Nontraumatic urethral dyssynergia in neonatally estrogenized male rats.

PURPOSE: Bladder outlet obstruction develops in estrogen treated males. Because of the lack of electromyography recordings, earlier studies have not clarified the failure mechanisms of voiding. We simultaneously recorded electromyography activity of the proximal rhabdosphincter in neonatally estrogenized rats with transvesical cystometry and urethral flow, followed by morphometric analysis of the urethral structure. MATERIALS AND METHODS: Rats treated neonatally with 10 microg. diethylstilbestrol daily on days 1 to 5 after birth were used in urodynamics and morphological studies at ages 5 to 6.5 months. Using anesthesia the bladder, anterior surface of the proximal rhabdosphincter and distal urethra were exposed to record simultaneously the high frequency oscillations of intraluminal bladder pressure, and the rates of intermittent flow from the distal urethra and electromyography activity of the proximal rhabdosphincter with a suction electrode. RESULTS: Neonatally estrogenized rats had higher mean maximal bladder pressure plus or minus standard deviation (42.1 +/- 6.4 versus 37.7 +/- 4.9 mm. Hg, p = 0.01), decreased mean flow (2.3 +/- 0.1 versus 4.1 +/- 1.6 ml. per minute, p < 0.0001) and mean increment of proximal rhabdosphincter electromyography depolarization amplitude (3.0 +/- 0.78 versus 2.6 +/- 0.87 mV., p = 0.02) compared with controls, while mean transient repolarization was absent or highly decreased (-0.3 +/- 0.61 versus 0.3 +/- 0.9 mV., p = 0.04). Morphologically the proximal rhabdosphincter was atrophied with increased connective tissue. CONCLUSIONS: Alterations in the structure and electromyography activity of the urethral musculature imply that neonatal exposure to diethylstilbestrol predisposes male rats to urethral atrophy and dyssynergia, evident as altered electromyography activity of the proximal rhabdosphincter.

Possible action of the proximal rhabdosphincter muscle in micturition of the adult male rat.

Micturition requires high bladder pressure and simultaneous opening of the urethra. In adult male rat, a rhabdosphincter (RB) is known to be electrically active when the bladder pressure is high. This indicates a closure rather than an opening of the urethra, which is inconsistent with the requirements of optimal urodynamics. In order to solve this problem, we simultaneously recorded electromyogram (EMG) of the proximal RB, bladder pressure, and flow rate. Micturition was evoked by an increased volume of saline in the bladder. A computer-based recording device was used with minimal filtering. The EMG was recorded with a monopolar flexible suction electrode. The suction electrode records action potentials resembling those obtained with a microelectrode technique. During the early high-frequency intraluminal pressure oscillation period (IPHFO), the increase of pressure initially associated with a decrease of potential of the RB. When the first flow peak appeared, the relationship of the bladder pressure and RB single EMG activities changed. The increasing pressure coincided with the positive potential wave (depolarisation). It was interrupted by a transient negative polarity period called transient repolarisation (TRP) coinciding with a flow rate peak, thus indicating an opening of the RB lumen. After the TRP, the depolarisation continued. Additional experiments employing different methods are needed for positive identification of the TRP mechanism.

8-bromo-cGMP reduces the myofilament response to Ca2+ in intact cardiac myocytes.

The role of cGMP in myocardial contraction is not established. Recent reports suggest that nitric oxide, released by endothelial cells or within myocytes, modifies myocardial contraction by raising cGMP. We studied the effects of 8-bromo-cGMP (8bcGMP, 50 mumol/L) on contraction (cell shortening) and simultaneous intracellular Ca2+ transients (indo 1 fluorescence ratio) in intact adult rat ventricular myocytes (0.5 Hz and 25 degrees C) 8bcGMP reduced myocyte twitch amplitude and time to peak shortening (-19.6 +/- 4.2% and -17.6 +/- 1.3%, respectively) and increased steady-state diastolic cell length (+0.6 +/- 0.1 microns, mean +/- SEM, n = 8; all P < .05) but had no effect on shortening velocity, systolic or diastolic fluorescence ratio, or time to peak fluorescence ratio (all P = NS). In 7 of 13 myocytes, this negative inotropic effect was preceded by a transient positive inotropic effect, with small increases in twitch amplitude, shortening velocity, and cytosolic Ca2+ transient. Analysis of 8bcGMP effects on both the dynamic and steady-state relation between cell shortening and intracellular Ca2+ (during twitch contraction and tetanic contraction, respectively) indicated reduction in the myofilament response to Ca2+ in all cases. These 8bcGMP effects were inhibited by KT5823 (1 mumol/L), an inhibitor of cGMP-dependent protein kinase, or by the presence of isoproterenol (3 nmol/L). 8bcGMP had no effect on cytosolic pH in cells (n = 4) loaded with the fluorescent probe carboxyseminaphthorhodafluor-1. These data indicate that cGMP may modulate myocardial relaxation and diastolic tone by reducing the relative myofilament response to Ca2+, probably via cGMP-dependent protein kinase.

