Management, treatment and prognostic significance of lateral lymph node metastases in rectal cancer-a regional cohort study.

PURPOSE: Lateral lymph node metastases in rectal cancer remain a clinical challenge. Different treatment regimens have been suggested. This retrospective regional cohort study examines outcome after combined oncological and surgical treatment of MRI-positive lateral lymph nodes (LLN). METHODS: Data from the Swedish Colorectal Cancer Registry (SCRCR) and patient records were used for retrospective analysis of resected high-risk rectal cancers between 2009 and 2014. The aim was to compare tumour characteristics, neoadjuvant therapy, recurrence and outcome after lateral lymph node dissection. RESULTS: One thousand and one hundred nineteen cases were identified and after exclusion 344 patients with cT3-T4 ≤ 10 cm from the anal verge were analysed. Thirty (8.7%) patients with MRI-positive LLN were identified. Synchronous distant metastases were associated with MRI-positive LLN (p-value 0.019). Long-course chemoradiotherapy was clinical practice in cases of MRI-positive LLN. No differences in local (p-value 0.154) or distant (p-value 0.343) recurrence rates between MRI-positive LLN patients and MRI-negative patients were detected. Only four patients underwent lateral lymph node dissection (LLND). There was no significant difference in overall survival during follow-up between the MRI-negative (CI at 95%; 99-109 months) and MRI-positive group (CI at 95%; 69-108 months; p-value 0.14). CONCLUSION: Lateral lymph node metastases present a challenging clinical situation. The present study shows that combination of neoadjuvant therapy and selective LLND is an applicable strategy in cases of MRI-positive LLN.

Impact of rectal perforation on recurrence during rectal cancer surgery in a national population registry.

BACKGROUND: Incidental perforation in rectal cancer surgery is considered a risk factor for poorer oncological outcome. Most studies emanate from the era before total mesorectal excision when staging, neoadjuvant treatment and surgical technique were suboptimal. This study assessed the impact of incidental perforation on oncological outcome in a cohort of patients with optimized management. METHODS: Data from the Swedish Colorectal Cancer Registry for patients undergoing R0 abdominal surgery for TNM stage I-III rectal cancer between 2007 and 2012, with 5-year follow-up, were analysed. Multivariable analysis was performed. RESULTS: In total, 6176 patients were analysed (208 with and 5968 without perforation). The local recurrence rate was increased after perforation (7·2 per cent (15 of 208) versus 3·2 per cent (188 of 5968); P = 0·001), but there were no differences in rates of distant metastasis (16·3 per cent (34 of 208) versus 19·8 per cent (1183 of 5968); P = 0·215) and overall recurrence (20·7 per cent (43 of 208) versus 21·0 per cent (1256 of 5968); P = 0·897). The 5-year overall survival rate was lower after perforation (66·4 versus 75·5 per cent; P = 0·002), but the 5-year relative survival rate was no different (79·9 versus 88·2 per cent; P = 0·083). In multivariable analysis, perforation was a risk factor for local recurrence (hazard ratio 2·10, 95 per cent c.i. 1·19 to 3·72; P = 0·011), but not for the other outcomes. CONCLUSION: Incidental perforation remains a significant risk factor for LR, even with optimized management of rectal cancer. This must be considered when discussing adjuvant treatment and follow-up.

ANTECEDENTES: La perforación incidental durante la cirugía de cáncer de recto se considera un factor de riesgo de un peor resultado oncológico. La mayoría de los estudios proceden de la era previa a la exéresis total del mesorrecto cuando la estadificación, el tratamiento neoadyuvante y la técnica quirúrgica eran subóptimos. En este estudio se evalúa el impacto de la perforación incidental en el resultado oncológico en una cohorte de pacientes con un tratamiento óptimo. MÉTODOS: Se analizaron los datos del Registro Sueco de Cáncer Colorrectal para pacientes sometidos a cirugía abdominal R0 en estadios TNM I-III entre 2007-2012 con un seguimiento de 5 años. Se realizó un análisis multivariable. RESULTADOS: En total, se analizaron 6.176 pacientes (208 con perforación, 5.968 sin perforación). La tasa de recidiva local (local recurrence, LR) aumentó después de la perforación (7,2% (15/208) versus 3,2% (188/5.968); P = 0,001)), pero no se detectaron diferencias con respecto a las tasas de metástasis a distancia (16,3% (34/208) versus 19,8% (1.183/5.968); P = 0,215)) ni de recidiva global (20,7% (43/208) versus 21,0% (1.256/5.968); P = 0,897)). La tasa de supervivencia global a los 5 años fue menor después de la perforación (66,4% versus 75,5%; P = 0,002), pero la tasa de supervivencia relativa a los 5 años fue similar (79,9% versus 88,2%; P = 0,083). En el análisis multivariable, la perforación fue un factor de riesgo para la LR (cociente de riesgos instantáneos, hazard ratio, HR 2,10 (i.c. del 95% 1,19-3,72); P = 0,011], pero no fue un factor de riesgo para los otros resultados. CONCLUSIÓN: La perforación incidental sigue siendo un factor de riesgo significativo para la LR incluso con el tratamiento optimizado del cáncer de recto, lo que debe tenerse en cuenta al discutir la indicación de tratamiento adyuvante y el tipo de seguimiento.

