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S M Sims

Publications and source records attributed to S M Sims.

69 records · Page 4Linked to original sources
PubMed1991

Direct effects of platelet-activating factor on isolated rat osteoclasts. Rapid elevation of intracellular free calcium and transient retraction of pseudopods.

Platelet-activating factor (PAF, 1-O-alkyl-(2R)-acetylglycero-3-phosphocholine) is a potent inflammatory mediator whose actions on bone cells have not been investigated previously. In this study, we examined effects of PAF on osteoclast morphology and intracellular free calcium. Osteoclasts, the large multinucleated cells responsible for bone resorption, were isolated from neonatal rat long bones, and the cytosolic free calcium concentration ([Ca2+]i) of individual fura-2-loaded cells was monitored by microspectrofluorimetry. In one series of experiments, PAF was applied focally to single, isolated osteoclasts (1 nM to 1 microM racemic mixture, in an application micropipette). Within 10 s of PAF application, [Ca2+]i increased from basal levels of 74 +/- 6 nM to peak levels of 209 +/- 28 nM (mean +/- S.E. of 24 cells responding). These results indicate that PAF acted directly on osteoclasts. In more than 75% of cells tested, PAF, at concentrations greater than or equal to 10 pM (final concentration, in the bath), induced biphasic elevation of [Ca2+]i. This response was highly specific for PAF, in that vehicle, lyso-PAF (the biologically inactive precursor/metabolite of PAF), and (S)-PAF (the inactive enantiomer of PAF) all failed to change [Ca2+]i. Moreover, [Ca2+]i elevation was blocked by the specific PAF antagonist CV-3988. To determine the source of Ca2+, cells were bathed in Ca(2+)-free medium, where PAF still caused an increase in [Ca2+]i, establishing that the response to PAF arose, at least in part, by release of Ca2+ from internal stores. In addition to changes in [Ca2+]i, PAF caused retraction followed by respreading of peripheral pseudopods. These findings indicate that rat osteoclasts respond to PAF by release of internal calcium and alterations in cell morphology and suggest that PAF may regulate resorption in inflammatory bone diseases.

Animals↗
PubMed1991

K+ and Cl- currents in freshly isolated rat osteoclasts.

Membrane electrical properties of freshly isolated rat osteoclasts were studied using patch-clamp recording methods. Characterization of the passive membrane properties indicated that the osteoclast cell membrane behaved as an isopotential surface. The specific membrane capacitance was 1.2 +/- 0.3 microF/cm2 (mean +/- SD), with no difference between cells plated on glass and those adhering to a permeable collagen substrate. The current/voltage (I/V) relationship of all cells showed inward rectification and I/V curves shifted 51 mV positive per tenfold increase of [K+]out, indicating an inwardly rectifying K+ conductance. The voltage dependence of the K+ chord conductance (gK) also shifted positive along the voltage axis, and the maximum conductance increased, with elevation of [K+]out. gK for cells bathed in 4.7 mM [K+]out increased e-fold per 12 mV hyperpolarization, and half-maximal activation was at -89 mV. Approximately 18% (50 pS/pF) of the maximum gK was active at -70 mV. Inward single-channel currents were recorded in cell-attached patches at hyperpolarizing potentials. With symmetrical K+, channel conductance was 25 +/- 3 pS and reversal was close to the K+ equilibrium potential, consistent with this K+ channel underlying the whole-cell K+ currents. With both conventional whole-cell and perforated-patch recording, no voltage-activated Ca2+ current was detected. In approximately 30% of osteoclasts studied, an outwardly rectifying current was observed, which was reversibly blocked by 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS). This DIDS- and SITS-sensitive current reversed direction at the chloride equilibrium potential. We conclude that an inwardly rectifying K+ current is present in all rat osteoclasts and that some osteoclasts also exhibit an outwardly rectifying Cl- current. Both these membrane conductances may play an important physiological role by dissipating the potential that arises from the electrogenic transport of H+ across the ruffled membrane of the osteoclast.

Animals↗
PubMed1991

Somatostatin activates an inwardly rectifying K+ conductance in freshly dispersed rat somatotrophs.

