In 2005, our center started a donation after cardiac death (DACD) program, by which patients who present an irreversible cardiac arrest outside hospital are brought to our center with the purpose of organ donation. We reviewed the outcomes of our program of kidney transplants from DACD. METHODS: We conducted a retrospective study of the DACD, and we reviewed the procedures carried out in our institution between July 2005 and December 2010 and descriptively analyzed the results obtained for kidney donation. RESULTS: One hundred and fifty-two of 274 potential donors were transferred to our hospital. Of them, 126 (82.8%) were connected to cardiopulmonary bypass machine, and organs were procured in 113 donors (74.3%). The discarded grafts were mainly due to inadequate perfusion. One hundred and fifty-six kidneys were transplanted (51.3%). Over a median follow-up period of 18 ± 13.7 months, the median creatinine clearance was 78.2 ± 10.2 ml/min. 8.6% of the grafts had no primary function, and 85% had a delayed graft function. Recipient survival and graft survival were 98% and 87%, respectively. CONCLUSIONS: DACD is an adequate source of organs for kidney transplantation. Our functional and survival results are encouraged in the short term, although further work is required to increase the program's benefits.

Saturation biopsies for prostate cancer detection: effectiveness, safety and predictive factors.

OBJECTIVES: To determine the effectiveness and safety of saturation biopsies for prostate cancer detection of and to identify predictive variables for cancer. METHODS: We conducted a retrospective transversal study in which we analyzed 144 saturation biopsies (January '06 - July '09). INCLUSION CRITERIA: at least two sets of biopsies without evidence of malignancy and Prostate Specific Antigen (PSA)levels >10 ng/ml or PSA kinetics suggestive of malignancy (rate >0.75 ng/ml/year)and patients with atypia in a previous biopsy.The variables analyzed were: age, abnormal digital rectal examination (DRE), total PSA, free/total PSA ratio, prostate volume, PSA density, previous histopathology, number of cylinders obtained and complications. Statistical analysis was performed using the Chi-square test, Student's t-test and logistic regression. RESULTS: Mean age was 66 years (SD ± 6.4), mean total PSA 14.4 ng/ml (SD ± 12.6), mean free/total PSA ratio 0.09 (SD ± 0.09), mean prostate volume 61.6 cc (SD ± 27.4), mean PSA density 0.27 (SD ± 0.26) and mean number of cylinders obtained 30.45 (SD ± 3.8). We diagnosed 32% of the patients with prostatic adenocarcinoma. We observed PSA density was higher in the prostate cancer group, 0.39 (SD ± 0.36), compared to 0.21 (SD ± 0.18) in patients without cancer (p=0.003). Adenocarcinoma was found in 58% of the biopsies in patients with suspicious DRE, compared to 28% with normal DRE (p=0.009). Mean prostate volume in the prostate cancer group was 52.5 (SD ± 24.7)compared to 66.0 (SD ± 27.7)in the group without cancer (p=0.006). In the multivariate analysis, the PSA density (p=0.02; 95% CI 1.36 - 37.36) was the only variable that independently predicted the presence of adenocarcinoma. No statistically significant differences were found in either univariate or multivariate analysis for the remaining variables analyzed. The incidence of complications was similar to that described in the literature for other series. CONCLUSIONS: Saturation biopsy is safe and effective for detection of prostate cancer. PSA density was the only factor that was shown to be independent predictive variable for tumor diagnosis.

Biopsias de saturación para la detección de cáncer de próstata: efectividad, seguridad y factores predictivos / Saturation biopsies for prostate cancer detection: effectiveness, safety and predictive factors

OBJETIVO: Determinar la rentabilidad y seguridad de las biopsias de saturación para la detección del cáncer de próstata (CaP), e identificar variables relacionadas con la presencia del tumor.MÉTODOS: Revisamos de forma transversal y retrospectiva 144 biopsias de saturación (enero/06-julio/09). Los criterios de inclusión fueron: al menos 2 sets de biopsias sin evidencia de malignidad y cifras de Antígeno Prostático Específico (PSA) > 10 ng/ml o cinética de PSA sugestiva de malignidad (velocidad > 0,75 ng/ml/año) y pacientes con atipias en biopsia/s previa/s. Las variables analizadas fueron: edad, tacto rectal sospechoso (TRS), PSA total, cociente PSA libre/total, volumen prostático, densidad de PSA, anatomía patológica previa, número de cilindros obtenidos y complicaciones. Se analiza estadísticamente mediante test de CHI-2, t de Student y regresión logística(AU)

