Simultaneous measurements of changes in sarcoplasmic reticulum and cytosolic.

1. The role of the sarcoplasmic reticulum (SR) was investigated in spontaneous and agonist-induced uterine Ca2+ transients, by combining low- (mag-fluo-4) and high-affinity (fura-2) indicators to measure intraluminal SR ([Ca2+]L) and cytosolic ([Ca2+]i) calcium concentration, simultaneously, in single smooth muscle cells from pregnant rat uterus. 2. Carbachol or ATP, in the absence of extracellular Ca2+, decreased [Ca2+]L and increased [Ca2+]i. Although some replenishment (around 50 %) occurred in its absence, extracellular Ca2+ was required for full replenishment of the SR Ca2+. 3. In 4/15 cells, ATP evoked oscillations of [Ca2+]i. These were accompanied by successive release and re-uptake of SR Ca2+. Inhibition of the SR Ca2+-ATPase with thapsigargin abolished the oscillations and luminal changes. 4. Spontaneous [Ca2+]i transients produced no detectable changes in [Ca2+]L. The larger [Ca2+]i transients evoked by high-K+ depolarisation increased [Ca2+]L. Spontaneous activity was inhibited when [Ca2+]L was increased. 5. These data show that it is possible to simultaneously measure SR and cytosolic [Ca2+], and to investigate their response to agonist application and spontaneous activity.

The endoplasmic reticulum and mitochondria as elements of the mechanism of intracellular signaling in the nerve cell.

Experimental data obtained in our laboratory from studies of intracellular signals arising within nerve cells during excitation are summarized. Measurements of transmembrane ion currents in conditions of fixed membrane potential and intracellular free Ca ion concentrations, using fluorescent probes, yielded the time and spatial characteristics of transient elevations in the Ca concentration (the "calcium signal") in various types of mouse and rat neurons. These studies showed that intracellular structures-the endoplasmic reticulum and mitochondria-had significant roles in forming these signals; these structures can take up Ca from the cytosol and liberate Ca into the cytosol; the contribution of these processes was extremely variable, depending on the internal organization of different functional types of neurons.

The role of the sarcoplasmic reticulum as a Ca2+ sink in rat uterine smooth muscle cells.

1. The mechanisms responsible for removing calcium ions from the cytoplasm were investigated in single rat uterine myocytes using indo-1. 2. Trains of depolarizing voltage-clamp pulses increased [Ca2+]i. The rate of decay of [Ca2+]i was slowed by inhibition of the sarcoplasmic reticulum (SR) Ca2+-ATPase with cyclopiazonic acid (CPA). However, if the sarcolemmal Na+-Ca2+ exchanger and Ca2+-ATPase were inhibited then recovery of [Ca2+]i was abolished showing that the SR Ca2+-ATPase alone cannot produce decay of [Ca2+]i. 3. In another series of experiments, Ca2+ release from the SR was induced with carbachol in a Ca2+-free solution. Under these conditions responses to repeated applications of carbachol could be obtained. In the presence of CPA, however, only the first application was effective. This suggests that the SR Ca2+-ATPase sequesters a significant amount of Ca2+ into the SR. 4. CPA slowed the rate of decay of [Ca2+]i following carbachol addition by > 50 %. Again, however, after a brief transient fall, decay was abolished when the Na+-Ca2+ exchanger and sarcolemmal Ca2+-ATPase were inhibited. 5. These data show that, although the SR Ca2+-ATPase contributes to the decay of [Ca2+]i, it cannot function effectively in the absence of Ca2+ removal from the cell. These data are discussed in the context of the superficial buffer barrier model in which Ca2+ is taken up into the SR and then released very close to sarcolemmal Ca2+ extrusion sites, i.e. the SR acting in series with the surface membrane extrusion mechanisms. We also suggest that the amount of filling of the SR influences the rate of Ca2+ removal.

Properties of voltage-activated [Ca2+]i transients in single smooth muscle cells isolated from pregnant rat uterus.