1. Somatotrophs from enzymatically dispersed anterior pituitary glands of rats, enriched to greater than 94% purity by density gradient centrifugation, were studied within 16 h of isolation using patch clamp recording methods in the conventional whole-cell and the perforated-patch configurations. 2. Rhythmic oscillations of membrane potential gave rise to action potentials in thirty-six of fifty-two cells studied with the perforated-patch technique. Membrane potential oscillated between approximately -70 mV and approximately -25 mV with an average frequency (mean +/- S.D.) of 0.9 +/- 0.9 s-1. 3. The current-voltage (I-V) relationship of cells was linear at negative potentials with outward rectification at potentials positive to -40 mV. Evidence that the outward current was due to K+ channels came from the deactivation tail currents, which reversed direction close to the K+ equilibrium potential (EK). The reversal potential shifted 60 mV per tenfold change of external K+ concentration ([K+]o), as expected for K+ current. 4. Suppression of outward current by tetraethylammonium (TEA) provided additional evidence for K+ current. Cd2+ reduced outward current, suggesting the presence of Ca(2+)-activated K+ conductance. 5. Depolarizing commands elicited transient inward Na+ current and a sustained Ca2+ current (ICa). ICa was recorded in isolation with Cs+ and TEA in the recording pipette and 10 mM-Ba2+ as the charge carrier. Activation of ICa began at approximately -40 mV, with peak inward current at 0 to +10 mV. The half-inactivation potential was approximately -35 mV. In addition, ICa was blocked by nifedipine. These characteristics indicate the presence of L-type Ca2+ channels in somatotrophs. 6. Somatostatin caused hyperpolarization and suppressed the spontaneous bursts of action potentials. Under voltage clamp, somatostatin activated an inwardly rectifying current that reversed direction near EK. When EK was altered by elevation of [K+]o, the reversal potential of the somatostatin-induced current shifted 55 mV per tenfold change of [K+]o, as predicted for a K+ current by the Nernst relation. The somatostatin-induced conductance (gK) was greater at more negative potentials, and the activation range shifted positive with elevation of [K+]o. 7. We conclude that freshly isolated rat somatotrophs possess Na+, Ca2+ and K+ currents. A large proportion of the cells exhibit spontaneous bursts of action potentials. Somatostatin activates an inwardly rectifying K+ conductance, causing hyperpolarization and cessation of spontaneous action potential activity, actions that would contribute to suppression of growth hormone release.

Action Potentials↗
PubMed1990

Dual regulation of M current in gastric smooth muscle cells: beta-adrenergic-muscarinic antagonism.

The effects of the beta-adrenergic agent isoproterenol on membrane currents were studied in freshly dissociated gastric smooth muscle cells of Bufo marinus. Voltage-clamp experiments were carried out with patch pipettes in the tight-seal, whole-cell recording mode or with conventional microelectrodes. Isoproterenol induced a current identified as M current by the following criteria: the induced current is outward and carried by K+ ions, is suppressed by muscarine or acetylcholine, remains steadily activated, turns off with hyperpolarization, and exhibits slow relaxations in response to voltage jumps. In contrast to endogenous M current, isoproterenol-induced M current usually exhibited slower relaxations on hyperpolarizing voltage commands and displayed a steady-state conductance/voltage relationship that was shifted in the negative direction along the voltage axis. M current was also induced by either forskolin or phosphodiesterase-resistant cAMP analogs. In all cases, muscarinic agonists suppressed the M current, apparently by acting at a locus downstream from regulation of cAMP levels by adenylate cyclase and phosphodiesterase. beta-Adrenergic agents may act to increase the number of M channels available to be opened and also modify their kinetics.

Acetylcholine↗
PubMed1990

Membrane currents and cholinergic regulation of K+ current in esophageal smooth muscle cells.

The tight-seal whole cell recording technique with patch pipettes was used to study membrane currents of smooth muscle cells freshly dissociated from the esophagus of cats. Under voltage clamp with K+ in the pipette, depolarizing commands elicited an initial inward current followed by a transient outward current that peaked and then declined to reveal spontaneous outward currents (SOCs). SOCs were evident at -60 mV and more positive potentials. The reversal of SOCs at the K+ equilibrium potential and their suppression by tetraethylammonium chloride lead to the conclusion that they represent the activity of K+ channels. Acetylcholine (ACh) caused reversible contraction of these cells and had two successive effects on membrane currents, causing transient activation of K+ current followed by suppression of SOCs. Both of these effects were blocked by atropine. Consistent with these observations, in current clamp, ACh caused a transient hyperpolarization followed by depolarization. The inward current activated by depolarization was blocked by external Cd2+, consistent with the inward current being a voltage-activated calcium current. Two types of Ca2+ current could be distinguished on the basis of voltage-activation range, time course of inactivation and "run-down" during whole cell recording.