Cytosolic calcium and myofilaments in single rat cardiac myocytes achieve a dynamic equilibrium during twitch relaxation.

1. Single isolated rat cardiac myocytes were loaded with either the pentapotassium salt form or the acetoxymethyl ester (AM) form of the calcium-sensitive fluorescent probe, Indo-1. The relationship of the Indo-1 fluorescence transient, an index of the change in cytosolic calcium [Ca2+]i concentration, to the simultaneously measured cell length during the electrically stimulated twitch originating from slack length at 23 degrees C was evaluated. It was demonstrated that even if the Ca2+ dissociation rate from Indo-1 was assumed to be as slow as 10 s-1, the descending limb ('relaxation phase') of the Indo-1 fluorescence transient induced by excitation under these conditions is in equilibrium with the [Ca2+]i transient. Additionally, the extent of Indo-1 loading employed did not substantially alter the twitch characteristics. 2. A unique relationship between the fluorescence transient and cell length was observed during relaxation of contractions that varied in amplitude. This was manifest as a common trajectory in the cell length vs. [Ca2+]i phase-plane diagrams beginning at the time of cell relengthening. The common trajectory could also be demonstrated in Indo-1 AM-loaded cells. The Indo-1 fluorescence-length relation defined by this common trajectory is steeper than that described by the relation of peak contraction amplitude and peak fluorescence during the twitch contractions. 3. The trajectory of the [Ca2+]i-length relation elicited via an abrupt, rapid, brief (200 ms) pulse of caffeine directly onto the cell surface or by 'tetanization' of cells in the presence of ryanodine is identical to the common [Ca2+]i-length trajectory formed by electrically stimulated contractions of different magnitudes. As the [Ca2+]i and length transients induced by caffeine application or during tetanization in the presence of ryanodine develop with a much slower time course than those elicited by electrical stimulation, the common trajectory is not fortuitous, i.e. it cannot be attributed to equivalent rate-limiting steps for the decrease of [Ca2+]i and cell relengthening. 4. The [Ca2+]i-length relation defined by the common trajectory shifts appropriately in response to perturbations that have previously been demonstrated to alter the steady-state myofilament Ca2+ sensitivity in skinned cardiac fibres. Specifically, the trajectory shifts leftward in response to an acute increase in pH or following the addition of novel myofilament calcium-sensitizing thiadiazinone derivatives; a rightward shift occurs in response to an acute reduction in pH or following the addition of butanedione monoxime.(ABSTRACT TRUNCATED AT 400 WORDS)

Spontaneous sarcoplasmic reticulum Ca2+ release leads to heterogeneity of contractile and electrical properties of the heart.