Risk of local recurrence of rectal cancer and circumferential resection margin: population-based cohort study.

BACKGROUND: A circumferential resection margin (CRM) of 1·0 mm or less after rectal cancer surgery is thought to increase the risk of local recurrence (LR). This retrospective population-based study examined how CRM distance affects the LR risk. METHODS: Data from the Swedish Colorectal Cancer Registry were used in a retrospective analysis of rectal cancers resected between 2005 and 2013. The primary endpoint was LR. RESULTS: A total of 12 146 patients were identified, of whom 8392 were included in the analysis; 739 patients had a CRM of 1·0 mm or less and 7653 had a CRM larger than 1·0 mm. The mean follow-up time was 51 months. There were 66 LRs (8·9 per cent) in the group with a CRM of 1·0 mm or less, and 256 (3·3 per cent) among patients with a CRM larger than 1·0 mm. The LR rate was 17·0 per cent (27 of 159), 6·7 per cent (39 of 580), 1·9 per cent (2 of 103) and 3·4 per cent (254 of 7550) when the CRM was 0, 0·1-1·0, 1·1-1·9 and at least 2·0 mm respectively. The risk of LR among patients with a CRM of 0 mm was higher than that in all other subgroups with a larger CRM (P < 0·050). There was no difference in LR between the subgroups with CRM 1·1-1·9 mm and at least 2·0 mm. LR was diagnosed earlier when the CRM was 1·0 mm or less. CONCLUSION: LR risk is related to exact CRM, with the highest risk in patients with a CRM of 0 mm. Close monitoring of patients with no measurable clear margin may allow early detection of LR.

ANTECEDENTES: Se cree que un margen de resección circunferencial (circumferential resection margin, CRM) de ≤1,0 mm tras la cirugía de cáncer de recto aumenta el riesgo de recidiva local (local recurrence, LR). Este estudio retrospectivo de base poblacional evaluó cómo la distancia del CRM afectaba al riesgo de LR. MÉTODOS: Se utilizaron los datos del Registro Sueco de Cáncer Colorrectal (SCRCR) para el análisis retrospectivo de los cánceres de recto resecados entre 2005 y 2013. El objetivo primario fue la LR. RESULTADOS: Se identificaron 12.146 pacientes, con 8.666 pacientes analizados después de las exclusiones. Un total de 739 pacientes tenían CRM de ≤ 1,0 mm y 7.653 pacientes CRM de > 1,0 mm. El tiempo medio de seguimiento fue de 51 meses. Hubo 66 (8,9%) casos de LR en grupo de CRM de ≤ 1,0 mm y 256 (3,3%) casos de LR en el grupo de CRM de > 1,0 mm. La tasa de LR fue del 17% (n = 27/159), 6,7% (n = 39/580), 1,9% (n = 2/103) y 3,4% (n = 254/7550) cuando el CRM fue de 0,0 mm, 0,1-1,0 mm, 1,1-1,9 mm y CRM ≥ de 2 mm, respectivamente. El riesgo de LR en CRM de 0,0 mm fue mayor en comparación con todos los otros grupos con CRM mayores (P < 0,05). No se observó diferencia en LR entre CRM de 1,1-1,9 mm y ≥ 2 mm. La LR se diagnosticó más precozmente cuando el CRM era de ≤ 1,0 mm. CONCLUSIÓN: El riesgo de LR está relacionado con el CRM exacto, con un riesgo más alto en pacientes con CRM de 0,0 mm. La monitorización estrecha de pacientes sin un margen claro medible puede permitir la detección temprana de LR.