Acetylcholine↗
PubMed1989

Transdermal iontophoretic drug delivery: mechanistic analysis and application to polypeptide delivery.

Three factors are of primary importance in determining the iontophoretic flux of a charged solute: the electrochemical potential gradient across the skin, an increase in skin permeability to passive transport due to iontophoresis (loosely defined as skin damage), and a current-induced water flux. The latter two factors can also affect the transport of uncharged solutes during iontophoresis. A method of correcting for the skin damage effect is introduced. The contributions of the water transport effect relative to that of the applied voltage drop for charged solutes is estimated. It is shown that the water transport contribution is generally lower than the contribution due to the applied voltage drop. The observed iontophonetic flux of the enhancement factors due to the applied voltage drop alone are compared with the theoretical predictions based on the constant field assumption. It is shown that the theoretical predictions are higher than the experimental observations. This work also examines, for the first time, a synergism of iontophoresis and pretreatment with a chemical penetration enhancer as a means for delivering high molecular weight polypeptides. It is shown that a 2-h pretreatment with absolute ethanol followed by iontophoresis dramatically increases the permeability coefficient of insulin through human skin.

Administration, Cutaneous↗
PubMed1989

Inwardly rectifying K+ current in osteoclasts.

Membrane properties of freshly isolated rat osteoclasts were studied using the whole cell patch-clamp recording technique. The membrane potential could switch between two stable levels, approximately -70 and -15 mV. Voltage-clamp studies indicated that osteoclasts exhibited marked inward rectification, with hyperpolarizing voltage commands from -70 mV activating large inward currents. No voltage-dependent currents were observed in response to depolarization. An increase in external K+ concentration shifted the current-voltage relationship positive in a manner predicted for K+ current. Furthermore, barium and cesium reversibly suppressed the inward current. Thus the dominant current evident in osteoclasts was inwardly rectifying K+ current, resembling that found in a number of cell types, including cardiac and skeletal muscle and oocytes. The current-voltage relationship of osteoclasts was "N-shaped" and could intersect the zero-current level at three potentials, accounting for two stable membrane potentials. Switching of membrane potential between these two levels may regulate a number of the cellular processes involved in bone resorption.

Animals↗
PubMed1988

Antagonistic adrenergic-muscarinic regulation of M current in smooth muscle cells.

The beta-adrenergic agonist isoproterenol and analogs of adenosine 3',5'-monophosphate (cAMP) induced a potassium current, M current, in freshly dissociated gastric smooth muscle cells. Muscarinic agonists suppress this current, apparently by acting at a locus downstream from regulation of cAMP levels by adenylate cyclase and phosphodiesterase. Thus, M current can be induced by an agent and regulated in antagonistic fashion by beta-adrenergic and muscarinic systems.

Acetylcholine↗
PubMed1986

Substance P and acetylcholine both suppress the same K+ current in dissociated smooth muscle cells.

The effect of substance P on freshly dissociated gastric smooth muscle cells was examined electrophysiologically. Substance P caused depolarization, associated with a membrane conductance decrease, which led to the generation of action potentials and contraction. When the membrane potential was held constant under voltage clamp, substance P induced a net inward current, also associated with a conductance decrease. The net inward current resulted from suppression of an outward K+ current, one which resembled the acetylcholine-sensitive M-current in these cells. When substance P maximally suppressed this outward K+ current, acetylcholine (ACh) had no additional effect. Conversely, when ACh fully suppressed the M-current, substance P was without additional effect. These results indicate that substance P suppresses the same outward K+ current affected by ACh. Suppression of M-current by substance P was observed in approximately half (44 of 85) of the cells studied in these experiments. In those cells that did not respond to substance P, ACh was nevertheless capable of suppressing the M-current. Thus both substance P and cholinergic agonists appear to exert their excitatory effects on smooth muscle cells by inhibiting a common K+ current.

Acetylcholine↗
PubMed1985

Cholinergic agonists suppress a potassium current in freshly dissociated smooth muscle cells of the toad.

