Effect of stimulatory and inhibitory drugs on uterine electrical activity measured noninvasively from the abdominal surface of pregnant rats.

OBJECTIVE: Our purposes were (1) to identify and analyze parameters of uterine electrical activity that change during active term and preterm labor in response to stimulatory (oxytocin) or inhibitory (terbutaline) agents and (2) to correlate the information obtained from abdominal surface measurement of electrical activity with intrauterine pressure and with the electrical activity measured directly from the uterine surface in vivo. STUDY DESIGN: Electromyographic activity was acquired simultaneously from the uterine wall and the abdominal surface by means of unipolar electrodes. Electromyographic activity was recorded in the 0.3 to 50-Hz range and digitized at 200 samples per second. Intrauterine pressure was measured via an intrauterine catheter. The effect of cumulative doses of oxytocin and terbutaline on power density spectrum, amplitude, number and duration of electromyographic bursts, and intrauterine pressure was recorded in anesthetized rats during spontaneous active term labor (n = 7) and induced preterm labor (n = 6). RESULTS: Bursts of electromyographic activity recorded from the abdominal surface mirrored those from the uterine wall, albeit at a lower amplitude. During active term labor, lower concentrations of oxytocin did not significantly affect power-density-spectrum energy, amplitude, or number of bursts per unit time. The duration of electromyographic bursts increased dose dependently. Myometrial contractions were phasic, with return to the baseline between phases. As the concentration of oxytocin increased, the energy, amplitude, and number of bursts per unit time declined while the intrauterine pressure continued to rise until the contraction became tetanic, without return to the baseline. In rats with induced preterm labor, terbutaline inhibited uterine contractility by decreasing the intrauterine pressure. This was accompanied by a progressive decrease in the power density spectrum, amplitude, number, and duration of the uterine wall and abdominal surface electrical bursts. CONCLUSIONS: First, uterine electromyographic activity measured noninvasively from the abdominal surface reflects changes in uterine electrical activity and intrauterine pressure measured directly and invasively in term and preterm labor, as well as during treatments to stimulate or inhibit labor. Second, this noninvasive method may be useful in monitoring uterine activity in vivo. Third, clinical studies to evaluate this technology in human subjects are warranted.

Uterine activity during pregnancy and labor assessed by simultaneous recordings from the myometrium and abdominal surface in the rat.

OBJECTIVES: The aim of this study was to analyze records of uterine electrical activity made from the abdominal surface of pregnant and laboring rats to examine whether similar quantitative information can be extracted as from direct recording from the uterine muscle. STUDY DESIGN: Electrical activity during pregnancy (nonlabor, days 18 to 22), term labor (day 22), and preterm labor (onapristone injected on day 18, delivery on day 19) was measured with use of electrodes attached to the uterine wall and to the abdominal surface. The fast Fourier transform and wavelet transforms were obtained for representative electromyographic bursts. Power spectra were generated. Intrauterine pressure was also measured. RESULTS: (1) Several parameters have been identified for use in following up the progressive increase in uterine activity that occurs in preparation for and during labor. Analyses of amplitude, frequency, and percent time active represent a convenient method for objectively determining the efficiency of uterine contraction. (2) Most of the changes in these parameters appear in the last 24 hours before delivery. (3) Although the uterine electromyographic signals on the abdominal surface are attenuated, the abdominal surface signals are generally similar to the signals obtained from the uterine muscle during pregnancy and during preterm and term labor. (4) The characteristics of onapristone-induced preterm labor are generally similar to those of spontaneous term labor. CONCLUSIONS: Recording of uterine electromyographic activity from the abdominal surface may be useful in following the progression of pregnancy and in predicting and diagnosing labor.

Gap junction currents in cultured muscle cells from human myometrium.

OBJECTIVE: The electrophysiologic properties of gap junctions between human myometrial smooth muscle cells were studied. STUDY DESIGN: Double whole-cell patch clamp recordings were made on pairs of cells from primary cultures of myometrial cells from women undergoing cesarean section. Macroscopic gap junction currents were measured as the change in current in a cell held at a constant voltage while the other member of a pair was subjected to a test pulse of voltage. The blockade by halothane was examined. RESULTS: Mean junctional conductance between pairs of cells was 23 +/- 14 nanosiemens (n = 57). Instantaneous gap junction conductance was constant as a function of transjunctional voltage. For transjunctional voltages of < or = 50 mV, currents were constant during a 5-second test pulse. For larger voltages, however, the currents showed a time-dependent decay. The currents were blocked completely and reversibly with 3.5 mmol/L halothane. Single-channel conductances of 60 picosiemens and 15 picosiemens were observed. CONCLUSION: This first study of gap junction currents in human myometrial cells confirms that connexin43 is the major functional constituent. Functional studies of myometrial gap junction channels may suggest new strategies for controlling uterine contractility.