The cytosolic Ca2+ (Cai) oscillation generated by the sarcoplasmic reticulum (SR) in response to an action potential (AP) occurs relatively synchronously within and among cells. The SR can also generate spontaneous Cai oscillations (S-CaOs), i.e., not triggered by sarcolemmal depolarization. The local increase in Cai due to S-CaOs is equivalent to that induced by an AP. Heterogeneity of diastolic Cai caused by asynchronous S-CaOs among cells within myocardial tissue leads to heterogeneous myofilament activation, the summation of which produces a Ca(2+)-dependent component to diastolic tone. The local increases in Cai due to S-CaOs also cause oscillatory sarcolemmal depolarizations due to Ca2+ modulation of the Na/Ca exchanger and of non-specific cation channels. Thus, inhomogeneous levels of diastolic Cai may lead to heterogeneity in cell coupling and thus may also affect the impulse conduction. The magnitude of the S-CaOs induced diastolic tonus and membrane depolarization varies with the extent to which S-CaOs are synchronized; partially synchronized S-CaOs following an AP induced SR Ca2+ release produce an aftercontraction and after depolarization. When local S-CaOs is sufficiently synchronized within the cell the resultant depolarization summates and can be sufficient to trigger spontaneous AP. Inhomogeneity of diastolic SR Ca2+ loading and sarcomere lengths within individual cardiac cells due to S-CaOs leads to inhomogeneous systolic Cai levels and sarcomere length inhomogeneities in response a subsequent AP; this heterogeneity compromises the systolic contraction amplitude. Heterogeneity of systolic Cai among cells due to diastolic S-CaOs also leads to heterogeneity of AP repolarization times, due, to heterogeneous Cai modulation of the Na/Ca exchanger, the non-specific cation channel and of the L type sarcolemmal Ca2+ channel. S-CaOs occurrence during a long AP plateau may also modulate the removal of voltage inactivation of L type Ca2+ channels and affect the likelihood of the occurrence of "early after depolarizations." Thus, as a single entity, S-CaOs may be implicated in diverse manifestations of heart failure--impaired systolic performance, increased diastolic tonus and an increased probability for the occurrence of arrhythmias.

The cytosolic calcium transient modulates the action potential of rat ventricular myocytes.

1. The modulation of the action potential by the cytosolic Ca2+ (Cai2+) transient was studied in single isolated rat ventricular myocytes loaded with the acetoxymethyl ester form of the Ca(2+)-sensitive fluorescent dye Indo-1. Stimulation following rest and exposure to ryanodine were used to change the amount of Ca2+ released from the sarcoplasmic reticulum and thus the size of the Cai2+ transient. The Cai2+ transient was measured as the change, upon stimulation, in the ratio of Indo-1 fluorescence at 410 nm to that at 490 nm (410/490) and action potentials or membrane currents were recorded using patch-type microelectrodes. 2. When stimulation was initiated following rest, the magnitude of the Cai2+ transient decreased in a beat-dependent manner until a steady state was reached. The negative staircase in the Cai2+ transient was accompanied by a similar beat-dependent decrease in the duration of the action potential, manifested primarily as a gradual loss of the action potential plateau (approximately -45 mV). A slow terminal phase of repolarization of a few millivolts in amplitude was found to parallel the terminal decay of the Cai2+ transient. 3. The terminal portion of phase-plane loops of membrane potential (Vm) vs. Indo-1 ratio from all of the beats of a stimulus train followed a common linear trajectory even though the individual beats differed markedly in the duration and amplitude of the action potential and Cai2+ transient. 4. When the stimulation dependence of the Cai2+ transient was titrated away with submaximal exposure to ryanodine, the stimulation-dependent changes in the action potential plateau and terminal phase of repolarization were also eliminated. The same effect was noted in cells which, fortuitously, did not show a staircase in the Cai2+ transient following a period of rest. 5. When action potentials were triggered immediately following spontaneous release of Ca2+ from the sarcoplasmic reticulum, which results in a small depolarization at the resting potential, phase-plane loops (Vm vs. Indo-1 ratio) of the spontaneous events followed the same linear trajectory as the terminal phase of repolarization in the loops of the stimulated beats. 6. Following repolarization from brief voltage clamp pulses (to minimize time and voltage-dependent currents associated with depolarization), an inward current was observed that rose and fell in phase with the Cai2+ transient. This current was present at -70 mV, near the resting potential, and at -40 mV, a potential relevant to the plateau of the action potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Sustained subthreshold-for-twitch depolarization in rat single ventricular myocytes causes sustained calcium channel activation and sarcoplasmic reticulum calcium release.

