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Reg (regenerating gene) family proteins are known to be overexpressed in gastrointestinal (GI) tissues under conditions of inflammation. However, the pathophysiological significance of Reg family protein overexpression and its regulation is still unclear. In the present study, we investigated the profile ofRegfamily gene expression in a colitis model and focused on the regulation of Reg IIIβand IIIγ, which are overexpressed in inflamed colonic mucosa. C57BL/6 mice were administered 2% dextran sulfate sodium (DSS) in drinking water for five days, and their colonic tissues were investigated histopathologically at interval for up to 12 weeks. Gene expression of theRegfamily and cytokines (IL-6,IL-17, andIL-22) was evaluated by real-time RT-PCR, and Reg IIIβ/γexpression was examined by immunohistochemistry. The effects of cytokines on STAT3 phosphorylation and HIP/PAP (type III REG) expression in Caco2 and HCT116 cells were examined by Western blot analysis. AmongRegfamily genes,Reg IIIβandIIIγwere alternatively overexpressed in the colonic tissues of mice with DSS-induced colitis. The expression of STAT3-associatedcytokines(IL-6,IL-17, andIL-22) was also significantly increased in those tissues, being significantly correlated with that ofReg IIIβ/γ. STAT3 phosphorylation and HIP/PAP expression were significantly enhanced in Caco2 cells upon stimulation with IL-6, IL-17, and IL-22. In HCT116 cells, those enhancements were also observed by IL-6 and IL-22 stimulations but not IL-17. The link between type III Reg and STAT3-associated cytokines appears to play a pivotal role in the pathophysiology of DSS-induced colitis.

Aim: To describe the longitudinal changes in canal length at 16–35 weeks' gestation in cases of twin pregnancy with preterm labor and delivery. Methods: We studied 22 cases of twin pregnancy that were delivered at

Aim: The aim of this study was to investigate the influence of preoperative hormonal therapy before laparoscopic cystectomy of ovarian endometriomas. We identified differences in follicle loss and surgical difficulties with or without preoperative hormonal therapy. Methods: Ninety-six patients with ovarian endometrioma underwent a laparoscopic cystectomy. Patients were divided into three groups: control group A (53 patients, 57 cysts) with no preoperative hormonal therapy; group B (34 patients, 40 cysts) who received gonadotropin-releasing hormone agonist therapy; and group C (9 patients, 11 cysts) who received danazol therapy before surgery. The medical and videotape records of all patients were retrospectively reviewed. The specimens of endometriomas were histologically evaluated. Results: Mean diameters of endometriomas before hormonal therapy in groups B and C were significantly greater than those in the control group. There were no significant differences in the following: the mean diameter of removed cysts, the revised-American Society of Reproductive Medicine scores, the number of capsules containing follicle(s), and the mean number of follicles attached to a cyst. However, the number of capsules showing fibrosis significantly increased in the preoperative hormonal therapy groups (P

Objective: The aim of this study was to clarify the changes in lipid metabolism in postmenopausal Japanese women during continuous combined hormone therapy (HT) in detail by using capillary isotachophoresis (cITP). Design: Twenty-three postmenopausal Japanese women with climacteric symptoms were recruited. Blood samples were collected from all participants before HT and after 3 and 6 months of HT, and changes in total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and apolipoprotein (apo) A-I, apoB, and lipoprotein(a) were assessed. The same blood samples were analyzed for charge-based lipoprotein subfractions using cITP. Results: Total cholesterol (-8.5%), LDL-cholesterol (-16.2%), and lipoprotein(a) (-25.5%) decreased and apoA-I (9.5%) increased significantly from the baseline level at 6 months after starting continuous combined HT. HDL-cholesterol increased nonsignificantly (+2.4%). cITP revealed that the fast HDL subfraction (+24.7%), which contains apoA-I and may be antiatherogenic, significantly increased. Conversely, the fast LDL subfraction (+14.4%), which may possess atherogenic potential, increased during HT. Conclusions: The changes in lipid metabolism in postmenopausal Japanese women receiving HT are inconclusive regarding atherogenicity, ie, it increased fast HDL and simultaneously increased fast LDL. cITP is useful for clarifying the changes in lipid metabolism during HT.

Electrophysiological characteristics were compared among four cloned human α1H isoforms transcripted by alternative splicings of exons 25B and 26 [Δ25B/+26 (native form; α1H-a), Δ25B/Δ?6 (α1H-b), +25B/Δ26, and +25B/+26] in the intracellular loop between domains III and IV (III-IV linker) of a human T-type calcium channel (Cav3.2). The native isoform Δ25B/+26 predominated in ovary and non-pregnant uterus, while isoform Δ25B/Δ26 (α1H-b) predominated in pregnant uterus and testis. Expressions of the newly identified +25B/Δ26 and +25B/+26 isoforms were greater in the uterus at gestation than in the non-pregnant uterus. When expressed in Xenopus laevis oocytes, all isoforms produced transient inward currents with low voltage-dependent activation and inactivation characterized in typical T-type Ca2+ currents. Each isoform possessing exon 25B (+25B/?Δ26 or +25B/+26) showed current activation and inactivation at a more negative membrane potential than the respective isoform (Δ25B/Δ26 or Δ25B/+26) lacking it. Moreover, the current activation and inactivation rates were faster for the two isoforms possessing exon 25B than for the respective isoforms lacking it. By itself, exon 26 seemed not to affect any electrophysiological characteristics. Increasing the net positive charge (relative to the native form), as occurred in isoforms Δ25B/Δ26, +25B/Δ26, and +25B/+26, caused recovery from short-term inactivation to become faster. Our results show that molecular-structure variations within the III-IV linker influence the voltage-dependence and kinetics of both activation and inactivation. Although the role of T-type Ca2+ channels in uterine tissue remains unknown, changes in the uterine expression of these α1H isoforms may influence physiological functions during pregnancy.

