Yoshihito Inoue

Endothelin augments unitary calcium channel currents on the smooth muscle cell membrane of guinea‐pig portal vein.

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.

Oestradiol-induced relaxation of rabbit basilar artery by inhibition of voltage-dependent Ca channels through GTP-binding protein

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 μm‐100 μm) relaxed arterial tissue pre‐contracted by excess [K]0 solution (30 mM) in a concentration‐dependent manner. In Ca‐free solution, histamine (10 μm) and caffeine (20 mM) each produced a phasic contraction, but oestradiol (10 μm) did not significantly affect their amplitude. 3 Oestradiol (≥ 100 μm) 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 μm). 4 Oestradiol (1 μm‐100 μm) inhibited the voltage‐dependent Ba current in a concentration‐dependent manner. Oestradiol (100 μm) inhibited the Ba current observed in the presence of nicardipine (1 μm) more than that in the absence of nicardipine (to 31.0% vs 62.0% of control). 5 GTPγS (30 μm) in the pipette enhanced the inhibitory actions of oestradiol on the Ba current. On the other hand, with GDPβS (1 mM) in the pipette, oestradiol failed to inhibit the Ba current. Pertussis toxin (PTX 3 μg ml−1) in the pipette totally prevented the inhibitory action of oestradiol on the Ba current. 6 Oestradiol (≷ 100 μm) 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.

Some electrical properties of human pregnant myometrium

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.

Dual action of FRC8653, a novel dihydropyridine derivative, on the Ba2+ current recorded from the rabbit basilar artery.

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)

A normal uterus communicating with a double cervix and the vagina: a müllerian anomaly without any present classification

OBJECTIVEnTo report a rare uterine anomaly consisting of a normal uterus, a double cervix, and a double vagina.nnnDESIGNnCase report.nnnSETTINGnUniversity hospital.nnnPATIENT(S)nA 28-year-old nulligravida patient referred for evaluation of primary infertility and a suspected müllerian anomaly.nnnINTERVENTION(S)nClinical and surgical evaluation of the anomaly.nnnMAIN OUTCOME MEASURE(S)nDescription and treatment for a rare uterine anomaly and a subsequent literature search.nnnRESULT(S)nSuccessful resection of vaginal septum and subsequent pregnancy.nnnCONCLUSION(S)nThis extremely rare anomaly is not explained by classic embryologic teachings, and it does not fit into the classification system currently used to describe müllerian anomalies.

Identification and electrophysiological characteristics of isoforms of T-type calcium channel Ca(v)3.2 expressed in pregnant human uterus.

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.

The chronological change of vertebral bone loss following oophorectomy using dual energy X-ray absorptiometry: the correlation with specific markers of bone metabolism.

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.

Modulation produced by nifedipine of the unitary Ba current of dispersed smooth muscle cells of the rabbit ileum

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 μM) 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 theL-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<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>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.

Oxytocin enhances action potentials in pregnant human myometrium—A study with microelectrodes ☆ ☆☆ ★ ★★

OBJECTIVEnOur purpose was to quantitatively assess the effects of oxytocin on membrane properties in the pregnant human myometrium.nnnSTUDY DESIGNnSpecimens 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.nnnRESULTSnTwo 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.nnnCONCLUSIONnOxytocin augments the excitability of pregnant human myometrial cells by multiple actions on the membrane, affecting both frequency and amplitude of action potentials.

Amphiregulin is much more abundantly expressed than transforming growth factor-alpha and epidermal growth factor in human follicular fluid obtained from patients undergoing in vitro fertilization–embryo transfer

OBJECTIVEnTo identify the most important epidermal growth factor (EGF) receptor ligand in the LH or hCG signal pathway in human ovary.nnnDESIGNnA retrospective clinical study.nnnSETTINGnTertiary university hospital.nnnPATIENT(S)nNinety-eight infertile patients who underwent IVF-embryo transfer.nnnINTERVENTION(S)nSera and follicular fluid were collected at the time of oocyte retrieval. The levels of EGF, transforming growth factor-alpha (TGFalpha), and amphiregulin (AR) were measured in follicular fluid and sera by using ELISA.nnnMAIN OUTCOME MEASURE(S)nThe relationships between the level of AR and level of hCG, fertilization rate, and embryo quality.nnnRESULT(S)nAmphiregulin was abundantly expressed in follicular fluid after hCG stimulation. Although large differences were found between AR and both EGF and TGFalpha 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.nnnCONCLUSION(S)nAmphiregulin 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.