Electrical activity of the human uterus during pregnancy as recorded from the abdominal surface.

OBJECTIVE: To validate the possibility that human uterine electrical events (electromyographic signals) can be recorded and characterized from the abdominal surface during pregnancy. METHODS: The gestational ages ranged from 20 to 43 weeks. The study included patients at term but not in labor, patients in active labor (term and preterm), postpartum patients, and patients followed monthly during their pregnancy (n = 40). Uterine electrical activity in the frequency range of 0.3-50 Hz was recorded using bipolar electrodes placed on the abdominal surface. In some patients, intrauterine pressure also was measured. Power spectral analysis was performed using the fast Fourier transform. RESULTS: Throughout most of pregnancy, uterine electrical activity was minimal, consisting of infrequent and low-amplitude electromyographic bursts. When bursts occurred before labor, they often corresponded to perceptions of contractility by the patient. During term and preterm labor, bursts of electromyographic activity were frequent and of large amplitude and were correlated with large transient changes in the intrauterine pressure and with pain. Fast Fourier transform analysis of the bursts during active term labor demonstrated a peak frequency of 0.71 +/- 0.05 Hz, compared with 0.48 +/d- 0.03 Hz before labor. Spectral analysis also showed a fivefold increase in the peak energy levels of the bursts during term labor (60.2 +/- 13.87 mu Vs) and preterm labor (62.3 +/- 22.93 mu Vs) compared with earlier in gestation (11.36 +/- 4.03 mu Vs at 27-36 weeks; P < .05). CONCLUSION: Recording of uterine electromyographic activity from the abdominal surface is a reliable method to follow the evolution of uterine contractility during pregnancy and during term and preterm labor. Further studies will define the usefulness of this noninvasive technology in the prediction and management of labor.

Voltage-clamp studies of gap junctions between uterine muscle cells during term and preterm labor.

Gap junctions between myometrial cells increase dramatically during the final stages of pregnancy. To study the functional consequences, we have applied the double-whole-cell voltage-clamp technique to freshly isolated pairs of cells from rat circular and longitudinal myometrium. Junctional conductance was greater between circular muscle-cell pairs from rats delivering either at term (32 +/- 16 nS, mean +/- SD, n = 128) or preterm (26 +/- 17 nS, n = 33) compared with normal preterm (4.7 +/- 7.6 nS, n = 114) and postpartum (6.5 +/- 10 nS, n = 16); cell pairs from the longitudinal layer showed similar differences. The macroscopic gap junction currents decayed slowly from an instantaneous, constant-conductance level to a steady-state level described by quasisymmetrical Boltzmann functions of transjunctional voltage. In half of circular-layer cell pairs, the voltage dependence of myometrial gap junction conductance is more apparent at smaller transjunctional voltages (< 30 mV) than for other tissues expressing mainly connexin-43. This unusual degree of voltage dependence, although slow, operates over time intervals that are physiologically relevant for uterine muscle. Using weakly coupled pairs, we observed two unitary conductance states: 85 pS (85-90% of events) and 25 pS. These measurements of junctional conductance support the hypothesis that heightened electrical coupling between the smooth muscle cells of the uterine wall emerges late in pregnancy, in preparation for the massive, coordinate contractions of labor.

Voltage-dependent Na+ channel mRNA expression in pregnant myometrium.

The mechanisms responsible for the onset of vigorous contractile activity in uterine muscle at the time of parturition, reversing the obligatory quiescence of pregnancy, remain poorly understood. We here describe the expression in myometrium from human and from timed-pregnant rats of an mRNA species encoding a voltage-sensitive sodium channel. The apparent concentration of this mRNA species, as measured by reverse-transcription polymerase chain reaction, underwent an approximately four-fold decline between mid-gestation and term. These results complement earlier reports of increased sodium current expression in mid- and late pregnancy and further support a role for smooth muscle sodium current in late pregnancy and/or labor.

