Bilan des activités d'Anesthésie au service de Gynécologie - Obstétrique du Centre de Santé Gaspard Kamara, Dakar (Sénégal)

Objectifs : Faire le bilan des activités d'anesthésie au Centre de Santé Gaspard Kamara et proposer des recommandations pour améliorer la qualité des soins.Patientes et méthodes :Il s'agissait d'une étude rétrospective, descriptive et analytique allant du 1er Janvier au 31 Décembre 2012 et incluant toutes les patientes ayant bénéficié d'une anesthésie et dont les dossiers étaient retrouvés et exploitables. Le personnel du service d'Anesthésie-Réanimation était composé d'un médecin anesthésiste ­ réanimateur et de 5 techniciens supérieurs d'anesthésie dont 3 vacataires à temps partiel. Les paramètres étudiés étaient le profil épidémiologique des patientes, l'indication de l'anesthésie, les données de la consultation pré anesthésique, l'anesthésie peropératoire, la prise en charge postopératoire, les complications et les facteurs influençant le choix de l'anesthésie. L'exploitation statistique des données était effectuée à l'aide du logiciel SPSS version 20.0.Résultats : Durant la période d'étude, nous avons pris en charge 5147 patientes. Parmi celles-ci, 1256 ont bénéficié d'une anesthésie, soit un taux de 24,4%. Nous avons exploité 1033 dossiers (82,2%) qui répondaient aux critères d'inclusion. Le profil épidémiologique était celui d'une femme âgée en moyenne de 28 ans, primipare (50,4%), avec une parité moyenne de 2 et ayant déjà bénéficié d'une anesthésie. Près de la moitié des patientes (46,7%) étaient classées ASA 1. La césarienne était l'indication d'anesthésie la plus fréquente (91,2%) et la majorité des interventions étaient pratiquées dans un contexte d'urgence (87%). L'anesthésie peropératoire était réalisée par les techniciens supérieurs d'anesthésie dans 95,8% des cas. La rachianesthésie était la technique la plus pratiquée (86,9%). L'analgésie postopératoire associait le plus souvent le paracétamol au tramadol (76,8%). Des complications peropératoires étaient dominées par l'hypotension artérielle (10,8%). Un seul décès (0,1%) était enregistré et il n'était pas directement lié à l'anesthésie.Conclusion : La pratique de l'anesthésie est fréquente en Gynécologie Obstétrique. La faible incidence des complications observées dans notre série ne doit pas masquer les difficultés rencontrées au quotidien dans notre pratique. Pour améliorer la qualité des soins en anesthésie, nous devons augmenter l'effectif des ressources humaines qualifiées en anesthésie-réanimation et relever le plateau technique

Argan Oil Exerts an Antiatherogenic Effect by Improving Lipids and Susceptibility of LDL to Oxidation in Type 2 Diabetes Patients.

In this study, we investigate the effect of argan oil consumption on serum lipids, apolipoproteins (AI and B), CRP, and LDL susceptibility to oxidation in type 2 diabetic patients which are known to have a high level of cardiovascular risk due to lipid abnormalities and lipid peroxidation. For that, 86 type 2 diabetic patients with dyslipidemia were randomized to one group consuming 25 mL/day of argan oil during 3 weeks and control group consuming 20 g/day of butter in breakfast. After argan oil intervention, serum triglycerides decreased by 11.84%, (P = 0.001), total chol by 9.13%, (P = 0.01), and LDL-chol by 11.81%, (P = 0.02). However, HDL-chol and Apo AI increased (10.51%, P = 0.01 and 9.40%, P = 0.045, resp.). Susceptibility of LDL to lipid peroxidation was significantly reduced by increasing of 20.95%, (P = 0.038) in lag phase after argan oil consumption. In conclusion, we show for the first time that consumption of argan oil may have an antiatherogenic effect by improving lipids, and the susceptibility of LDL to oxidation in type 2 diabetes patients with dyslipidemia, and can therefore be recommended in the nutritional management of type 2 diabetes.

Factors influencing the awareness of diabetic foot risks.