High risk of permanent stoma after anastomotic leakage in anterior resection for rectal cancer.

AIM: This study investigates how often bowel continuity was restored after anastomotic leakage in anterior resection for rectal cancer and assesses the clinical factors associated with permanent stoma. METHOD: The Swedish Colorectal Cancer Registry was used to identify cases of anastomotic leakage registered in southern Sweden between January 2001 and December 2011. Patient characteristics, surgical details and clinical information about the anastomotic leakages were retrieved from medical records. RESULTS: Of the 1442 patients operated on with anterior resection in 11 hospitals, 144 (10%) were diagnosed with anastomotic leakage after anterior resection for rectal cancer. After a median follow-up of 87 months (range 21-165), the overall rate of permanent stoma among patients with anastomotic leakage was 65%. Age ≥ 70 years (P = 0.02) and re-laparotomy (P < 0.001) were independently related to permanent stoma. Compared with nondefunctioned patients with anastomotic leakage, defunctioned patients with anastomotic leakage at the index procedure less often required re-laparotomy at some point during the entire clinical course (P < 0.001), but nondefunctioned and defunctioned patients with anastomotic leakage both had permanent stoma to the same extent (67% and 62%, respectively). CONCLUSION: Anastomotic leakage is highly associated with permanent stoma after anterior resection, especially in patients aged ≥ 70 years. In this cohort of patients with anastomotic leakage, 65% had permanent stoma at long-term follow-up. A defunctioning stoma ameliorates the clinical course but does not affect the end result of bowel continuity in established anastomotic leakage after anterior resection.

Late leakage after anterior resection: a defunctioning stoma alters the clinical course of anastomotic leakage.

AIM: Anastomotic leakage (AL) is common after anterior resection (AR). Long term clinical outcomes of AL including late presenting leakage (LL) are not well studied. This study was undertaken to assess clinical features of LL with respect to incidence, association with predisposing factors and need for re-intervention. METHODS: The Swedish Colorectal Cancer Registry (SCRCR) was explored for AL cases after AR for rectal cancer in patients operated in the south of Sweden from 1 January 2001 to 31 December 2011. Demographic data, surgical technical details, number of postoperative days (POD) until diagnosis of AL, presenting symptoms, methods of diagnosis and treatment were retrieved from medical records. LL was defined according to different cut-offs as leakages occurring after hospital discharge (LLAHD), after 30 POD (LL ≥ POD 30) and after 90 POD (LL ≥ POD 90). RESULTS: In total, 1442 patients were operated on with AR of whom 144 cases of AL (10%) were identified. Median time from operation to follow-up was 87 months (range 21-162). LLAHD, LL ≥ POD 30 and LL ≥ POD 90 were present in 51%, 24% and 9% respectively. All categories of LL were associated with a defunctioning stoma. Relaparotomy was significantly less often employed in LLAHD, but not in other categories of LL. CONCLUSION: LL constitutes a substantial portion of all AL after AR for rectal cancer. The large proportion of LLAHD calls for awareness in the outpatient setting.

Gender aspects of survival after surgical treatment for rectal cancer.

AIM: Longer survival in women than men after rectal cancer surgery has been reported. Our hypothesis was that after correction for their longer life expectancy a survival benefit for women would still remain. METHOD: We studied 2792 patients diagnosed with rectal cancer in the southern part of Sweden between 1996 and 2006. The following parameters were included in a prespecified multivariable Cox regression analysis: age at diagnosis, gender, preoperative radiotherapy, stage, year and type of surgery. In addition to overall survival, relative survival was calculated using the Hakulinen approach utilizing an age-, gender- and calendar year-matched Swedish control cohort. RESULTS: Female patients were significantly older, received neoadjuvant treatment less often and were more often operated on by local excision. Overall survival was significantly longer in women. In the multivariable analysis of relative survival, controlling for neoadjuvant treatment, Dukes stage and year and type of surgery, no significant effect of gender [hazard ratio (HR) 1.10 for men, P = 0.114] was found, whereas an improved relative survival with increased age (HR 0.96 per year, P < 0.001) was seen. In contrast, using the same multivariable model with no correction for underlying mortality in the population, male gender (HR 1.38, P < 0.001) and greater age (HR 1.05 per year, P < 0.001) increased the risk of death. CONCLUSION: The results show that after correction for the underlying longer survival in women and some known confounders, survival after surgical treatment for rectal cancer appears to be gender neutral.