Cloning and expression of cDNA and genomic clones encoding three delayed rectifier potassium channels in rat brain.

Rat brain cDNA and genomic clones encoding three K+ channels, Kv1, Kv2, and Kv3, have been isolated by screening with Shaker probes and encode proteins of 602, 530, and 525 amino acids. Each of the deduced protein sequences contains six hydrophobic domains (including an S4-type region characteristic of many voltage-gated channels) and are 68%-72% identical to each other overall. Transcripts of approximately 3.5, approximately 6.5, and approximately 9.5 kb encode Kv1, Kv2, and Kv3, respectively. The Kv2 mRNA is expressed only in brain, whereas the Kv1 and Kv3 transcripts are found in several other tissues as well. There is a marked increase in the amount of Kv1 mRNA in cardiac tissue during development and a similar, but less pronounced, increase of both this mRNA and the Kv2 transcript in brain. RNAs synthesized in vitro from the three clones induce voltage- and time-dependent, delayed rectifier-like K+ currents when injected into Xenopus oocytes, demonstrating that they encode functional K+ channels.

Estrogen induction of a small, putative K+ channel mRNA in rat uterus.

Estrogen causes dramatic long-term changes in the activity of the uterus. Here we report the molecular cloning of a small (700 base) uterine mRNA species capable of inducing a slow K+ current in Xenopus oocytes. The 130 amino acid protein encoded by this mRNA species has a predicted structure that does not resemble that of previously described voltage-dependent channels from mammalian sources. It is, however, similar to structural motifs found in certain prokaryotic ion channels. The induction of this mRNA by estrogen is rapid; this uterine mRNA species is not detectable in uteri from estrogen-deprived rats, but is substantially induced after 3 hr of estrogen treatment. These results support a critical role for regulation of ion channel expression by estrogen in the uterus.

Primary structure and functional expression of a mammalian skeletal muscle sodium channel.

We describe the isolation and characterization of a cDNA encoding the alpha subunit of a new voltage-sensitive sodium channel, microI, from rat skeletal muscle. The 1840 amino acid microI peptide is homologous to alpha subunits from rat brain, but, like the protein from eel electroplax, lacks an extended (approximately 200) amino acid segment between homologous domains I and II. Northern blot analysis indicates that the 8.5 kb microI transcript is preferentially expressed in skeletal muscle. Sodium channels expressed in Xenopus oocytes from synthetic RNA encoding microI are blocked by tetrodotoxin and mu-conotoxin at concentrations near 5 nM. The expressed sodium channels have gating kinetics similar to the native channels in rat muscle fibers, except that inactivation occurs more slowly.

Microinjected Xenopus oocytes secrete mature, biologically active parathyroid hormone.

Xenopus oocytes have been shown to faithfully translate, process, and secrete a number of secretory proteins after the injection of heterologous mRNAs. The oocyte has the capacity to perform a variety of posttranslational protein modifications but has been reported to be incapable of carrying out certain two-step cleavages which proceed via propeptide intermediates. We examined the ability of the oocyte to process preproPTH after the injection of parathyroid mRNA. Microinjected oocytes secreted material which could be detected in a sensitive cytochemical bioassay for PTH. This activity paralleled that of the PTH standard in the assay and was entirely eliminated by a competitive inhibitor of PTH binding, by preincubation with an anti-PTH antiserum, and by coinjecting oocytes with an oligonucleotide mixture complementary to PTH sequences. Immunoprecipitable proPTH and PTH were present in oocyte homogenates, but oocyte-conditioned medium contained only mature PTH(1-84). We conclude that the Xenopus oocyte is capable of accurately processing preproPTH to the mature secretory form of the peptide.

Hormonal regulation of K+-channel messenger RNA in rat myometrium during oestrus cycle and in pregnancy.