Single rat ventricular myocytes, voltage-clamped at -50 to -40 mV, were depolarized in small steps in order to define the mechanisms that govern the increase in cytosolic [Ca2+] (Cai) and contraction, measured as a reduction in myocyte length. Small (3-5 mV), sustained (seconds) depolarizations that caused a small inward or no detectable change in current were followed after a delay by small (less than 2% of the resting length), steady reductions in cell length measured via a photodiode array, and small, steady increases in Cai measured by changes in Indo-1 fluorescence. Larger (greater than -30 and less than -20 mV), sustained depolarizations produced phasic Ca2+ currents, Cai transients, and twitch contractions, followed by a steady current and a steady increase in Cai and contraction. Nitrendipine (or Cd, verapamil, or Ni) abolished the steady contraction and always produced an outward shift in steady current. The steady, nitrendipine-sensitive current and sustained increase in Cai and contraction exhibited a similar voltage dependence over the voltage range between -40 and -20 mV. 2 microM ryanodine in the presence of intact Ca2+ channel activity also abolished the steady increase in Cai and contraction over this voltage range. We conclude that when a sustained depolarization does not exceed about -20 mV, the resultant steady, graded contraction is due to SR Ca2+ release graded by a steady ("window") Ca2+ current. The existence of appreciable, sustained, graded Ca2+ release in response to Ca2+ current generated by arbitrarily small depolarizations is not compatible with any model of Ca2(+)-induced Ca2+ release in which the releasing effect of the Ca2+ channel current is mediated solely by Ca2+ entry into a common cytosolic pool. Our results therefore imply a distinction between the triggering and released Ca2+ pools.

Simultaneous measurement of Ca2+, contraction, and potential in cardiac myocytes.

A system is described that can simultaneously record cytosolic Ca2+ concentration ([Ca2+]i), cell length, and either membrane potential or current in single cardiac myocytes loaded with the fluorescent Ca2+ indicator indo-1. Fluorescence is excited by epi-illumination with 3.8-microsecond flashes of 350 +/- 5 nm light from a xenon arc. Indo-1 fluoresence is measured simultaneously in spectral windows of 391-434 nm and 457-507 nm, and the ratio of indo-1 emission in the two bands is computed as a measure of [Ca2+]i for each flash. With cells loaded with the permeant acetoxymethyl ester of indo-1, quantitation of [Ca2+]i is not precise, owing to subcellular compartmentation of indo-1; however, the instrument would allow full quantitation if indo-1 free acid was introduced by microinjection. Simultaneously, cell length is measured on-line from the bright-field image of the cell. Because fluorescence collection is time gated during the brief flash, and red light (650-750 nm) is used for the bright-field image, cell length and [Ca2+]i measurements are obtained simultaneously without cross talk. Membrane potential or current can be recorded simultaneously with indo-1 fluorescence and cell length via standard patch-clamping techniques.

Electrical activity in the human oviduct after the menopause.

Electrical activity was recorded in 18 morphologically normal human oviducts excised from 17 women during hysterectomy. All women were in the first five years after menopause. The first part of the oviduct to become inactive after menopause was the isthmus near the uterus or the ampullary-isthmic junction. The region which exhibited electrical activity last was the ovarian end of the tube. The typical wave-form of electrical activity in post-menopausal oviducts resembled that recorded during the follicular phase in fertile women: a single smooth wave lasting 3-5s. In three cases, over a bulbous swelling of the ampulla, this typical activity was replaced in continuous, high frequency activity. A hypothesis is provided that the observed changes in electrical activity may be related to certain clinical phenomena such as the increased incidence of tubal pregnancy in the pre-menopause.

Myoelectrical activity in the human oviduct with tubal pregnancy.

Myoelectrical activity and progesterone (P) and 17 beta-estradiol (E2) concentrations were measured in 12 human oviducts with tubal pregnancy. Spikes were frequently superimposed on a single, smooth, slow wave. The region above a large conceptus (P dominance) was inactive in eight of 10 oviducts. The myoelectrical activity spread toward these conceptuses from both sides, apparently holding the conceptus in place. In three cases with small placentas, the spread of weak activity tended to push the conceptus out of the oviduct. A steep decline of P and E2 levels distally in both directions from the conceptus was coincident with frequency of peaks.

Smooth muscle of the quail oviduct functions as a stretch receptor during ovum transport.