1. The intracellular calcium concentration ([Ca2+]i) was measured at 35 degrees C using the fluorescent indicator indo-1 in patch-clamped, single uterine myocytes from pregnant rats to investigate the relationship between depolarization, Ca2+ current (ICa) and [Ca2+]i. 2. Membrane depolarization activated ICa and produced a [Ca2+]i transient. The rapid increase in [Ca2+]i occurred at the same time as the inward ICa. Both ICa and the increase in [Ca2+]i were abolished by nifedipine (10 microM). 3. When the membrane potential was held at -80 mV the threshold depolarization for an increase in [Ca2+]i was about -55 to -50 mV. As the magnitude of the depolarization was increased to about 0 mV there was an increase in the size of both ICa and the increase in [Ca2+]i. As the magnitude of the depolarization was further increased both ICa and the [Ca2+]i increase declined. 4. When the depolarizing pulses were applied at 3 Hz to mimic normal action potentials then the individual [Ca2+]i transients did not fully relax and a tetanic rise of [Ca2+]i was observed. Under these conditions, there was not a simple relationship between the magnitude of the Ca2+ response and Ca2+ entry. When pairs of depolarizing pulses were applied, the increase in [Ca2+]i produced by the second pulse was larger (in relation to the magnitude of the L-type Ca2+ current) than that produced by the first pulse. This facilitation was abolished by both ryanodine and cyclopiazonic acid suggesting a role for release from intracellular stores. 5. We conclude that the L-type Ca2+ current is the major source of Ca2+ ions entering the cell to produce the [Ca2+]i transient on depolarization. The magnitude of the increase in [Ca2+]i may, however, be amplified by Ca2+-induced Ca2+ release.

[Endoplasmic reticulum and mitochondria as elements of mechanism of intracellular signaling in neurons]. / Endoplasmaticheskii retikulum i mitokhondrii kak élementy mekhanizma vnutrikletochnoi signalizatsii v nervnoi kletke.

The temporal and spatial characteristics of a transitory increase in free Ca2+ ("calcium signals") concentration were determined in various types of the mice and rat neurones. Intracellular structures: endoplasmatic reticulum and mitochondria, were shown to play a major part in formation of these signals, the structures being able to absorb the Ca2+ ions from cytosol and release them back. The contribution of these processes proves rather varying depending on internal organisation and functional assignment of a neurone.

Changes of pH affect calcium currents but not outward potassium currents in rat myometrial cells.

Spontaneous contraction of uterine smooth muscle is enhanced by alkalinization and depressed by acidification. We have investigated the ionic currents responsible for this in single myometrial cells. Intracellular acidification (20 mM butyrate) at constant external pH depressed the magnitude of the calcium current to 58+/-6% of control, but had little effect on outward currents. Similar but slower effects were also observed when the extracellular pH was lowered to 6.9 (56+/-9% of control). Correspondingly, when the intracellular or extracellular pH was elevated (20 mM NH4Cl or pH 7.9 respectively) the calcium current magnitude increased (165+/-15% in NH4Cl; 136+/-2% at pH 7.9) and there was, again, no effect on the outward currents. These observations are consistent with the effects of pH on spontaneous contractile activity being due to an effect on the membrane calcium current.

[The functional status of the small intestine in rats in the dynamics of a bone marrow form of acute radiation sickness]. / Funktsional'noe sostoianie tonkoi kishki krys v dinamike kostno-mozgovoi formy ostroi luchevoi bolezni.

A study was made of 75Se-methionine absorption rate and contraction and choline-reactivity of smooth muscle cells of rat jejunum longitudinal layer. Whole-body X-irradiation with a dose of 5.8 Gy (LD50/30) caused a sharp decrease in the rate of absorption of 75Se-methionine by the blood from the intestine on days 3--4 after irradiation followed by an incomplete recovery of absorption by the 14th day. At the height of acute radiation sickness there was an 8- to 10-fold decrease in the sensitivity of the smooth muscle cell membrane to acetylcholine the contractile capacity being retained. This was perhaps the cause of the intestine motor activity inhibition. It is concluded that rat intestine functions are suppressed considerably with the bone marrow form of acute radiation sickness.

[Effect of neurotensin and substance P on the active state of the gastric smooth muscles]. / Vliianie neirotenzina i substantsii P na aktivnoe sostoianie gladkikh myshts zheludka.

Activating effect of neurotensin applied to muscle strips taken from fundal and antral portions of the guinea pig stomach was found to be due to depolarization of the muscle cells membrane and activation of the electrically controlled calcium channels. Activating effect of substance P seems to be associated with the activation of the chemo-controlled calcium channels as the contraction following the substance P application develops with no apparent changes in the membrane potential. Substance P increases the amplitude of the potassium contracture tonic component. The dynamics of active state of the muscle contractile element does not change under these conditions.