Effects of several Cl− channel blockers on ionic currents in mouse embryos were studied using whole-cell patch-clamp and microelectrode methods. Microelectrode measurements showed that the resting membrane potential of early embryonic cells (1-cell stage) was −23 mV and that reduction of extracellular Cl− concentration depolarized the membrane, suggesting that Cl− conductance is a major contributor for establishing the resting membrane potential. Membrane currents recorded by whole-cell voltage clamp showed outward rectification and confirmed that a major component of these embryonic currents are carried by Cl− ions. A Cl− channel blocker, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), suppressed the outward rectifier current in a voltage- and concentration-dependent manner. Other Cl− channel blockers (5-nitro-2-[3-phenylpropyl-amino] benzoic acid and 2-[3-(trifluoromethyl)-anilino] nicotinic acid [niflumic acid]) similarly inhibited this current. Simultaneous application of niflumic ...

The properties of hyperpolarization-activated current in pregnant rat uterus (17-19 days gestation) were investigated using microelectrode and patch-clamp techniques, and isometric tension recording. The resting membrane potentials were -58.4 mV and -48.5 mV in longitudinal and circular muscle cells, respectively. Application of hyperpolarizing current pulses produced a time-dependent anomalous inward rectification of membrane potential only in circular muscle cells. Under voltage-clamp conditions, inward currents (Ih) were activated by long hyperpolarizing pulses below -60 mV in circular but not in longitudinal muscle cells. Application of extracellular but not intracellular Cs+ reduced the amplitude of I(h) in a concentration-dependent manner (an IC50( of 0.15 mM). The reversal potential for Ih was -26.2 mV and the slope conductance was 5 nS/pF. Changes in [K+]o and [Na+]o shifted the reversal potential, and Ih amplitude increased with excess [K+]o and decreased with low [Na+]o. The steady-state activation of Ih was well fitted by a Boltzmann equation with a half-activation potential of -84.3 mV and a slope factor of 9.6 mV. Time courses of activation and deactivation of the current strongly depended on membrane potential, and were well fitted by a single exponential function. The activation time constant of Ih was dependent on temperature. In isometric tension recording, application of extracellular Cs+ to the circular muscles reduced the frequency, but not the amplitude, of spontaneous contractions in a concentration-dependent manner. It is concluded that in pregnant rat uterus Ih channels are predominantly distributed in smooth muscle cells from the circular layer. Since Ih is activated at the resting membrane potential, it is likely that this current contributes to the maintenance of resting membrane potential and spontaneous activity in circular smooth muscle cells of late pregnant rats.

The purpose of this study was to investigate the actions of 17beta-estradiol on the electrical activity of pregnant rat myometrium. The longitudinal layer of the myometrium was dissected from pregnant rats (17 to 19 days of gestation), and single cells were isolated by enzymatic digestion. Calcium currents and potassium currents were recorded by the whole-cell voltage-clamp method, and the single calcium-dependent potassium current was recorded by the outside-out patch-clamp method. The effects of 17beta-estradiol on these currents were investigated. When a myometrial cell was held at -50 mV, depolarization to a potential more positive than -30 mV produced an inward current followed by a slowly developing outward current. Application of tetraethylammonium inhibited the outward current while the inward current was completely abolished in a calcium-free solution. Estradiol at high concentrations (> 3 microM) inhibited both inward and outward currents in a voltage-dependent manner. Removal of estradiol restored the amplitude of the outward but not of the inward current. Estradiol (30 microM) also inhibited the activity of single calcium-dependent potassium channels without changing single channel conductance. In conclusion, estradiol at high concentrations inhibited: (1) voltage-dependent calcium, (2) calcium-dependent potassium and (3) voltage-dependent potassium currents. These actions of estradiol would prevent action potential generation and after-hyperpolarizations. Suppression of the after-hyperpolarization might further prevent spike generation due to slowing of the calcium channel's recovery from the inactivated state.

Objective: We previously reported that 0.625 mg/day of conjugated equine estrogen (CEE) could not prevent acute bone loss in the first year after oophorectomy. The effect of additional administration of ipriflavone on bone mineral density (BMD) and biochemical indices of bone remodeling were studied to investigate whether concurrent use of CEE and ipriflavone prevent acute bone loss in the early stages following surgical menopause. Methods: One-hundred and sixteen oophorectomized women were randomly divided into four groups according to treatment; group 1: placebo, n=30; group 2: CEE (0.625 mg/day), n=29; group 3: ipriflavone (600 mg/day), n=30; group 4: CEE (0.625 mg/day) plus ipriflavone (600 mg/day), n=27. Vertebral BMD was measured using dual energy X-ray absorptiometry (DEXA) and two biochemical indices of bone metabolism, urinary pyridinoline (Pyr) and serum intact human osteocalcin (hOC), were also measured before, 24 weeks, and 48 weeks after initiation of treatment. Results: BMD was reduced 48 weeks after treatment by 6.1, 3.9 and 5.1% in groups 1–3, respectively, but by only 1.2% in group 4. Pyr decreased by 49.5, 32.0 and 41.5% in groups 2–4, respectively. hOC also decreased by 45.2 and 21.6% in groups 2 and 4, but increased by 40.5% in group 3, suggesting an inhibitory action of CEE and ipriflavone on the turnover of bone metabolism and stimulatory action of ipriflavone on bone formation. Conclusion: Concomitant use of ipriflavone with CEE from an early stage after oophorectomy inhibited bone loss and was considered to be effective in maintaining bone mass after oophorectomy.

Objective: The effects of conjugated equine estrogen (CEE) on bone mineral density (BMD) and biochemical indices of bone remodeling in oophorectomized women were studied for 3 years during estrogen replacement therapy (ERT) to investigate whether 0.625 mg/day of CEE alone prevent acute bone loss in the early stage of surgical menopause. Methods: We divided the subjects into three groups according to interval between oophorectomy and the start of ERT (group 1: less than 2 years after surgery, n = 31; group 2: 2–5 years after surgery, n = 29; and group 3: more than 5 years after surgery, n = 27). Vertebral BMD was measured using dual energy X-ray absorptiometry (DEXA). Two biochemical indices of bone metabolism, urinary deoxypyridinoline (DPyr) and serum intact human osteocalcin (hOC) were also measured. Results: In group 1, continuous ERT with 0.625 mg/day of CEE could not prevent a BMD decrease within the first year. However, by the end of the second year, BMD was restored to the pre-ERT. The same dosage of CEE significantly increased BMD in groups 2 and 3 by the end of the first year. DPyr and hOC levels both decreased dramatically in the initial 6 months of therapy and were stable thereafter. Conclusion: In the initial 2-year period after oophorectomy, 0.625 mg/day of CEE alone could not prevent acute bone loss suggesting that additional therapy for the prevention of osteoporosis may be needed.