Split-thickness skin graft as a treatment for voluminous vaginal fluid discharge after surgery due to vesico-intestino-vaginal fistulation: A case report and review of the literature.

•Repeated pelvic surgery in irradiated tissue increases the risk for vaginal rupture.•We present a rare case with heavy secretion from the ruptured vagina.•Split skin grafting was used as an unusual treatment for this complication.

Postoperative non-steroidal anti-inflammatory drug use and oncological outcomes of rectal cancer.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) are known to suppress the inflammatory response after surgery and are often used for pain control. This study aimed to investigate NSAID use after radical surgical resection for rectal cancer and long-term oncological outcomes. METHODS: A cohort of patients who underwent anterior resection for rectal cancer between 2007 and 2013 in 15 hospitals in Sweden was investigated retrospectively. Data were obtained from the Swedish Colorectal Cancer Registry and medical records; follow-up was undertaken until July 2019. Patients who received NSAID treatment for at least 2 days after surgery were compared with controls who did not, and the primary outcome was recurrence-free survival. Cox regression modelling with confounder adjustment, propensity score matching, and an instrumental variables approach were used; missing data were handled by multiple imputation. RESULTS: The cohort included 1341 patients, 362 (27.0 per cent) of whom received NSAIDs after operation. In analyses using conventional regression and propensity score matching, there was no significant association between postoperative NSAID use and recurrence-free survival (adjusted hazard ratio (HR) 1.02, 0.79 to 1.33). The instrumental variables approach, including individual hospital as the instrumental variable and clinicopathological variables as co-variables, suggested a potential improvement in the NSAID group (HR 0.61, 0.38 to 0.99). CONCLUSION: conventional modelling did not demonstrate an association between postoperative NSAID use and recurrence-free survival in patients with rectal cancer, although an instrumental variables approach suggested a potential benefit.

Nonsteroidal anti-inflammatory drugs and the risk of anastomotic leakage after anterior resection for rectal cancer.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been widely used in colorectal surgery due to their opioid-sparing effect. However, several studies have indicated an increased risk of anastomotic leakage following NSAID treatment, although conflicting results exist. The primary goal of this study was to further examine whether postoperative NSAIDs are independently associated with anastomotic leakage after anterior resection for rectal cancer. METHODS: Patients who underwent anterior resection for rectal cancer during 2007-2013 in 15 different hospitals in three healthcare regions in Sweden were included in the study. Registry data and information from patient records were retrieved. The association between NSAID treatment (for at least two days in the first postoperative week) and symptomatic anastomotic leakage (within 90 days) was evaluated with multiple logistic regression, with adjustment for pertinent confounding factors. RESULTS: Some 1495 patients were included in the study. Of these, 27% received postoperative NSAIDs for at least two days in the first postoperative week. Symptomatic anastomotic leakage occurred in 11% and 14% in the NSAID and non-NSAID group, respectively. With adjustment for confounders, the odds ratio for leakage among patients who received NSAIDs compared with those who did not was 0.88 (95% CI 0.65-1.20). No differences were seen between non-selective and COX-2-selective NSAIDs. CONCLUSION: Postoperative NSAID treatment does not seem to increase the risk of symptomatic anastomotic leakage after anterior resection for rectal cancer. NSAID use appears to be safe, but a well-powered randomized clinical trial is warranted.

Immunocytochemical demonstration of oestrogen receptor beta in blood vessels of the female rat.

The role of oestrogen receptor (ER) beta in vascular function remains unclear. With the use of a specific ERbeta antibody we have now, using immunocytochemistry, visualized ERbeta in different parts of the vascular tree. In about 70% of medial smooth muscle cells of female rat aorta, tail artery and uterine artery, nuclear immunoreactivity to ERbeta was observed. In these vessels endothelial cells also expressed ERbeta. Vascular expression of the ERalpha subtype was lower than that of ERbeta. In aorta and tail artery, no immunoreactivity towards ERalpha was observed, while in uterine vessels occasional medial smooth muscle and endothelial cells expressed this ER subtype. ERbeta and alpha expression in uterine vessels was independent of the stage of the oestrous cycle, suggesting that variations in uterine blood flow occurring during the cycle are independent of ER density. The regional distribution of ERalpha, as determined by immunocytochemistry, was supported by measurements of ERalpha levels by enzyme immunoassay. In the uterine artery, the level of ERalpha was several times higher (P<0.001) than that of aorta and tail artery (10.1+/-1.7 fmol/mg protein in the uterine artery vs 3.3+/-1.0 and 0.5+/-0.5 fmol/mg protein in aorta and tail artery respectively). Thus, a prominent nuclear expression of ERbeta was observed in the vascular wall of several parts of the vascular tree, while ERalpha predominantly was expressed in uterine vessels, suggesting that ERbeta and alpha may have different roles in vascular function.