The present experiments were conducted to test the hypothesis that ovum transport in the quail oviduct is regulated by a time-dependent, stretch-mediated feedback cycle which alters the frequency of contractions. According to this hypothesis, a ligature preventing the forward movement of ovum should reverse the direction of the feedback cycle and an artificial ovum should be transported like the normal ovum. When the ligature was placed in the borderline between magnum and isthmus, it caused the reversal of transport direction after a delay of several minutes. Once the direction had changed, it persisted until the ovum was expulsed through the fimbrial end or until a second reversal was caused by either a second ligature or a minor mechanical impediment at the proximal end of the magnum. The ovum was transported between the ligatures at the mean speed of 1.7 +/- 0.17 mm/min (n = 7) until the ovum broke. An artificial ovum placed in the proximal magnum from which the natural ovum had been removed, was transported like the natural ova. Myoelectrical activity recorded with suction electrodes was statistically similar in both types of experiments and the direction of the frequency gradient changed when the transport direction was reversed. The frequency of the electrical activity of oviductal smooth muscle was significantly higher behind the ovum than in its front whether ova were transported in the direction of shell gland or infundibulum; in the segment maximally stretched by the ovum the activity was significantly lower than in other segments. These observations confirmed the hypothesis and suggest that the quail oviduct functions like a stretch receptor.

The membrane potential of the Japanese quail's oviductal smooth muscle during ovum transport.

Previously it has been shown that in the quail's oviductal smooth muscle there exists coordinated electrical and mechanical activity. It is not known how the contractions are regulated; the oviduct probably functions as a stretch receptor and the mechanical stimulus is produced by the ovum itself. The cellular basis for stretch-induced contractions is not known. In strips (n = 8) from the magnum, stretches of 50% changed the membrane potential. Compared with the resting length L0 (= 100%), stretches to 150% and 200% significantly (p less than 0.001) hyperpolarized the membrane potential. Analyses of variance, however, revealed that at the length 150% both the level of stretch and single experiments differed significantly in relation to the membrane potential: F = 7.1, p less than 0.5; F = 14.6, p less than 0.01, respectively. At the length 200%, there were highly significant differences between the groups 100% and 200% (F = 36.3, p less than 0.001) but the differences could be explained by the highly significant (F = 13.9, p less than 0.001) interaction. Membrane potential measured from isolated, intact oviducts (n = 7) which had ovum in the magnum was significantly depolarized by 8-9 mV on the region over the ovum. On the other hand, the membrane potentials were the same in the different segments of the oviduct which did not contain an ovum: 52 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane potential oscillations in enzymatically isolated rat myocardial cells.

Enzymatical isolation was used for preparation of single myocardial cells, contractions of which were mainly of slow phasic type but normal fast contractions also occurred. Membrane potential (MP) changes of these cells were measured by conventional microelectrode techniques. Initial MP level of these cells varied between -60 to -90 mV (74.4 +/- 1.9). In the majority of the cells the MP rapidly dropped to -20 to -40 mV and continued to decline while oscillating. In some cells MP recovered from -20 to -40 back to a more negative potential, and the MP oscillation was strongest (up to 20 mV) between -30 to -50 mV. In about 3% of the impalements the MPs did not degenerate at all, but stayed at initial values for several minutes. The MP oscillations were dependent on the MP level and connected to the slow phasic contractions. In connection to the normal fast contractions, two types of action potentials (AP) could be registered. Near -50 mV the cells often generated slow APs of duration 200-400 ms and dV/dt less than 3 V/s but at higher MP level normal fast APs up to 120 mV (100-300 ms and dV/dt greater than 40 V/s) were generated. The slow phasic contractions were never connected to either type of AP.

Adrenergic and cholinergic activity of the Japanese quail oviduct in vitro.

The responses to isoprenaline, phenylephrine, and carbachol in different segments of isolated intact oviduct were studied. Smooth muscle electrical activity was recorded with suction electrodes placed, one electrode in each, in the infundibulum, proximal magnum, distal magnum, isthmus, shell gland, and vagina. The drugs were added cumulatively. Only in the magnum did isoprenaline cause a significant change in the frequency of spike discharges; the decrease was about 20%. The effects of phenylephrine were not so clear, although the frequency of the electrical activity increased 30% in the shell gland. With the exception of the infundibulum, carbachol significantly increased the frequency of the oviductal electrical activity. It was concluded that adrenergic nerves have only a minor role in controlling quail oviductal smooth muscle because the oviduct was rather insensitive to adrenergic drugs whereas the cholinergic drug carbachol caused a strong response.