Most patients with Asherman's syndrome present with infertility and menstrual problems. In this retrospective clinical study, we analyzed patients with Asherman's syndrome who underwent hysteroscopic adhesiolysis to examine their associated symptoms, disease etiologies, and fertility outcomes.Twenty-seven patients with Asherman's syndrome that were diagnosed using hysteroscopy were recruited. The chief complaints were infertility, hypomenorrhea, and amenorrhea. Each case of Asherman's syndrome was classified according to the American Fertility Society classification. Hysteroscopic adhesiolysis was performed in all cases and concomitant transabdominal ultrasonography was conducted in cases with extensive and dense adhesions.There were no complications associated with the hysteroscopic procedure. Normal menstrual cycles resumed in all cases. Of the 16 infertile patients, 9 conceived. Three patients achieved term deliveries and one patient is currently pregnant. None of the patients had obstetric complications. Two patients had spontaneous abortions, one had an ectopic pregnancy, one had an abortion at 16 weeks' gestation due to cervical incompetence, and one had a molar pregnancy and required uterine artery embolization for uncontrolled hemorrhaging during a dilatation and curettage procedure.Hysteroscopic adhesiolysis with transabdominal ultrasonography is a suitable treatment method for Asherman's syndrome. Subfertile patients with Asherman's syndrome undergoing adhesiolysis should be appropriately informed about the risk of associated life-threatening complications and preterm delivery.

1. Effects of oestradiol on the electrical and mechanical properties of the rabbit basilar artery were investigated by use of microelectrode, patch-clamp and isometric tension recording methods. 2. Oestradiol (10 nM-100 microM) relaxed arterial tissue pre-contracted by excess [K]o solution (30 mM) in a concentration-dependent manner. In Ca-free solution, histamine (10 microM) and caffeine (20 mM) each produced a phasic contraction, but oestradiol (10 microM) did not significantly affect their amplitude. 3. Oestradiol (< or = 100 microM) did not change the resting membrane potential of the artery whether in the presence or absence of TEA (10 mM). Action potentials observed in the presence of 10 mM TEA were abolished by oestradiol (100 microM). 4. Oestradiol (1 microM-100 microM) inhibited the voltage-dependent Ba current in a concentration-dependent manner. Oestradiol (100 microM) inhibited the Ba current observed in the presence of nicardipine (1 microM) more than that in the absence of nicardipine (to 31.0% vs 62.0% of control). 5. GTP gamma S (30 microM) in the pipette enhanced the inhibitory actions of oestradiol on the Ba current. On the other hand, with GDP beta S (1 mM) in the pipette, oestradiol failed to inhibit the Ba current. Pertussis toxin (PTX 3 micrograms ml-1) in the pipette totally prevented the inhibitory action of oestradiol on the Ba current. 6. Oestradiol (< or = 100 microM) had no significant effect on the outward K currents evoked by a membrane depolarization. 7. These results strongly suggest that oestradiol relaxes arterial tissue by inhibition of voltage-dependent Ca channels and that it inhibits both nicardipine-sensitive and -resistant Ca currents via a PTX-sensitive GTP-binding protein. The main target of oestradiol among the arterial Ca channels seems to be the nicardipine-resistant Ca channel, rather than the nicardipine-sensitive one.

Two kinds of outward K+ currents were examined in single smooth muscle cells from pregnant rat uterus, using whole-cell voltage clamp. The first and faster component was more sensitive to 4-aminopyridine (4-AP), whereas the second and slower (delayed) component was more sensitive to tetraethylammonium (TEA). A possible third K+ component (Ca activated K+ current) was not recorded as the pipette solution included EGTA. Forskolin inhibited the outward current in a concentration-dependent manner (50% inhibition occurred at about 30 microM); it affected the delayed component rather than the fast component. 8-Bromo-cAMP did not alter the outward current. In addition, inhibitors of protein kinase A and GDP-beta S and GTP-gamma S did not affect the forskolin-induced inhibition. These results indicate that forskolin inhibition of the delayed component of the outward current is independent of cAMP generation in the pregnant rat myometrial cells. Therefore, forskolin seems to directly inhibit specific K+ channels, as was reported for several other cell types.

To describe the longitudinal changes in canal length at 16-35 weeks' gestation in cases of twin pregnancy with preterm labor and delivery.We studied 22 cases of twin pregnancy that were delivered at36 weeks and/or that underwent preterm labor requiring tocolysis. We also studied 44 cases of twin pregnancy delivered ator = 36 weeks without tocolysis (non-tocolysis twin pregnancy). Controls were 82 cases of normal singleton pregnancy. Canal length was longitudinally measured using transvaginal ultrasonography. The observational period of 16-35 weeks was divided into 4-week periods for analysis.From 28 to 31 weeks onwards the canal length of non-tocolysis twin pregnancies was shorter than that of normal singleton pregnancies (P0.05). The canal length of twin pregnancies with preterm labor and delivery was shorter than that of non-tocolysis twin pregnancies at 16-19 weeks and decreased rapidly until 24-27 weeks (P0.01).A short canal length at 16-19 weeks followed by rapid canal length shortening in the second trimester are specific characteristics in preterm labor and delivery of twin pregnancies. Sequential measurements of canal length in the second trimester starting at20 weeks may be a suitable parameter to predict preterm labor and delivery in twin pregnancies.

Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L type) Ca2+ channels and fast (T type) Ca2+ channels. Whole-cell voltage clamp was done on single smooth muscle cells isolated from the longitudinal layer of the 18-day pregnant rat uterus. Depolarizing pulses, applied from a holding potential of −90 mV, evoked two types of inward current, fast and slow. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by tetrodotoxin (TTX) (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o (or Ba2+), and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. We conclude that the ion channels that generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells that possess fast Na+ channels. The Ca2+ channel current density was also higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and we suggest that the fast Na+ current may be involved in spread of excitation. Isoproterenol (β-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 = 12 mM) and nifedipine (K0.5 = 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of β-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions cannot be explained by a stimulation of ICa(s).Key words: sodium current, fast sodium current, calcium currents, myometrial smooth muscle cells, pregnant uterine muscle.