Role of K+ channels in spontaneous electrical and mechanical activity of smooth muscle in the guinea-pig mesotubarium.

1. The spontaneous electrical and mechanical activity and the efflux rate of 86Rb+ in the guinea-pig mesotubarium were studied in the presence of agents interacting with K+ channels. 2. Tetraethylammonium (TEA, 10 mM) increased the amplitude of the action potentials while having no consistent effect on the frequency or amplitude of spontaneous contractions. 3. 4-Aminopyridine (4-AP, 1-5 mM) caused a graded increase in the duration of the contractions and of the electrical slow waves, and a decrease in the duration of the relaxed period between contractions. At 4 mM-4-AP or more the cell was unable to repolarize from the slow wave and the membrane depolarized to -26 mV from the normal resting potential of -63 mV. The rate of 86Rb+ efflux in the presence of 5 mM-4-AP was higher than that at 60 mM-K+, where the membrane potential is -24 mV. 4. 4-AP (5 mM) evoked a contracture in Ca(2+)-free solution, containing 1 mM-EGTA, both at the normal [K+]o of 5.9 mM and at 60 mM-K+, suggesting release of intracellular Ca2+. 5. Apamin (0.1-1 microM) and charybdotoxin (1-10 nM), blockers of Ca(2+)-dependent K+ channels, were without effects on the spontaneous electrical and mechanical activity. 6. The K+ channel opener pinacidil (10 microM) inhibited the spontaneous contractions and hyperpolarized the membrane by about 7 mV. The permeability to 86Rb+ was increased by a factor of 1.4. 7. It is concluded that different K+ channels are involved in the generation of spikes and slow waves: one sensitive to TEA and responsible for repolarization of the individual action potential, and another sensitive to 4-AP and responsible for repolarization of the slow wave. The duration of the relaxed period can be influenced by activation of K+ channels sensitive to pinacidil.

Metabolic correlates to pacemaker activity in the smooth muscle of guinea-pig mesotubarium.

Oxygen consumption (FO2) and lactate production (Flac) were measured during spontaneous activity in the guinea-pig mesotubarium. During spontaneous contractions FO2 increased to maximally 0.270 +/- 0.025 mumol min-1g-1 (n = 23), followed by a rapid fall immediately upon relaxation. In the relaxed period (5-15 min) between spontaneous contractions FO2 continued to slowly decrease by about 25% towards a final value of 0.150 +/- 0.01 mumol min-1g-1. Flac showed no consistent variation during the relaxed period. Ouabain (10(-6)M) produced a contracture, which was abolished by the Ca2(+)-antagonist felodipine (10(-6)M). In the presence of felodipine, addition of ouabain caused depolarization and a decrease of oxygen consumption by 21% and of lactate production by 31%. Exchange of glucose in the physiological Krebs solution for beta-hydroxybutyrate did not influence spontaneous activity, while subsequent addition of cyanide (2 mM) abolished contractions and caused a hyperpolarization of 15 mV. Blockade of ATP-dependent K+ channels by addition of glibenclamide (10(-7)M) to the relaxed muscle in this situation caused spontaneous contractile activity to reappear. In glucose-containing Krebs solution glibenclamide had no effect on the spontaneous contractile and electrical activity and contractions persisted after addition of cyanide. The relaxing and hyperpolarizing effect of pinacidil could be counteracted by addition of glibenclamide. The results suggest that a decrease in electrogenic Na+/K(+)-pump activity in the course of the relaxed period between contractions could contribute to the pacemaker behaviour. ATP-dependent K+ channels, while having little influence on the spontaneous contractile activity under normal metabolic conditions, could be activated during blockade of aerobic and anaerobic metabolism, leading to inhibition of pacemaker activity.

Spontaneous electrical and contractile activity correlated to 86Rb+ efflux in smooth muscle of guinea-pig mesotubarium.