Description of the structural control systems of ovum transport in the quail oviduct.

Some electron microscropic and light microscopic aspects of the quail oviduct have been studied in relation to ovum transport. Previously it has been shown that in this smooth muscle there exist spontaneous coordinated electrical and mechanical activity which suggests a good electrical and mechanical coupling of the muscle cells. The structural basis for this coupling is not known. The majority of muscle cell contacts observed were simple appositions and intermediate junctions. Less numerous were attachments of the interdigitation type. No nexuses or tight junctions were seen. Mechanical stretching or contraction of cells induced with 10(-4)M carbachol did not affect the contacts. The fine structure of the muscle cells did not differ from that described for other smooth muscles. Electronmicroscopically the muscle cell bundles could not be distinguished into separate layers, in the light microscope the cell bundles were spirally arranged. Stretching of the oviductal strips to the length to which the ovum stretches the muscular wall during ovum transport caused re-orientation of the muscle cell bundles. One-directional stretching turned the axes of the muscle cells and the collagen bundles parallel, while stretching in two direction made the tissue look like a network. The re-orientation of muscle cell bundles may be of importance in producing forces in the muscular tunic during ovum transport. The nerve supply to the muscle cells was negligible. These and previous results show that structurally the muscular wall of the quail oviduct is a dynamic unity in which the ovum via stretch induces the electrical and mechanical activity throughout the tissue. Innervation may play a minor role in controlling the contractions.

Movements of the luminal contents in two different regions of the caput epididymidis of the rat in vitro.

Transport of the epididymal contents was studied in vitro by filming, for 1-2.5 h, the movements of tiny, stained oil droplets injected through a micropipette into two regions of the lumen of the caput epididymidis: the most proximal part, with the widest outer diameter (region I), and the neighbouring, narrowest portion (region II). The movements of the oil droplets were pendular. Displacement, caused by a contraction of the wall spreading in either direction, was followed by a shorter, usually passive reflux leading to a small net displacement, delta l. The distance of transport during 5 min periods varied between 0.09 and 16.79 mm (median 1.0 mm) in region I and 0.05 and 3.62 mm (median 0.42 mm) in region II. Transport divided into periods when little or no net transport took place (slow transport) and periods when the transport was effective (fast transport). Although the periods of fast transport were infrequent, their significance in transport towards the ductus deferens was high. During 5 min sampling periods of fast transport, the pendular movements were longer in both regions: delta l was longer in region I and the probability of delta l being in the direction of transport was higher than during slow transport in both regions. The mean probability of delta l being in the direction of the ductus deferens was 0.63 in region I and 0.57 in region II. Higher frequency of pendular movements, longer delta l values and higher probability of delta l being towards the ductus deferens in region I than II suggest that the transport speed is higher in region I than II. Transport consisting of short steps occurring with variable probabilities in both directions is a stochastic process.

Electrical activity in the human oviduct during the menstrual cycle.

Electrical activity in 25 isolated human oviducts on different days of the menstrual cycle was recorded with six simultaneous suction electrodes in at least 18 locations. During the follicular phase, electrical activity consisted of a smooth, single slow spike that lasted 3 to 6 seconds, and on which was superimposed a fast spike(s) in the ampulla immediately after menstruation. The shape of this activity changed at midcycle, first in the ampulla and later in the isthmus, to a burst of potentials; in the ampulla it sometimes changed to a slow wave on which was superimposed a series of fast spikes. The pacemakers were stable and their number few. The electrical activity spread with a velocity of 1 to 3 mm/sec. The probability of spread toward the uterus varied with the location in the oviduct and with the day of the cycle. After menstruation, electrical activity spread in the uterine direction. On cycle day 12, activity spread toward the ampullary-isthmic junction (AIJ) from both ends of the oviduct. On days 14 and 15, it spread a short distance from the ampulla to the isthmus, through the AIJ. On cycle day 18, spread toward the uterus covered the uterine half of the ampulla. AIJ, and the isthmus. Two to 5 days later, no constant features could be detected in the spread. These findings suggest that the human oviduct functions like the oviducts of other mammalian species, with the spread of electrical activity and the transport of ova being related.