OBJECTIVES The aim of this study was to evaluate the beta-adrenergic receptor (beta-AR) selectivity, organ specificity and efficacy of delaying the onset of spontaneous delivery of bedoradrine (KUR-1246), a novel uterine relaxant. METHODS beta-AR selectivity was evaluated in terms of the amount of cyclic adenosine monophosphate produced by bedoradrine, ritodrine and isoprenaline in Chinese hamster ovary cells expressing human beta(1)-, beta(2)-AR or beta(3)-AR. Inhibition of contractions of the atrium, trachea and proximal colon by bedoradrine were compared with those of the uterus in pregnant rats using an organ bath method. Finally, the delaying effect of bedoradrine on spontaneous labor was evaluated by an in vivo study using term pregnant rats. RESULTS EC(50) values of bedoradrine for cyclic adenosine monophosphate production in Chinese hamster ovary cells via beta(1)-, beta(2)- and beta(3)-AR were 2400 +/- 30, 2.9 +/- 0.10 and 363 +/- 3 nmol/L, respectively, indicating that bedoradrine had 832- and 126-fold higher selectivity for beta(2)-AR than for beta(1)- and beta(3)-AR. EC(50) values of bedoradrine for the uterus, atrium, trachea and proximal colon were 1.01 +/- 0.27, 2300 +/- 356, 1610 +/- 299 and 219 +/- 23.5 nmol/L, respectively. Thus, bedoradrine was 2280-, 1590- and 217-fold more specific for the uterus than for the atrium, trachea and proximal colon, respectively. Bedoradrine delayed the spontaneous delivery of 21-day-pregnant rats in a dose-dependent manner. CONCLUSIONS Bedoradrine is a promising drug for the treatment of preterm labor in obstetrical practice because it has better selectivity for beta(2)-AR and specificity for the uterus than currently used agents and may effectively delay spontaneous delivery.

Objective To report a rare uterine anomaly consisting of a normal uterus, a double cervix, and a double vagina. Design Case report. Setting University hospital. Patient(s) A 28-year-old nulligravida patient referred for evaluation of primary infertility and a suspected mullerian anomaly. Intervention(s) Clinical and surgical evaluation of the anomaly. Main Outcome Measure(s) Description and treatment for a rare uterine anomaly and a subsequent literature search. Result(s) Successful resection of vaginal septum and subsequent pregnancy. Conclusion(s) This extremely rare anomaly is not explained by classic embryologic teachings, and it does not fit into the classification system currently used to describe mullerian anomalies.

Actions of FRC8653 on the macroscopic and unitary Ba2+ currents were studied using the rabbit basilar artery. Application of (+/-)-FRC8653 (less than 1 microM) increased the amplitude of the inward current when depolarization pulses more negative than -10 mV were applied but inhibited it when depolarization was more positive than 0 mV (in each case from a holding potential of -80 mV). At a holding potential of -40 mV, (+/-)-FRC8653 (greater than 0.1 nM) consistently inhibited the inward current. (-)-FRC8653 (greater than 1 nM) inhibited the amplitude of the inward current evoked by a depolarizing pulse more positive than -10 mV (the holding potential being -80 mV). At the holding potential of -80 mV, but not at -40 mV, (+)-FRC8653 (1 microM) enhanced the current amplitude evoked by a depolarizing pulse more negative than -10 mV but inhibited the current evoked by a pulse more positive than 0 mV. (+/-)-FRC8653 shifted the voltage-dependent inhibition curves to the left, and the slope of the curve became steeper (test pulse of +10 mV). Two types of single Ca2+ channel currents (12 and 23 pS) were recorded from the basilar artery by the cell-attached patch-clamp method. Opening of the 12-pS channel occurred with a depolarizing pulse (-20 mV) from a holding potential of -80 mV, but not from one of -60 mV. (+)-FRC8653 activated, and (-)-FRC8653 inhibited, the 23-pS channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane properties of the human pregnant myometrium were investigated with the conventional microelectrode and patch clamp methods. The majority of preparations produced spontaneous action potentials at a very low frequency, and action potentials were inhibited in sodium-deficient or calcium-free solutions. With the patch clamp technique with 120 mmol/L cesium-20 mmol/L tetraethylammonium in the pipette, the inward current was evoked by a depolarizing pulse above -40 mV from a holding potential of -60 mV and maximum amplitude was obtained at 0 mV. At a holding potential of -100 mV, the inward current could be evoked with less positive depolarizing pulses, and the membrane potential that evoked the maximum amplitude of inward current was shifted to a more negative potential. Single-channel current recording revealed that two types of calcium channels existed in human pregnant myometrium, with single-channel conductances of 12 and 29 pS. One of the calcium channels (12 pS) was inactivated at a holding potential of -60 mV.

Actions of porcine endothelin (ET) on the electrical and mechanical activity of the rat portal vein were investigated by means of the intracellular microelectrode and isometric tension recording techniques, ET (greater than 0.1 nM) enhanced the amplitude and frequency of the spontaneous contractions which ceased in the presence of 100 nM dihydropyridine derivatives (nifedipine or nicardipine). ET (0.15 nM) increased the frequency of the spontaneous action potentials, with no change in the basal membrane potential. Higher concentrations of ET (greater than or equal to 0.3 nM) further depolarized the membrane potential and increased the spike frequency. After blocking the spontaneous action potentials with nifedipine (100 nM), ET still depolarized the membrane. The depolarization was associated with a reduction in the electrotonic potential and was blocked in a Na-deficient solution (15.5 mM) but not in Ca-free, K-deficient or Cl-deficient solutions. In a Na-deficient solution, ET still evoked action potentials without depolarization. In Ca-free solution, ET depolarized the membrane potential with small oscillations, which were blocked by nifedipine (100 nM). The results indicate that in the rat portal vein, ET enhances electrical and mechanical responses through activation of the dihydropyridine-sensitive and voltage-dependent Ca channels. Acceleration of the Ca entry induced by ET can occur with or without depolarization of the membrane and can enhance the pacemaking mechanism.