1. The spontaneous mechanical activity of guinea-pig mesotubarium consists of fused tetanic contractions lasting about 6 min, with a frequency of about four per hour. The muscle is completely relaxed between the contractions. Stretching the relaxed muscle elicits a contraction of the same appearance as the spontaneous ones. Comparison of preparations from oestrus (day 1 of the hormonal cycle), dioestrus (days 9-11) and prooestrus (days 14-15) showed no variation in the pattern of mechanical activity. 2. The resting membrane potential, measured by intracellular microelectrodes, did not differ with hormonal phase (prooestrus: -63.5 +/- 0.84 (n = 16); oestrus: -63.7 +/- 1.6 (n = 5); dioestrus: -61.6 +/- 0.77 (n = 17]. In most recordings a depolarization of a few millivolts occurred during the relaxation period (5-10 min), but in a few cells a more pronounced spontaneous depolarization of 10-15 mV was found. Ouabain (1 microM) caused depolarization by about 9 mV, both in pro- and dioestrus, leading to the initiation of maintained repetitive spiking. 3. Contraction is preceded by a depolarization lasting 10-30 s, and when a threshold is reached a train of slow waves and spikes is elicited. The frequency of slow waves and the number of spikes on each slow wave progressively decrease during the contraction, until spiking eventually ceases. 4. The spikes are resistant to tetrodotoxin (0.5 microM) and disappear in Ca2+-free medium, which also causes membrane depolarization. The duration of contractions increased with Ca2+ concentration in the range 1-5 mM. 5. The rate of 86Rb+ efflux, expressed as fractional release in 2 min intervals, showed a consistent variation during the contractile cycle in preparations with regular spontaneous activity. Relative to the value at the end of the relaxed period the efflux rate increased by about twofold during the contraction. From the beginning of the relaxed period after the contraction the efflux rate decreased by about 25% until the beginning of the next contraction. 6. It is concluded that the contractile activity in the mesotubarium, as opposed to that of the fallopian tube (Lydrup & Hellstrand, 1986a), is independent of the hormonal phase, including the period around ovulation. The mechanism for initiation of the trains of action potentials associated with spontaneous contractions may involve a gradual decrease of permeability of K+ channels or activity of the Na+-K+ pump during the relaxed period.

Rate of oxidative and glycolytic metabolism in the guinea-pig oviduct in relation to contractility and hormonal cycle.

The rates of oxygen consumption and lactate production in the guinea-pig oviduct were measured together with registration of contractile activity during three phases of the hormonal cycle. In pro-oestrus (high oestrogen, low progesterone levels) and oestrus (time of ovulation, high oestrogen and progesterone) the rate of O2 consumption was higher than in dioestrus (low oestrogen, high progesterone). The frequency of spontaneous contractions was higher in oestrus than in the other phases. No significant differences in the proportion of the cross-sectional area occupied by smooth muscle were found between oviducts in di- and pro-oestrus. Stimulation by phenylephrine caused decreased frequency and increased amplitude of contractions in dioestrus but not in pro-oestrus, suggesting hormonal modulation of adrenergic mechanisms. The rate of relaxation of high-K+ contractures was higher in pro- than dioestrus. Lactate production and contents of ATP, ADP and phosphocreatine showed no significant variation with hormonal state. The increased rate of oxidative metabolism under oestrogenic dominance could in part reflect changes in ionic transport mechanisms, such as intracellular Ca2+ handling.

Acute and long-term effects of 17 beta-oestradiol on agonist-stimulated force in rat tail artery.

The effects of 17 beta-oestradiol on the force responses to KCI and noradrenaline were investigated in rings of the rat tail artery. Incubation with 10 microM 17 beta-oestradiol for 100-295 min reduced the force amplitude after 5 min in high-K+ (140 mM) to 10% of the control value. The inhibitory effect of the steroid was unaffected by the NO-synthase inhibitor L-NAME. Rings activated by an intermediate degree of depolarization (60 mM K+) were less affected by the steroid (58% of control force). The sustained force response to 1 microM noradrenaline was reduced in the presence of 17 beta-oestradiol to 60% of control value. Lower concentrations of 17 beta-oestradiol (0.1 and 1 microM) were without acute effects on force development. However, longterm effects of 17 beta-oestradiol on vessel reactivity were found at these low concentrations. Rings were cultured for 3-7 days in the absence or in the presence of the steroid before they were stimulated with agonists. Cultured rings developed an increased sensitivity to noradrenaline compared with freshly prepared ones. Cocaine (30 microns) shifted the noradrenaline concentration-response curve to the left in freshly prepared rings while it had no effect in cultured ones, indicating that the increased sensitivity to noradrenaline in cultured rings depends on loss of noradrenaline uptake. Rings cultured for 7 days in the presence of 0.1 microM 17 beta-oestradiol developed a more pronounced supersensitivity to noradrenaline (EC50 for noradrenaline was 0.13 +/- 0.03 microM in steroid exposed rings vs. 0.38 +/- 0.09 microM in control rings). Thus, prolonged treatment with 17 beta-oestradiol results in a potentiation of noradrenaline evoked force, in contrast to the acute effect of the steroid.