RESULTADOS: La edad media fue de 66 años (DS±6,4), el PSA total medio 14,4 ng/ml (DS±12,6), el cociente medio PSA libre/total 0,09 (DS±0,09), el volumen prostático medio 61,6 cc (DS±27,4), la densidad media de PSA 0,27 (DS±0,26) y el número medio de cilindros obtenidos de 30,45 (DS±3,8).Diagnosticamos un 32% de adenocarcinoma prostático. La densidad de PSA fue mayor en el grupo con CaP, 0,39 (DS±0,36) frente a 0,21 (DS±0,18) en los pacientes sin cáncer (p=0,003). En el 58% de los pacientes con TRS se observó adenocarcinoma en la biopsia, frente a un 28% con TR normal (p=0,009). El volumen prostático medio del grupo con CaP fue de 52,5 (DS±24,7) frente a 66 (DS±27,7) del grupo sin cáncer (p=0,006).En el análisis multivariante, la densidad de PSA (p=0,02; IC 95% 1,36 - 37,36) es la única variable que predice de forma independiente la existencia de adenocarcinoma.Para el resto de variables analizadas, tanto en el análisis univariante como en el multivariante, no se hallaron diferencias estadísticamente significativas.La incidencia de complicaciones derivadas del procedimiento fue similar a la descrita en la literatura para otras series.CONCLUSIONES: La biopsia de saturación es efectiva y segura para determinar la presencia de cáncer de próstata. La densidad de PSA es el único factor que se han mostrado como variable predictiva independiente para el diagnóstico de tumor(AU)

OBJECTIVES: To determine the effectiveness and safety of saturation biopsies for prostate cancer detection of and to identify predictive variables for cancer.METHODS: We conducted a retrospective transversal study in which we analyzed 144 saturation biopsies (January ‘06 - July ‘09). Inclusion criteria: at least two sets of biopsies without evidence of malignancy and Prostate Specific Antigen (PSA) levels >10ng/ml or PSA kinetics suggestive of malignancy (rate >0.75ng/ml/year) and patients with atypia in a previous biopsy.The variables analyzed were: age, abnormal digital rectal examination (DRE), total PSA, free/total PSA ratio, prostate volume, PSA density, previous histopathology, number of cylinders obtained and complications. Statistical analysis was performed using the Chi-square test, Student’s t-test and logistic regression.RESULTS: Mean age was 66 years (SD ± 6.4), mean total PSA 14.4 ng/ml (SD ± 12.6), mean free/total PSA ratio 0.09 (SD ± 0.09), mean prostate volume 61.6 cc (SD ± 27.4), mean PSA density 0.27 (SD ± 0.26) and mean number of cylinders obtained 30.45 (SD ± 3.8).We diagnosed 32% of the patients with prostatic adenocarcinoma. We observed PSA density was higher in the prostate cancer group, 0.39 (SD ± 0.36), compared to 0.21 (SD ± 0.18) in patients without cancer (p=0.003). Adenocarcinoma was found in 58% of the biopsies in patients with suspicious DRE, compared to 28% with normal DRE (p=0.009). Mean prostate volume in the prostate cancer group was 52.5 (SD ± 24.7) compared to 66.0 (SD ± 27.7) in the group without cancer (p=0.006).In the multivariate analysis, the PSA density (p=0.02; 95% CI 1.36 - 37.36) was the only variable that independently predicted the presence of adenocarcinoma. No statistically significant differences were found in either univariate or multivariate analysis for the remaining variables analyzed. The incidence of complications was similar to that described in the literature for other series(AU)

CONCLUSIONS: Saturation biopsy is safe and effective for detection of prostate cancer. PSA density was the only factor that was shown to be independent predictive variable for tumor diagnosis(AU)