INTRODUCTION: The aim of the present study was to identify factors influencing diabetic patients' awareness of the risk of foot problems. METHODS: We performed a prospective study of diabetic patients hospitalized or seen in consultation. Various factors were analyzed in order to identify those related to the patients' level of awareness of risk factors in diabetic foot. RESULTS: Ninety-one patients were included (mean age: 48; male/female gender ratio: 0.63). Over 50% of the study population was not aware of the risks of diabetic foot. Educational level and socioeconomic status had an impact on awareness of good foot health and care. Poor knowledge of the degenerative complications of diabetes was associated with age, a low educational level and low socioeconomic status. DISCUSSION: Our results revealed low levels of patient awareness concerning the potential severity of diabetic foot and the means of preventing foot problems. The patients gave a range of explanations for this marked lack of awareness; including a lack of information and financial constraints. Hence, patient education is still a major aspect of prevention in diabetes. CONCLUSION: In diabetes, there is still a need for easily assimilated, locally provided patient education.

Idiopathic corporeal hemihypertrophy associated with hemihypertrichosis.

The hemihypertrophy or hemihyperplasy is a rare congenital abnormality, characterized by an asymmetric growth of the limbs, the trunk, and the face or half of the entire body. It may be isolated or be part of several syndromes including Beckwith-Wiedemann syndrome, Klippel-Trenaunay-Weber syndrome, Silver-Russell syndrome and Proteus syndrome. In its isolated form, it is called idiopathic. The latter may be associated with several anomalies including dermatological and urogenital abnormalities with increased risk of developing embryonal tumors. We report the case of a 22-month-old infant, who was referred by his pediatrician at the age of 15 months for a corporeal hemihypertrophy associated with hemihypertrichosis. In his medical history, a second degree parental consanguinity and a hypospadias in the father and a paternal uncle were found. Clinical examination found a weight and a size greater than chronological age (3 standard deviations), a hemihypertrophy of entire left side with a difference of length and diameter between the left and right limbs of 2 cm. The hemihypertrichosis was widespread in the left body and the genital examination found a hypospadias. Biological and radiological assessments did not show any abnormality, with the exception of an initially high plasma testosterone level, which gradually normalized. Thus, the diagnosis of idiopathic hemihypertrophy with congenital hemihypertrichosis was retained. This is the fourth case reported in the literature. Its management is similar to all hemihypertrophies, which consists of an initial assessment to detect an embryonic tumor, followed by a monitoring protocol including an abdominal and renal ultrasound every 6 months until the age of 8, determination of alpha-feto-protein every 6 to 12 weeks until the age of 4 years to track the development of the two most frequent tumors: Wilms tumor and hepatoblastoma. The hemihypertrophy associated with hemihypertrichosis has been exceptionally reported and the cause of this association has not been identified to date.

Parathyroid incidentaloma. Literature review about three case reports.

BACKGROUND: Parathyroid incidentaloma is not a well-known entity. The aim of this study was to show its incidence and to discuss its management. METHODS: This was a prospective study analyzing cases of enlarged parathyroid glands discovered during thyroid surgery. The records of patients with parathyroid incidentaloma were reviewed. We also reviewed all cases of primary hyperparathyroidism (HPTPs) operated during the same period for comparison. RESULTS: Three cases of enlarged parathyroid were found. No clinical or biochemical features led us to suspect hyperparathyroidism before surgery, but a macroscopically enlarged parathyroid gland was discovered during the dissection and was removed in all three patients. CONCLUSIONS: Enlarged parathyroid glands discovered at the time of surgery may represent an early pathological stage responsible for overt primary hyperparathyroidism. In absence of major risk for recurrent nerve palsy, we recommend removal of any enlarged parathyroid discovered during neck surgery in order to avoid the risks of future surgical procedures, preserving in the same time at least one normal parathyroid gland.

[Amyloid goiter: first sign of systemic amyloidosis]. / Goitre amyloïde : première manifestation d'une amylose systémique.