Effects of extracellular K+ and Ca2+ on membrane potential, contraction and 86Rb+ efflux in guinea-pig mesotubarium.

The effects of varying extracellular concentrations of K+ and Ca2+ [K+]o and [Ca2+]o on force development and membrane potential were investigated in the guinea-pig mesotubarium. At [K+]o up to 40 mM, spontaneous action potentials were present, while higher [K+]o gave sustained contractures at a stable membrane potential (-24 to -12 mV for [K+]o from 60 to 120 mM). Tension decreased successively with increasing [K+]o from 30 to 120 mM. The relaxing potency of the dihydropyridine Ca2+ antagonist, felodipine, increased as the membrane was depolarized with increasing [K+]o and action potentials ceased. These results are compatible with the existence of Ca2+ channels showing voltage-dependent affinity with dihydrophyridines. Increasing [Ca2+]o from 2.5 to 10 mM caused membrane hyperpolarization by about 11 mV and was accompanied by a lower frequency of spontaneous contractions and a longer duration of the relaxation between contractions. 86Rb+ efflux measurements in 60 mM K+ in the absence and presence of felodipine revealed a Ca2(+)-dependent component of the voltage-activated efflux. In normal solution (5.9 mM K+), efflux in the presence of felodipine was similar to the minimal value during normal spontaneous activity. The results indicate regulation of the permeability of K+ channels by the intracellular Ca2+ concentration ([Ca2+]i) and suggest participation of such channels in the generation of the regularly occurring bursts of action potentials characteristic of spontaneous activity in the mesotubarium.

Effect of glibenclamide on membrane response to metabolic inhibition in smooth muscle of rat portal vein.

Vascular smooth muscle tone is dependent on oxidative metabolism, a phenomenon of potential importance for the metabolic regulation of blood flow to tissues. The response of the rat portal vein to inhibition of cell respiration by cyanide (0.1-1 mM) is a reduction of its spontaneous myogenic activity. The trains of action potentials triggering phasic contractions are reduced in duration, while the frequency of trains is often somewhat increased as the resting membrane potential in the intervals between spike trains is less negative by 6.5 mV. Glibenclamide (10(-7) M) did not affect the resting membrane potential or spontaneous mechanical activity of oxygenated portal veins, but partly restored the depressed myogenic activity in the presence of cyanide (0.5 mM). The spike trains were longer, while the membrane was depolarized by 3 mV compared with the effects of cyanide alone. Inhibition of both oxidative and glycolytic metabolism by 2 mM NaCN in a medium where glucose was replaced by beta-hydroxybutyrate caused a hyperpolarization which was abolished by 10(-7) M glibenclamide. The relaxing effect of the K+ channel opener cromakalim (5 x 10(-9) to 6.25 x 10(-7) M) was partly antagonized by glibenclamide. Basal cytosolic [Ca2+] was increased by cyanide, while the Ca2+ transients associated with phasic contractions were reduced in duration. This latter effect was partially reversed by glibenclamide. The effect of cyanide on high-K+ contractures, which are associated with sustained membrane depolarization and not dependent on repetitive spike activity, was not influenced by 10(-7) M glibenclamide. The effects of inhibited cell respiration on spontaneous electrical activity seem to reflect a depolarizing drive caused by inhibited active ion exchange mechanisms, modified by a repolarizing drive, possibly from ATP-regulated K+ channels, causing reduced duration of the spike trains. While glibenclamide affects spontaneous activity at all levels of oxidative blockade, glibenclamide-sensitive hyperpolarization is seen only when both oxidative and glycolytic metabolism is inhibited.