Single micro-electrode voltage-clamp and current-clamp techniques were used to study cholinergic responses in single freshly isolated gastric smooth muscle cells from the toad Bufo marinus. Acetylcholine (ACh) or muscarine caused membrane depolarization, which sometimes gave rise to action potentials and contractions. The agonist-induced depolarization is due to the suppression of a voltage-dependent K+ conductance, a conclusion based on the following observations. Depolarization was accompanied by an apparent membrane conductance decrease, seen as the increased size of voltage deflexions in response to constant current pulses. The conductance decrease was confirmed under voltage clamp, where current deflexions in response to constant voltage jumps were smaller in the presence of cholinergic agonists. Muscarine induced net inward currents at potentials positive to the K+ equilibrium potential (EK), and net outward currents at potentials negative to EK. In experiments where external K+ concentration ([K+]o) ranged from 20 to 90 mM the reversal potentials shifted 58 mV positive per tenfold elevation of [K+]o, as expected for a K+ current. The steady-state current-voltage relationship revealed that the K+ current inhibited by muscarine was larger at more positive potentials than expected from driving force considerations alone. Therefore, the underlying conductance suppressed by cholinergic agonists was voltage dependent, with almost complete deactivation at potentials more negative than approximately -70 mV and exhibiting a sigmoidal activation curve upon depolarization. The deactivation of this voltage-dependent K+ conductance caused slow current relaxations to occur in response to hyperpolarizing voltage commands from depolarized holding potentials. In experiments where [K+]o ranged from 3 to 30 mM, these current relaxations reversed direction at potentials near EK and the reversal potential shifted 52 mV positive per tenfold elevation of [K+]o, indicating that K ions carry most of the charge. The current relaxations that occurred in response to hyperpolarizing voltage commands were suppressed by ACh, muscarine and oxotremorine. The effects of muscarine persisted in nominally Ca2+-free solutions containing Mn2+. Ba2+ mimicked the effects of muscarinic agonists. Thus, isolated smooth muscle cells exhibit a K+ current resembling the M-current of sympathetic and other neurones, which is reversibly suppressed by cholinergic agonists. The existence of a cholinergic K+ conductance decrease is of interest because it has not previously been demonstrated in smooth muscle.

Action Potentials↗
PubMed1982

Improved electrical coupling in uterine smooth muscle is associated with increased numbers of gap junctions at parturition.

We have studied some passive electrical properties of uterine smooth muscle to determine whether a change in electrical parameters accompanies gap junction formation at delivery. The length constant of the longitudinal myometrium increased from 2.6 +/- 0.8 mm (X +/- SD) before term to 3.7 +/- 1 mm in tissues from delivering animals. The basis of the change was a 33% decrease in internal resistance and a 46% increase in membrane resistance. Axial current flow in an electrical syncytium such as myometrium is impeded by the cytoplasm of individual cells plus the junctions between cells. Measurement of the longitudinal impedance indicated that the specific resistance of the myoplasmic component was constant at 319 +/- 113 omega . cm before term and 340 +/- 93 omega . cm at delivery. However, a decrease in junctional resistance was apparent from 323 +/- 161 omega . cm to 134 +/- 64 omega . cm at delivery. 1.5-2 d after delivery, the junctional resistance was increased, as was the myoplasmic resistance. Thin-section electron microscopy of some of the same muscle samples showed that gap junctions were present in significantly greater numbers in the delivering tissues. Therefore, our results support the hypothesis that gap junction formation at delivery is associated with improved electrical coupling of uterine smooth muscle.

Action Potentials↗
PubMed1978

Possible role of gap junctions in activation of myometrium during parturition.

Gap junctions between smooth muscle cells of the myometrium of pregnant rats were found only immediately prior to, during and immediately after parturition by quantitative thin-section and freeze-fracture microscopy. Ovariectomy of 16- to 17-days-pregnant rats resulted in premature termination of pregnancy and the appearance of gap junctions. Methods that prolonged normal pregnancy in rats or maintained pregnancy in ovariectomized animals (progesterone treatment) prevented the appearance of gap junctions. Gap junctions formed in tissues incubated for 24--96 h in vitro without any hormonal influence. We propose that gap junctions are essential for normal labor and delivery for synchronous contraction of the muscle of the uterus. We present a model for control of parturition that may apply to other animals including humans. The model proposes: 1) the possible roles progesterone, prostaglandins, or estrogens may play in initiating gap-junction formation; 2) that the formation of gap junctions is a necessary step in activation of the myometrium leading to labor; and 3) that agents used to stimulate or inhibit labor may do so by affecting gap junctions.

Animals↗