Although amyloid infiltration of the thyroid gland is an uncommon but well-known phenomenon, the appearance of a goiter secondary to amyloid deposits is rare. The goiter enlarges rapidly and progressively, often becoming compressive like thyroid cancer. The diagnosis is rarely suggested clinically even in the presence of known amyloidosis. We describe the case of a 45-year-old patient who presented an amyloid goiter as the first manifestation of systemic amyloidosis, probably secondary to bronchiectasis.

Is Ramadan fasting safe in type 2 diabetic patients in view of the lack of significant effect of fasting on clinical and biochemical parameters, blood pressure, and glycemic control?

The study objective was to determine if Ramadan fasting was safe in patients with type 2 diabetes mellitus (T2D), based upon a determination of the effect of fasting on a broad range of physiological and clinical parameters, including markers of glycemic control and blood pressure. The study was carried out in Ramadan 1422 (December 2001-January 2002) at the Diabetology Services, Hopital Ibn Sina, Rabat, Morocco. One hundred and twenty T2D Moroccan patients (62 women, 58 men), aged 48-60 yrs with well-controlled diabetes through diet and/or oral hypoglycemic drugs (OHD), received dietary instructions and readjustment of the timing of the dose of OHD (gliclazide modified release) according to the fasting/eating periods. Anthropometric indices and physiological parameters (blood pressure, lipid, hematological, and serum electrolyte profiles, as well as markers of glycemic control, nutrition, renal and hepatic function) were measured on the day before Ramadan and then on the 15(th) and 29(th) day of fasting and thereafter 15 days later. Statistical analysis was done by standard methods. Ramadan fasting had no major effect on energy intake, body weight, body mass index, blood pressure, and liver enzymes. Fasting and post-prandial glucose levels decreased, while insulin levels increased. Diabetes was well controlled, as indicated by HbA1c, fructosamine, C-peptide, HOMA-IR, and IGF-1 values. There were fluctuations in some lipid and hematological parameters, creatinine, urea, uric acid, total protein, bilirubin, and electrolytes; however, all values stayed within the proper physiological range. In conclusion, diabetes was well-controlled in patients with dietary/medical management, without serious complications. With a regimen adjustment of OHD, diet control, and physical activity, most patients with T2D whose diabetes was well-controlled before Ramadan can safely observe Ramadan fasting.

[Characterization of RET proto-oncogene C634Y mutation in a Moroccan family with multiple endocrine neoplasia type 2A]. / Caractérisation de la mutation C634Y du proto-oncogène RET dans une famille marocaine de néoplasie endocrinienne multiple de type 2A.

Medullary thyroid carcinoma (MTC) is a rare cancer which originates from the calcitonin producing "C" cells of thyroid gland. It presents in as isolated form or as part of the multiple endocrine neoplasia type 2 (MEN 2). The familial form of MTC which frequency remains underestimated, account for 25 to 40% of all MTC presentations. All hereditary forms are transmitted in an autosomal dominant manner and are due to proto-oncogene RET germ line mutations. Although MCT is relatively rare, preclinical or prebiological diagnosis can be achieved with genetic screening with high specificity and sensitivity. Early diagnosis is crucial for disease prevention. In this study we identified the first RET mutation underlying NEM 2A in Morocco. The C634Y mutation was present in the heterozygous state in a Moroccan family with MEN 2A. Genetic screening showed that six asymptomatic members of this family were not C364Y carriers. This report should contribute to the development of genetic screening for NEM 2 and F-MTC in Morocco.

[Pure gonadal dysgenesis XX and XY: observations in fifteen patients]. / Dysgénésies gonadiques pures XX et XY: à propos de 15 cas.