Objective: To identify the most important epidermal growth factor (EGF) receptor ligand in the LH or hCG signal pathway in human ovary. Design: A retrospective clinical study. Setting: Tertiary university hospital. Patient(s): Ninety-eight infertile patients who underwent IVF–embryo transfer. Intervention(s): Sera and follicular fluid were collected at the time of oocyte retrieval. The levels of EGF, transforming growth factor-alpha (TGFa), and amphiregulin (AR) were measured in follicular fluid and sera by using ELISA. Main Outcome Measure(s): The relationships between the level of AR and level of hCG, fertilization rate, and embryo quality. Result(s): Amphiregulin was abundantly expressed in follicular fluid after hCG stimulation. Although large differences were found between AR and both EGF and TGFa in follicular fluid, no significant difference was detected in the levels of the three EGF receptor ligands in sera. The level of AR was inversely correlated with the fertilization rate and hCG level, whereas little significant association was observed between the level of AR and embryo quality. Conclusion(s): Amphiregulin was expressed most dominantly among EGF receptor ligands tested and may mediate the hCG signal in human oocyte maturation. Elaborate interaction between AR and hCG may be required for an optimal oocyte maturation. (Fertil Steril� 2009;91:1035–41. � 2009 by American Society for Reproductive Med

Left ventricular outflow obstruction may result from preserving the anterior leaflet after mitral valve replacement. A 79-year-old woman, who had a mitral valve replacement with the native mitral leaflets left intact 16 years before, was admitted to our hospital with severe dyspnea due to heart failure. Echocardiography showed systolic anterior motion of preserved anterior mitral leaflet, and continuous wave Doppler detected severe left ventricular outflow tract jets during systole without mitral chordal rupture. Surgical incising of the anterior mitral leaflet through the aortic root relieved the obstruction without removing the prosthetic mitral valve.

Electrophysiological characteristics were compared among four cloned human alpha1H isoforms transcripted by alternative splicings of exons 25B and 26 [Delta25B/+26 (native form; alpha1H-a), Delta25B/Delta?6 (alpha1H-b), +25B/Delta26, and +25B/+26] in the intracellular loop between domains III and IV (III-IV linker) of a human T-type calcium channel (Ca(v)3.2). The native isoform Delta25B/+26 predominated in ovary and non-pregnant uterus, while isoform Delta25B/Delta26 (alpha1H-b) predominated in pregnant uterus and testis. Expressions of the newly identified +25B/Delta26 and +25B/+26 isoforms were greater in the uterus at gestation than in the non-pregnant uterus. When expressed in Xenopus laevis oocytes, all isoforms produced transient inward currents with low voltage-dependent activation and inactivation characterized in typical T-type Ca2+ currents. Each isoform possessing exon 25B (+25B/?Delta26 or +25B/+26) showed current activation and inactivation at a more negative membrane potential than the respective isoform (Delta25B/Delta26 or Delta25B/+26) lacking it. Moreover, the current activation and inactivation rates were faster for the two isoforms possessing exon 25B than for the respective isoforms lacking it. By itself, exon 26 seemed not to affect any electrophysiological characteristics. Increasing the net positive charge (relative to the native form), as occurred in isoforms Delta25B/Delta26, +25B/Delta26, and +25B/+26, caused recovery from short-term inactivation to become faster. Our results show that molecular-structure variations within the III-IV linker influence the voltage-dependence and kinetics of both activation and inactivation. Although the role of T-type Ca2+ channels in uterine tissue remains unknown, changes in the uterine expression of these alpha1H isoforms may influence physiological functions during pregnancy.

The aim of this study was to evaluate the difference in the expression of mRNA of two types of calcium channels between longitudinal and circular muscle layer of rat myometrium during pregnancy. Changes in the expressions of the mRNA encoding L-type (alpha1C) and T-type (alpha1G, alpha1H, and alpha1I) calcium channels in longitudinal and circular smooth muscle cells of the rat myometrium were examined using a comparative kinetic RT/PCR method. During the course of pregnancy, alpha1C mRNA expression showed an N-shaped change in longitudinal muscle, but simply increased after mid-pregnancy in circular muscles. The mRNAs for alpha1G and alpha1H, but not that for alpha1I, were expressed in both longitudinal and circular smooth muscle. In longitudinal muscle, the change in alpha1H mRNA was similar to that in alpha1C mRNA during gestation, but the expression of alpha1G mRNA changed significantly only at term (day 22). In circular muscle, alpha1H mRNA expression was stable at any stage during pregnancy, but alpha1G mRNA significantly increased on day 15 and at term. No relationship was observed between voltage-dependent calcium-channel mRNA expressions and either proliferation or hypertrophy of circular muscle during pregnancy. These results show (a) that during pregnancy, the expression levels of L-type channels change dynamically, and it may contribute directly to the regulation of cell excitability, and (b) that the T-subtype that increases during pregnancy differs between longitudinal and circular muscle cells, although their functions remain unclear.

Effects of several Cl(-) channel blockers on ionic currents in mouse embryos were studied using whole-cell patch-clamp and microelectrode methods. Microelectrode measurements showed that the resting membrane potential of early embryonic cells (1-cell stage) was -23 mV and that reduction of extracellular Cl(-) concentration depolarized the membrane, suggesting that Cl(-) conductance is a major contributor for establishing the resting membrane potential. Membrane currents recorded by whole-cell voltage clamp showed outward rectification and confirmed that a major component of these embryonic currents are carried by Cl(-) ions. A Cl(-) channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), suppressed the outward rectifier current in a voltage- and concentration-dependent manner. Other Cl(-) channel blockers (5-nitro-2-[3-phenylpropyl-amino] benzoic acid and 2-[3-(trifluoromethyl)-anilino] nicotinic acid [niflumic acid]) similarly inhibited this current. Simultaneous application of niflumic acid with DIDS further suppressed the outward rectifier current. Under high osmotic condition, niflumic acid, but not DIDS, inhibited the Cl(-)current, suggesting the presence of two types of Cl(-) channels: a DIDS-sensitive (swelling-activated) channel, and a DIDS-insensitive (niflumic acid-sensitive) Cl(-) channel. Anion permeability of the DIDS-insensitive Cl(-) current differed from that of the compound Cl(-) current: Rank order of anion permeability of the DIDS-sensitive Cl(-) channels was I(-) = Br(-)Cl(-)gluconate(-), whereas that of the DIDS-insensitive Cl(-) channel was I(-) = Br(-)Cl(-)gluconate(-). These results indicate that early mouse embryos have a Cl(-) channel that is highly permeable to amino acids, which may regulate intracellular amino acid concentration.