Effects of metabolic inhibition on cytoplasmic calcium and contraction in smooth muscle of rat portal vein.

Contractions in the rat portal vein, evoked by spontaneous action potentials or depolarizing high-K+ solution, are rapidly and reversibly inhibited by hypoxia or respiratory blockade. Intracellular free calcium ([Ca2+]i) was measured using Fura-2 to evaluate the effects of metabolic blockade on excitation-contraction coupling. Spontaneous contractions were associated with transient increases in [Ca2+]i. During exposure to cyanide (0.2-0.4 mM) or 2,4-dinitrophenol (30 microM) the duration and amplitude of the Ca2+ transients were decreased, leading to a decreased mean time integral of the individual [Ca2+]i transient, and corresponding decrease in the duration and amplitude of the contraction. Basal [Ca2+]i was increased in the presence of the metabolic inhibitors. High-K+ (40 mM) contractions caused a sustained increase in [Ca2+]i, which was not inhibited by exposure to cyanide, although the amplitude of the associated contraction was greatly reduced. Together with the earlier demonstration of decreased 20 kD myosin light chain phosphorylation under these conditions, this indicates that the activation of contraction is influenced by metabolism via the energy dependence of the light chain phosphorylation reaction. Thus at least three steps in the excitation-contraction sequence are influenced by inhibition of oxidative metabolism: membrane excitation, light chain phosphorylation, and the cross-bridge cycle. This provides mechanisms for a high degree of metabolic sensitivity of vascular tone, of importance for the adaptation of blood flow to tissue metabolic demands.

O2 consumption, aerobic glycolysis and tissue phosphagen content during activation of the Na+/K+ pump in rat portal vein.

Oxygen consumption, lactate production and tissue contents of ATP, phosphocreatine (PCr) and lactate were measured following readdition of K+ to K+-depleted rat portal veins, in order to study the energy turnover associated with Na+/K+ pumping. During incubation in K+-free medium at 37 degrees C spontaneous contractions disappeared in 10-20 min. Readdition of K+ (5.9 mM) after 40 min K+-free incubation caused hyperpolarization of the cell membrane for the first 5-10 min and then gradual depolarization with return of spontaneous action potentials and contractions by 10-20 min. During the first 4-6 min after K+ readdition aerobic lactate production was about doubled and then gradually returned to the original level (0.17 mumol/min g) at about 20 min. The increase in glycolytic rate was prevented by 1 mM ouabain. In contrast, O2 consumption (in K+-free medium, 0.38 mumol/min g) rose by about 10% when K+ was added and this increase lasted about 5 min. By 8 min after K+ addition the increased glycolysis and oxidative phosphorylation had accounted for each about the same amount of extra ATP generation over that extrapolated from the steady rate before K+ addition. The average total increase in ATP turnover in the first 8 min was 15%. During this period there was no change in the cellular content of ATP, PCr, or extractable ADP. The results indicate that Na+/K+ pumping utilizes a relatively small share of the total energy turnover in the vascular smooth muscle but is to a large extent dependent on aerobic glycolysis and therefore a major site of carbohydrate usage.

Free cytosolic calcium during spontaneous contractions in smooth muscle of the guinea-pig mesotubarium.

The free intracellular calcium ion concentration ([Ca2+]i) was measured simultaneously with isometric force in strips of guinea-pig mesotubarium using the Fura-2 technique. During the relaxed period (5-15 min) between spontaneous contractions [Ca2+]i continues to decrease after full mechanical relaxation to reach a minimal level of 86 +/- 8 nM (n = 9) just before the start of the next contraction. During the spontaneous contractions (5-15 min) [Ca2+]i reached a maximum of 211 +/- 19 nM and then oscillated between 155 +/- 16 nM and 194 +/- 9 nM. Increased extracellular Ca2+ concentration to 10 mM from the standard concentration of 1.5 mM caused a decreased frequency of spontaneous contractions and an increase in [Ca2+]i both in the relaxed and contracted states. In 10 mM extracellular Ca2+, addition of AlF4-, as 1 mM NaF + 10 microM AlCl3, caused a sustained increase in [Ca2+]i and maintained force. Addition of verapamil (10 microM) in this situation decreased [Ca2+]i to the resting level. The results suggest that the cyclic appearance of trains of action potentials is related to variation in [Ca2+]i, possibly via inactivation of Ca2(+)-dependent K+ channels.