BACKGROUND: Pure gondal dysgenesis is characterized by impuberism with a female phenotype without genital ambiguity. The aim of the study is to describe the diagnostic and therapeutic patterns as well as the clinical features. PATIENTS AND METHODS: A retrospective study of 15 patients with pure gonadal dysgenesis (15 patients, 46 XX and two 46 XY). Clinical parameters, familial cases, serum gonadotropin levels, pelvic ultrasonography, endoscopic data, karyotype, analysis of SRY (sex determining Y chromosome) and therapeutic control and clinical course were recorded. RESULTS: Average age at diagnosis was 21+/-2.83 years. Primary amenorrhea was the most frequent reason for consultation. A familial case was found in five patients. The association of sensorineural deafness was noted in one patient, suggesting Perrault's syndrome. Serum gonadotropin levels were elevated. Celioscopic evaluation carried out for six patients confirmed the diagnosis. There was one case of uterine and vaginal aplasia association (Mayer-Rokytansky-Küster-Hauser syndrome). In one XY patient, SRY analysis was normal. Prophylactic gonadectomy was performed in both XY patients. Substitution therapy was initiated in 11 patients. Follow-up in 6 patients revealed development of secondary sexual characters. DISCUSSION: The clinical, biological and histological features of our patients presenting pure gonadal dysgenesis XX were in agreement with earlier reports in the literature. Familial cases suggest possible autosomal transmission. The lack of a mutation in XY patients suggests a post-transcription anomaly. Complete or parital dysgenesis can be identified by histological analysis of the gondads. CONCLUSION: Study of sex determining genes should provide new perspectives for earlier diagnosis and treatment of pure gondadal dysgenesis.

Gender-specific leptinemia and its relationship with some components of the metabolic syndrome in Moroccans.

The levels of the liporegulatory hormone leptin are increased in obesity, which contributes to the metabolic syndrome; the latter is associated with elevated cardiovascular risk and morbidity. Leptin may play a role in the metabolic syndrome since correlations have been observed between serum leptin levels and several components of the metabolic syndrome. The association of leptinemia and hypertension or diabetes is inconsistent. Leptin levels are higher in females versus males and obese versus lean individuals. We investigated if correlations exist between leptin levels and several indices of the metabolic syndrome in obese and lean Moroccan subjects with (63 males, 129 females) and without (123 males, 234 females) diabetes and/or hypertension. Plasma glucose and insulin and systolic and diastolic blood pressures were higher in obese versus lean individuals. Obesity had no effect on lipid profile, plasma IGF-1, or C-peptide levels. Leptin levels were higher in females versus males and in obese versus lean individuals. The levels correlated significantly with body mass index. Serum leptin concentration did not correlate with either systolic or diastolic blood pressure, although there was a trend for higher blood pressure with increased leptin levels in females. There was no significant difference in leptin levels between NIDDM patients and healthy controls. However, in hypertensive patients, leptin levels were significantly higher in both lean males and females with diabetes as compared to those without diabetes. Similarly, the higher leptin levels paralleled elevated insulin levels in obese nondiabetic males and females, and in male and female diabetics with hypertension. Correlations were observed between leptin levels and C-peptide (an estimate of endogenous insulin secretion), but not with serum IGF-1. The calculated values of HOMA-IR, a marker of insulin resistance, were somewhat higher, parallel with elevated leptin levels, in obese male and female individuals compared to their lean counterparts. There was no relationship between leptin levels and serum lipids. There was a trend for increased serum uric acid levels with higher leptin concentrations. Thus, leptinemia is related to some components of metabolic syndrome, and in turn, it may contribute to the syndrome. This study is novel in that relationships were determined between leptin levels and various indices of metaboli syndrome in a large population of the same ethnic/regional background.

Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+) channel cell surface expression.

The epithelial Na(+) channel (ENaC) plays an essential role in the regulation of whole body Na(+) balance and blood pressure. The cell surface expression of this channel, a complex of three subunits (alpha, beta and gamma ENaC), has been shown to be regulated by hormones such as aldosterone and vasopressin and by intracellular signaling, including ubiquitylation and/or phosphorylation. However, the molecular mechanisms involving phosphorylation in the regulation of ENaC are unclear. Here we show by expression studies in Xenopus laevis oocytes that the aldosterone-induced Sgk1 kinase interacts with the ubiquitin protein ligase Nedd4-2 in a PY motif-dependent manner and phosphorylates Nedd4-2 on Ser444 and, to a lesser extent, Ser338. Such phosphorylation reduces the interaction between Nedd4-2 and ENaC, leading to elevated ENaC cell surface expression. These data show that phosphorylation of an enzyme involved in the ubiquitylation cascade (Nedd4-2) controls cell surface density of ENaC and propose a paradigm for the control of ion channels. Moreover, they suggest a novel and complete signaling cascade for aldosterone-dependent regulation of ENaC.