Objective: The aims of this study were (1) to evaluate the usefulness of the new β 2 -adrenergic stimulant KUR-1246 as a tocolytic agent and (2) to clarify the mechanisms that underlie the diverse inhibitory effects of β 2 -stimulants that are seen in human myometria in vitro. Study Design: The displacement of tritiated ([ 3 H]) (−)CGP 12177 (0.4 nmol/L) by KUR-1246 and other β2-stimulants was examined with human β 1 - and β 2 -receptors present on membrane fractions. The inhibitory effects of these β 2 -stimulants on the term-pregnant human myometrium were compared with the use of isometric tension recording and microelectrode methods. Finally, the relationship between [ 3 H]dihydroaloprenolol binding and the magnitude of the tocolytic effect of isoproterenol was examined. Results: KUR-1246 was approximately 80 times and 7 times more selective for β 2 -receptors than isoproterenol and ritodrine, respectively. The inhibitory effect of KUR-1246 was as strong as the inhibitory effect of the conventional β 2 -adrenergic stimulants. A wide range of inhibitory effects was observed, even when high concentrations of isoproterenol or KUR-1246 were applied. There was a correlation between the degree to which isoproterenol suppressed contractions and the number of [ 3 H]dihydroaloprenolol binding sites on the membrane in each muscle strip. Conclusion: KUR-1246 should be a very useful β2-adrenergic stimulant for use as a tocolytic agent because of its high selectivity for the β 2 -receptor and its potent inhibitory effect. The diversity of the inhibitory effects that are induced by β 2 -stimulants is at least partly due to differences in β 2 -receptor density among term-pregnant human myometria. (Am J Obstet Gynecol 2002;186:997-1004.)

Two cases of androgen-producing tumors, including a Sertoli-Leydig cell tumor in a woman of reproductive age and a Leydig cell tumor in a postmenopausal woman, are reported herein. In both cases, only selective venous sampling was able to detect the presence of the androgen-producing ovarian tumors.

The purpose of this study was to investigate the actions of estradiol on spontaneous and evoked action potentials in the isolated longitudinal smooth muscle cells of the pregnant rat. Single cells were obtained by enzymatic digestion from pregnant rat longitudinal myometrium. Action potentials and currents were recorded by whole-cell current-clamp and voltage-clamp methods, respectively. The acute effects of 17β-estradiol on action potentials and inward and outward currents were investigated. The following results were obtained. The average resting membrane potential of single myometrial cells was -54 mV (n = 40). In many cells, an electrical stimulation evoked a membrane depolarization, and action potentials were superimposed on the depolarization. In some cells, spontaneous action potentials were observed. Estradiol (30 µM) slightly depolarized the membrane (ca. 5 mV) and attenuated the generation of action potentials by reducing the frequency and amplitude of the spikes. Afterhyperpolarization was also attenuated by estradiol (30 µM). On the other hand, in 5 of 35 cells, estradiol increased the first spike amplitude and action potential duration, while frequency of the spike generation and afterhyperpolarization were inhibited. In voltage-clamped muscle cells, estradiol inhibited both inward and outward currents. Acute inhibition or augmentation of spike generation by estradiol is due to the balance of inhibition of inward and outward currents. Inhibition of both currents also prevented afterhyperpolarization, causing potential-dependent block of Ca spikes.Key words: estradiol, progesterone, rat myometrium, action potential, channel current.

Objective: To assess the diffusion of gonadotropin into the follicular fluid (FF) and its relation to the results achieved in a human IVF-ET program. Design: Retrospective pharmacokinetic study. Setting: Fukuoka University Hospital, Japan. Patient(s): Eighty-seven infertile patients underwent 137 cycles of IVF-ET. Intervention(s): Serum and FF were collected at the time of oocyte recovery. The hCG ratio (between follicular hCG and serum hCG concentrations, measured by time-resolved fluoroimmunoassay) was evaluated as an index of the diffusion of exogenous gonadotropin. Main Outcome Measure(s): Relation between hCG ratio and the results and outcome of the IVF-ET program. Result(s): The hCG ratio decreased with the total dosage of hMG and increased with the serum E 2 level, the number of oocytes recovered, and the number of oocytes fertilized. Patients with a poor response showed a low hCG ratio, which was associated with a complete lack of fertilization. The mean hCG ratio in the pregnant cycles was significantly higher than that in the nonpregnant cycles. An hCG ratio >0.46 was seen in all pregnant cycles. Conclusion(s): The diffusion of exogenous gonadotropin into the FF may be an important predictor of IVF outcome.

Objective: Our purpose was to compare the functional roles of oxytocin in term and preterm labor in rats by both biochemical and pharmacologic means. Study Design: We determined the myometrial oxytocin receptor density and the maternal plasma concentrations of oxytocin and progesterone on gestational days 18, 20, and 22 (morning) and at the onset of delivery (day 22 afternoon) in rats with labor at term and at the onset of delivery (day 20 afternoon) in rats in preterm labor induced by the combined use of bilateral ovariectomy and estradiol injection. We also evaluated the effects of an oxytocin antagonist, L-366,509, on the initiation of both term and preterm labor. Results: The number of tritiated oxytocin binding sites in myometrial membranes rapidly increased on gestational day 22 (morning) in rats with term labor. Plasma progesterone level decreased in an inverse fashion. A rapid increase in circulating oxytocin concentration was observed at the onset of delivery in rats in labor at term. Both the plasma oxytocin concentration and the receptor density had the same values in rats with preterm labor as in rats with term labor. L-366,509 delayed the initiation of labor in rats with term and preterm labor in a dose-dependent manner. Conclusion: It is confirmed biochemically and pharmacologically that oxytocin plays an important role in the initiation of both term and preterm labor in rats. The oxytocin antagonist examined was able to delay term and preterm labor, so it might prove useful in clinical practice for the treatment of preterm labor. (Am J Obstet Gynecol 1999;180:621-7.)