The dramatic enhancement of smooth-muscle excitability in the uterus which occurs at oestrus and at term in pregnant rats is closely related to increased blood oestrogen concentrations. How oestrogen alters the electrical properties of myometrial cells is unclear, although electrical coupling between cells has been shown to increase. Many examples are known of changes in cellular excitability involving modification of existing ion channels by second-messenger pathways. Steroid hormones, in contrast, are generally thought to influence cellular processes mainly through effects on gene expression, inducing the synthesis of new proteins. Previous work, using an oocyte translation system, has shown that a very slowly activating, voltage-dependent K+ current can be expressed from the poly(A)+ RNA of oestrogen-treated rat uteri. We report here that the messenger RNA species producing this channel is rapidly and reversibly induced in the presence of oestrogen, as shown by the appearance and disappearance of this mRNA during the oestrous cycle, its emergence at the end of pregnancy, and its presence or absence following hormonal treatments. These results suggest that oestrogen controls the expression of a voltage-dependent ion channel in uterine smooth muscle cells.

Xenopus oocytes injected with rat uterine RNA express very slowly activating potassium currents.

Under the influence of estrogen, uterine smooth muscle becomes highly excitable, generating spontaneous and prolonged bursts of action potentials. In a study of the mechanisms by which this transition in excitability occurs, polyadenylated RNA from the uteri of estrogen-treated rats was injected into Xenopus oocytes. The injected oocytes expressed a novel voltage-dependent potassium current. This current was not observed in oocytes injected with RNA from several other excitable tissues, including rat brain and uterine smooth muscle from ovariectomized rats not treated with estrogen. The activation of this current on depolarization was exceptionally slow, particularly for depolarizations from relatively negative membrane potentials. Such a slowly activating channel may play an important role in the slow, repetitive bursts of action potentials in the myometrium.

Serotonin increases intracellular Ca2+ transients in voltage-clamped sensory neurons of Aplysia californica.

Noxious stimulation of the tail of Aplysia californica produces behavioral sensitization; it enhances several related defensive reflexes. This reflex enhancement involves heterosynaptic facilitation of transmitter release from sensory neurons of the reflex. The facilitation is stimulated by serotonin (5-HT) and involves suppression of a 5-HT-sensitive K+ current (the S current). Suppression of the S current broadens the action potential of the sensory neurons and is thought to enhance transmitter release by prolonging entry of Ca2+ in the presynaptic terminals. We now report a component of enhanced Ca2+ accumulation that is independent of changes in spike shape. We have measured intracellular free Ca2+ transients during long depolarizing steps in voltage-clamped sensory neuron cell bodies injected with the Ca2+-sensitive dye arsenazo III. The free Ca2+ transients elicited by a range of depolarizing voltage-clamp steps increase in amplitude by 75% following application of 5-HT. Since it is observed under voltage-clamp conditions, this increase in the free Ca2+ transients is not merely secondary to the changes in K+ current but must reflect an additional mechanism, an intrinsic change in the handling of Ca2+ by the cell. We have not yet determined whether this change in Ca2+ handling reflects an increase in Ca2+ influx, a reduction in intracellular Ca2+ uptake, or a release of Ca2+ from intracellular stores. Regardless of the underlying mechanism, however, it seems possible that the enhancement of Ca2+ accumulation and the reduction in K+ current act synergistically in producing short-term presynaptic facilitation. Alternatively, this additional modulation of Ca2+ by 5-HT might contribute to processes such as classical conditioning or long-term sensitization that may depend on Ca2+.

The number and size of neurons in the CNS of gastropod molluscs and their suitability for optical recording of activity.

Several factors that affect the suitability of opticaL methods for monitoring neuron activity were evaluated in several species of gastropod molluscs. The mean cell body diameter and the total number of cells in the central nervous system were determined In 6 preparations and qualitative evaluations were made for an additional 25 species. There was a factor of 10 difference in mean diameter between species with the smallest cells (prosobranchs) and those with the largest (certain opisthobranchs). Several opisthobranchs had about 5000 central neurons; we estimate that the prosobranchs and pulmonates had at least 5 times as many neurons. When the opacity to transmitted light was measured the percent transmissions of circumesophageal ganglia were between 4% and 40%. We attempted to measure optical signals associated with spike activity in 20 gastropod species; in most species signals were readily detected in single trials.

Improvements in optical methods for measuring rapid changes in membrane potential.

In an effort to increase the utility of optical methods for measuring membrane potential in excitable cells, an additional 369 dyes were tested on giant axons from the squid. Several promising dyes with relatively large absorption and fluorescence signals are described. In addition, a simple modification of the apparatus led to a sixfold increase in the size of dye-related birefringence signals. In preparations with a suitable geometry, these signals are as large as absorption signals but photodynamic damage and bleaching are eliminated when wavelengths longer than the absorption band are used.