Effects of 8-cpt-cAMP on the epithelial sodium channel expressed in Xenopus oocytes.

Vasopressin stimulates the activity of the epithelial Na channel (ENaC) through the cAMP/PKA pathway in the cortical collecting tubule, or in similar amphibian epithelia, but the mechanism of this regulation is not yet understood. This stimulation by cAMP could not be reproduced with the rat or Xenopus ENaC expressed in Xenopus oocyte. Recently, it was shown that the alpha-subunit cloned from the guinea-pig colon (alpha gp) could confer the ability to be activated by the membrane-permeant cAMP analogue 8-chlorophenyl-thio-cAMP (cpt-cAMP) to channels produced by expression of alpha gp, beta rat and gamma rat ENaC subunits. In this study we investigate the mechanism of this activation. Forskolin treatment, endogenous production of cAMP by activation of coexpressed beta adrenergic receptors, or intracellular perfusion with cAMP did not increase the amiloride-sensitive Na current, even though these maneuvers stimulated CFTR (cystic fibrosis transmembrane conductance regulator)-mediated Cl currents. In contrast, extracellular 8-cpt-cAMP increased alpha gp, beta rat and gamma rat ENaC activity but had no effect on CFTR. Swapping intracellular domains between the cpt-cAMP-sensitive alpha gp and the cpt-cAMP-resistant alpha rat-subunit showed that neither the N-terminal nor the C-terminal of alpha ENaC was responsible for the effect of cpt-cAMP. The mechanisms of activation of ENaC by cpt-cAMP and of CFTR by the cAMP/PKA pathway are clearly different. cpt-cAMP seems to increase the activity of ENaC formed by alpha gp and beta gamma rat by interacting with the extracellular part of the protein.

Functional analyses of a N-terminal splice variant of the alpha subunit of the epithelial sodium channel.

The amiloride-sensitive epithelial sodium channel (ENaC) is the limiting step for sodium absorption in epithelial cells of the distal nephron, distal colon, airways and excretory ducts of several glands. In vivo and in vitro studies showed that the alpha subunit of ENaC is necessary for the expression of functional channels. Using RT-PCR strategy, a novel N-terminal splice variant has been identified which deletes 49 amino acids in the N-terminal region of the mouse alphaENaC subunit. In oocytes expressing the alphaENaC splice variant, together with beta and gammaENaC subunits, amiloride-sensitive currents were less than 20% of values obtained with the wild type ENaC. The single channel conductance and the ionic selectivity were similar and there was only a minor decrease in the level of expression of the protein at the oocyte surface. These findings indicate that the deleted sequence in the N-terminal part of the mouse and rat alphaENaC subunit might play a role in the regulation of the activity of expressed ENaC channels.

Functional expression of a pseudohypoaldosteronism type I mutated epithelial Na+ channel lacking the pore-forming region of its alpha subunit.

The autosomal recessive form of type I pseudohypoaldosteronism (PHA-I) is an inherited salt-losing syndrome resulting from diminution-of-function mutations in the 3 subunits of the epithelial Na+ channel (ENaC). A PHA-I stop mutation (alpha(R508stop)) of the ENaC alpha subunit is predicted to lack the second transmembrane domain and the intracellular COOH-terminus, regions of the protein involved in pore function. Nonetheless, we observed a measurable Na+ current in Xenopus laevis oocytes that coexpress the beta and gamma subunits with the truncated alpha subunit. The mutant alpha was coassembled with beta and gamma subunits and was present at the cell surface at a lower density, consistent with the lower Na+ current seen in oocytes with the truncated alpha subunit. The single-channel Na+ conductance for the mutant channel was only slightly decreased, and the appearance of the macroscopic currents was delayed by 48 hours with respect to wild-type. Our data suggest novel roles for the alpha subunit in the assembly and targeting of an active channel to the cell surface, and suggest that channel pores consisting of only the beta and gamma subunits can provide significant residual activity. This activity may be sufficient to explain the absence of a severe pulmonary phenotype in patients with PHA-I.