OBJECTIVE: Our purpose was to quantitatively assess the effects of oxytocin on membrane properties in the pregnant human myometrium. STUDY DESIGN: Specimens were obtained from the lower uterine segment during cesarean section at term. Electrical activity was recorded from individual cells by a conventional microelectrode method and the membrane functions were analyzed. RESULTS: Two types of spontaneous action potentials were seen: a long plateau potential and a spike-like action potential. With no change in the resting membrane potential, low concentrations of oxytocin either evoked an action potential with a plateau phase, increased the amplitude and duration of the plateau potential, or increased the frequency of generation of action potentials. Oxytocin also lowered the threshold for evoking an action potential. Higher concentrations depolarized the membrane with an associated reduction in membrane resistance. CONCLUSION: Oxytocin augments the excitability of pregnant human myometrial cells by multiple actions on the membrane, affecting both frequency and amplitude of action potentials.

The changes of vertebral bone mineral density (BMD; g/cm2) following oophorectomy were studied, using dual energy X-ray absorptiometry (DEXA) in 38 premenopausal and 244 oophorectomized women. Two biochemical indices of bone remodeling, urinary deoxypyridinoline (DPyr) for bone resorption and serum intact human osteocalcin (hOC) for bone formation, were also measured at the same time. The rate of bone loss in the first year after oophorectomy was 10.7%, while that in the second year was 5.7% (rapid phase), followed by a slow phase at the rate of 1.1%. The bone mass finally reached an osteoporotic level (BMD < 0.767 g/cm2) at 12 years after oophorectomy. The DPyr increased to reach a peak level in the first year, whereas the hOC increased and reached its peak level in the second year after surgery. The maximal bone loss in the first year is considered to be caused by the remarkable increase of bone resorption and the biological delay of the maximal increase in bone formation.

Most smooth muscle (SM) cells normally do not possess fast Na+ channels, and inward current for the action potential (AP) is carried primarily through slow (L- type) Ca2+ channels. However, it has been demonstrated that fast Na+ channels appear in myometrial SM cells during the latter half of pregnancy in the rat, presumably under steroid hormone control (4, 7). In addition, we have recently found functioning fast Na+ channels in rat and human intestinal (colonic) SM cells (23) and in rat portal vein cells (Xiong & Sperelakis, unpublished) under normal conditions. This review-type article provides a brief summary of (a) the changes in fast Na+ channels (and slow Ca2+ channels) in myometrial SM cells (from longitudinal layer) during pregnancy in the rat (4, 7); (b) the existence of functional fast Na+ channels in colonic SM cells (from circular layer) of the proximal (ascending) colon in both adult rat and human (13) and (c) the presence of functional fast Na+ channels in vascular SM cells from the rat protal vein (Xiong & Sperelakis unpublished).

Most smooth muscle (SM) cells normally do not possess fast Na+ channels, and inward current for the action potential (AP) is carried primarily through slow (L-type) Ca2+ channels. The slow Ca2+ channel activity is regulated by several mechanisms, as is well known for myocardial cells. In myocardial cells, cyclic AMP and cAMP-dependent protein kinase (cA-PK) stimulate slow Ca2+ channel activity, whereas cyclic GMP and cGMP-dependent protein kinase (cG-PK) inhibit channel activity (reviewed in Sperelakis et al., 1992). Phosphorylation by PK-C also stimulates activity of the myocardial slow Ca2+ channels (Domenici & Rogers, 1988). Intracellular ATP also modulates slow Ca2+ channel activity in myocardial cells, ATP being obligatory for channel activity (Sperelakis & Schneider, 1976; O’Rourke et al., 1992). Acidosis rapidly, reversibly, and rather selectively inhibits the slow Ca2+ channels in cardiac muscle (Vogel & Sperelakis, 1977; Belardinelli et al., 1979; Irisawa and Sato, 1987). Gating of myocardial Ca2+ slow channels by Gs-protein (GTP-activated alpha subunit) has also been demonstrated (Yatani et al., 1988). Intracellular ATP also modulates activity of slow Ca2+ channels in SM cells (Ohya and Sperelakis, 1989a, b), and phosphorylation by either cAMP or cGMP-mediated pathways inhibit slow Ca2+ channel activity (Bkaily et al., 1988a).

The effects of oxytocin, a uterotonic polypeptide hormone, on the voltage-dependent slow calcium, fast sodium, and potassium channel currents were studied using whole-cell voltage clamp of freshly isolated cells from late pregnant (18–21 day) rat myometrium. The calcium current was rapidly inhibited by oxytocin (about 25% inhibition at 20 nM) in a dose-dependent manner, and this inhibitory effect was completely reversible by washout. However, inhibition was not observed when barium was used as the charge carrier. Sodium current and potassium current were not modified by oxytocin, thus sodium and potassium currents may not play important roles in oxytocin-induced augmentation of uterine contraction. It is concluded that oxytocin stimulates uterine contraction by mechanisms other than augmentation of the voltage-dependent calcium current, e.g., by release of Ca from sarcoplasmic reticulum (by inositol trisphosphate) or by activation of a receptor-operated Ca channel. The inhibition of the slow calcium current may be induced by the elevation of [Ca]i.Key words: oxytocin, ionic channels, uterine smooth muscle, whole-cell voltage clamp, pregnant rat myometrium.

Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L-type) Ca2+ channels and fast (T-type) Ca2+ channels. Using whole-cell voltage clamp of single smooth muscle cells isolated from the longitudinal layer of 18-day pregnant rat uterus, depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow [8]. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by TTX (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o, and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current, and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. Thus, the ion channels which generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells which possess fast Na+ channels, and it is suggested that the fast Na+ current may be involved in spread of excitation. The Ca2+ channel current density also was higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and may facilitate parturition. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 of 12 mM) and nifedipine (K0.5 of 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions is not due to stimulation of ICa(s). Figure 11 summarizes the possible mechanisms by which uterine contractility can be modulated. In contrast to vascular smooth muscle, neither ISO nor adenosine, which produce elevation of cyclic AMP, affected ICa and INa. Therefore, no arrow can be drawn between cA-PK/cG-PK and the Ca2+ slow channel.(ABSTRACT TRUNCATED AT 400 WORDS)

Visceral smooth muscle cells, including vascular smooth muscle cells, possess various types of Ca channels. The voltage-dependent Ca channel is commonly observed in many tissues and is thought to play an important role in the generation of action potentials. Neural transmitters, hormones, autacoids, peptides and other substances activate individual receptors and cause activation of the receptor-operated ion channels which are permeable to Na and Ca, and in some tissues, CI ion. Thus, receptor activation may induce an influx of Ca via activation of the receptor-operated channel and voltage-dependent Ca channel, and also induce release of Ca from the sarcoplasmic reticulum (SR) via synthesis of inositol 1,4,5-trisphosphate (IP3). In addition, the concentration gradient between extra- and intra-cellular Ca (2.5 mM and 100 nM, respectively) may promote the passive influx of Ca. However, analysis of this current has not yet been made in detail. In this chapter, we discuss mainly the features of the voltage-dependent Ca channel recorded from visceral smooth muscle cells using voltage- and patch-clamp procedures, and also compare their characteristics to those in cardiac muscle cells.

The effects of diltiazem and its derivative, TA3090 on the Ba-induced inward current were investigated using dispersed smooth muscle cells of the rabbit mesenteric artery. Diltiazem and TA3090 inhibited the inward current, in a concentration-dependent manner, and TA3090 was more potent than diltiazem (IC50 for diltiazem was 300 mumol/l and for TA3090, 30 mumol/l). The inward current completely recovered from the inhibitory actions of diltiazem (100 mumol/l) after its removal at holding potentials of -60 and -40 mV, though complete recovery did not occur after TA3090 (1-100 mumol/l). The voltage-dependent inactivation curves were shifted to the left by both drugs. Judging from the amplitudes of shift of V-half, the dissociation constants of inhibition for both diltiazem and TA3090 in the inactivated state were 17 mumol/l and 2.6 mumol/l, respectively. At a holding potential of -60 mV, diltiazem and TA3090 reduced the peak amplitude of the inward current, and diltiazem, but not TA3090, accelerated the decay of the current (300 ms command pulse duration). However, TA3090 did accelerate the decay of the current when a longer pulse (1 s) was applied, or when a depolarizing pulse was applied from a holding potential of -80 mV. Changes in the pH (range 6.8-7.7) of the bathing solution did not influence the decay of the current. In conclusion, TA3090 inhibits the Ca-channels of smooth muscle cells of the rabbit mesenteric artery, in a voltage-dependent manner, and its inhibitory actions are similar to those of diltiazem. However, TA3090 has much longer and stronger inhibitory actions on the Ba-inward current than diltiazem.

1. The effects of endothelin (ET) on the Ca2+ channel current in smooth muscle cells of the guinea-pig portal vein were investigated using the patch-clamp technique with whole-cell and cell-attached configurations. 2. ET augmented the macroscopic Ba2+ current in a dose-dependent manner; this effect was inhibited by nifedipine or Cd2+. Augmentation of the inward current by ET did not depend on the amplitude of the depolarizing pulse. Further, when the membrane potential was held at -60 mV, ET increased the amplitude of the Ba2+ inward current measured at the peak and end of the depolarizing pulse to the same extent. 3. By contrast, when the membrane potential was held at -80 mV, depolarizing pulses to potentials more negative than 0 mV produced greater augmentation of the inward current than did those more positive than 0 mV. Moreover, when a depolarizing pulse to below 0 mV was applied, ET increased the peak amplitude of the inward current more than the amplitude measured at the end of pulse. 4. Using the patch-clamp technique with cell-attached configuration, two types of unitary Ba2+ current with conductances of 22 and 12 pS were obtained in 50 mM-Ba2+ solution. Nifedipine inhibited both types of unitary channel current, but the sensitivity of the 22 pS Ca2+ channel to nifedipine was 20-fold higher than the 12 pS Ca2+ channel. 5. Bath application of ET prolonged the mean open time, reduced the number of sweeps in which no Ca2+ channel was opened ('blank' sweep), and increased the number of channel openings evoked by each depolarizing pulse without changes of conductance. As a consequence, ET increased the open probability of both channels. 6. Augmentation of the 12 pS channels by ET was seen only in the early phase of a depolarizing pulse (57 ms from the onset of 170 ms pulse), while augmentation of the 22 pS channels was seen during the entire period of a depolarizing pulse. 7. When ET was added to the pipette solution, the activity of both Ca2+ channels was increased. However, this effect was less frequently observed than when ET was applied in the bath. 8. These results suggest that ET augments both the nifedipine-sensitive and resistant Ca2+ channels in the smooth muscle cell membrane of the guinea-pig portal vein, but in different ways. Presumably, ET acts indirectly on the voltage-dependent Ca2+ channel.

Using a whole-cell clamp technique on longitudinal smooth muscle cells of the rabbit ileum, it was found that the rate of decay of the macroscopic Ba current evoked by depolarization (to +20 mV) was not modified by changing the holding potential from -60 mV to -30 mV. Cadmium (20 microM) left only a small transient inward current. Using the patch clamp technique with cell-attached configuration, only one type of unitary Ba current, with conductance of 25 pS was obtained in 100 mM Ba solution. The conductance depended on the external Ba concentration, had a dissociation constant of 19 mM and maximum conductance of 42 pS, suggesting that the Ca channel is of the L-type. Depolarizing pulses (to 0 mV and 150 ms duration) delivered at a frequency of 0.5 Hz (high frequency) evoked a unitary Ba current with low open probability (p less than 0.3). However 65-75% of depolarizing pulses evoked no unitary Ba current ("blank" sweep). On the other hand, depolarizing pulses at 0.033 Hz (low frequency) reduced the number of "blank" sweeps (20-50%), and increased the fraction of sweeps with high open probability (p greater than 0.5). Thus, the channel activity may depend on the stimulus frequency. Nifedipine, in both stimulus conditions, reduced the open probability of the channel due to an increase in the fraction of "blank" sweeps to a greater extent than to a decrease in the time constant of open-times.