Comparison of NovoPen 3 and syringes/vials in the acceptance of insulin therapy in NIDDM patients with secondary failure to oral hypoglycaemic agents.

This open, randomised, cross-over study compared the acceptance and safety of NovoPen 3 with that of conventional syringes and vials when initiating insulin treatment in 96 NIDDM patients with secondary failure to oral hypoglycaemic agents. These patients had not previously been treated with insulin. All patients used each insulin administration system for 12 weeks. Group A started therapy using NovoPen 3 and crossed over to syringe/vial administration; Group B started with syringe/vial administration followed by NovoPen 3. In total, 78 patients completed the study. Most patients in Group A initially found the insulin injections very easy or easy and many of those who found injections easy at first found them very easy by the end of week 12. During the first period, patients in Group B found insulin administration more difficult than those in Group A. Injection pain was significantly lower with NovoPen 3 than with syringes and vials (P = 0.0018). Patients in Group B reported a significantly lower level of injection pain after the switch to using NovoPen 3 (P = 0.0003). Acceptance of insulin injections was significantly higher by patients using NovoPen 3 than by those using syringes and vials (P = 0.0059). Setting and drawing up the dose of insulin was also easier for patients using NovoPen 3 (P = 0.0490). At the end of the study, most patients (89.5% (68/76 replies)) said that they preferred NovoPen 3 to syringes and vials. Glycaemic control improved compared with baseline after starting insulin therapy, with no differences between Groups A and B, or between the two injection systems. The number of reported hypoglycaemic episodes was very low and was not significantly different between Groups A and B, or between the two administration systems. No treatment-related adverse events were reported. We conclude that use of NovoPen 3 provides better acceptance of insulin injection than use of conventional syringes and vials during initiation of insulin therapy in NIDDM patients with secondary failure to treatment with oral hypoglycaemic agents.

Protease modulation of the activity of the epithelial sodium channel expressed in Xenopus oocytes.

We have investigated the effect of extracellular proteases on the amiloride-sensitive Na+ current (INa) in Xenopus oocytes expressing the three subunits alpha, beta, and gamma of the rat or Xenopus epithelial Na+ channel (ENaC). Low concentrations of trypsin (2 microg/ml) induced a large increase of INa within a few minutes, an effect that was fully prevented by soybean trypsin inhibitor, but not by amiloride. A similar effect was observed with chymotrypsin, but not with kallikrein. The trypsin-induced increase of INa was observed with Xenopus and rat ENaC, and was very large (approximately 20-fold) with the channel obtained by coexpression of the alpha subunit of Xenopus ENaC with the beta and gamma subunits of rat ENaC. The effect of trypsin was selective for ENaC, as shown by the absence of effect on the current due to expression of the K+ channel ROMK2. The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-gammaS, suggesting that this effect was not mediated by G proteins. Measurement of the channel protein expression at the oocyte surface by antibody binding to a FLAG epitope showed that the effect of trypsin was not accompanied by an increase in the channel protein density, indicating that proteolysis modified the activity of the channel present at the oocyte surface rather than the cell surface expression. At the single channel level, in the cell-attached mode, more active channels were observed in the patch when trypsin was present in the pipette, while no change in channel activity could be detected when trypsin was added to the bath solution around the patch pipette. We conclude that extracellular proteases are able to increase the open probability of the epithelial sodium channel by an effect that does not occur through activation of a G protein-coupled receptor, but rather through proteolysis of a protein that is either a constitutive part of the channel itself or closely associated with it.

An epithelial serine protease activates the amiloride-sensitive sodium channel.

Sodium balance, and ultimately blood pressure and extracellular fluid volume, is maintained by precise regulation of the activity of the epithelial sodium channel (ENaC). In a Xenopus kidney epithelial cell line (A6), exposure of the apical membrane to the protease inhibitor aprotinin reduces transepithelial sodium transport. Sodium-channel activity can be restored by subsequent exposure to the nonspecific protease trypsin. Using A6 cells and a functional complementation assay to detect increases in ENaC activity, we have cloned a 329-residue protein belonging to the serine protease family. We show that coexpression of this protein with ENaC in Xenopus oocytes increases the activity of the sodium channel by two- to threefold. This channel-activating protease (CAP1) is expressed in kidney, gut, lung, skin and ovary. Sequence analysis predicts that CAP1 is a secreted and/or glycosylphosphatidylinositol-anchored protein: ENaC activity would thus be regulated by the activity of a protease expressed at the surface of the same cell. This previously undiscovered mechanism for autocrine regulation may apply to other ion channels, in particular to members of the ENaC family that are present in neurons and epithelial cells.

Effects of internal pH on the nonselective cation channel from the mouse collecting tubule.

We investigated the effects of internal pH on Ca-activated, nucleotide-inhibited nonselective cation channels in the basolateral membranes of mouse collecting tubules, using the inside-out variant of the patch clamp technique. pH modulated the channel open probability (Po), giving a bell-shaped curve peaking at pH 6.8/7.0: Po at pH 6.0 was 11 +/- 6% of Po at pH 7.2 and 32 +/- 7% at pH 8.0. The open and closed time distributions, best fitted to the sum of two exponentials, were differently sensitive to acid and alkaline conditions. Low pH reduced the short and long open times to 38 and 24% of their pH 7.2 values, while high pH produced a 4-fold increase in the long closed time. As previously reported, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) induced a quasi-permanent opening of the channel. The inhibition of the channel produced by high pH disappeared in the presence of SITS, while the inhibition produced by low pH was unaffected. These results suggest that the pH dependence of the channel is due to two separate mechanisms. pH was without effect on the ATP-evoked inhibition of the channel, while high pH profoundly reduced the steepness of the AMP inhibition curve, without altering the half-maximal inhibitory AMP concentration.

A small-conductance Cl- channel in the mouse thick ascending limb that is activated by ATP and protein kinase A.

1. Chloride channels were identified in the basolateral membrane of isolated cortical thick ascending limbs (CTALs) of the mouse nephron by the patch-clamp technique. A channel with a conductance of 45 pS, previously shown to be Cl- selective, was detected in 21% of cell-attached patches when CTAL fragments were pre-incubated with 10 mumol l-4 forskolin for at least 15 min. The same channel was found in only 8.5% of cell-attached patches formed on unstimulated tubules. 2. Another channel with a smaller conductance (7-9 pS) was found in 42.8% of cell-attached patches and 57% of inside-out patches in unstimulated CTAL tubules, but in 82-87% of patches from forskolin-treated tubules. 3. The small channels was Cl- selective (Cl(-)-to-Na+ permeability ratio, PCl/PNa = 9.8) with the permeability sequence: NO3- > Br- > Cl- > F- > gluconate. Channel activity decreased (Br-) or disappeared (NO3-) at negative voltages. At 140 mmol l-1, I- completely inhibited channel activity at all voltages, but a PI/PCl ratio of 1.6 was estimated using a low I- concentration (10 mmol l-1). 4. Internal adenosine triphosphate (ATP) increased normalized current (nPo) in 48% of inside-out patches containing Cl- channels from unstimulated tubules and in 63% of patches from forskolin-treated CTAL tubules. The non-hydrolysable ATP analogue, adenosine 5'-adenylyl imidodiphosphate (AMP-PNP) did not increase channel activity. 5. Adding the catalytic subunit of protein kinase A to the bath in the presence of ATP increased the activity of the small channel in 58% of inside-out patches from unstimulated tubules, but it had no effect on the 45 pS channel. 6. The Cl- channel blockers 5-nitro-2-(3-phenylpropylamine)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) or glibenclamide, all at 0.1 mmol l-1, and diphenylamine-2-carboxylic acid (DPC), at 1 mmol l-1, inhibited the small channel activity by 80-100% in inside-out patches. 7. These results indicate that two Cl- channels with contrasting properties mediate the basolateral step of NaCl absorption in the thick ascending limb of the loop of Henle.