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N G Publicover

Publications and source records attributed to N G Publicover.

17 recordsLinked to original sources
PubMed1992

Spontaneous release of nitric oxide inhibits electrical, Ca2+ and mechanical transients in canine gastric smooth muscle.

1. In canine antrum, rhythmic electrical activity consists of a rapid upstroke phase followed by a plateau depolarization. In response to slow waves, cytosolic Ca2+ ([Ca2+]cyt) and tension increased. 2. Addition of sodium nitroprusside (SNP, 0.5 microM) decreased the amplitude of the plateau phase of slow waves without significant effects on the upstroke depolarization. SNP also inhibited changes in [Ca2+]cyt and tension associated with the plateau potential. SNP induced a negative chronotropic effect at concentrations above 0.1 microM. 3. Similar to the effects of SNP, illumination of muscles during slow waves with ultraviolet (UV) light caused premature repolarization. UV illumination is known to release NO in some tissues. 4. L-NG-monomethyl-arginine (L-NMMA, 300 microM), Methylene Blue (MB, 5 microM) and oxyhaemoglobin (oxy-Hb, 5 microM) increased the force of contractions. In contrast, L-arginine (L-Arg, 300 microM) decreased contractile force and antagonized the effects of L-NMMA. 5. During the upstroke phase, SNP caused a small reduction in [Ca2+]cyt and a large reduction in force, suggesting that SNP caused a decrease in Ca2+ sensitivity. 6. In muscles permeabilized by alpha-toxin, cyclic GMP (100 microM) and UV illumination inhibited Ca(2+)-induced contraction (at pCa 5.5). 7. These data suggest that NO or NO-related compounds are spontaneously released in gastric muscles. These agents have two effects on excitation-contraction coupling: (i) inhibition (directly and/or indirectly) of the voltage-dependent Ca2+ channels that participate in the plateau phase of slow waves, and (ii) reduction in the Ca2+ sensitivity of the contractile element.

Animals↗
PubMed1992

Cyclic AMP-mediated regulation of excitation-contraction coupling in canine gastric smooth muscle.

1. Agonists known to increase cyclic AMP levels in gastrointestinal smooth muscles were studied in isolated circular muscles of the canine antrum to investigate the mechanisms of the inhibitory effects of these agents. 2. Muscles were electrically active, generating typical slow wave activity. Cytosolic Ca2+ ([Ca2+]cyt; measured by Indo-1 fluorescence) and tension increased in response to slow waves. 3. Stimulation by isoprenaline (via beta 2-receptors) or forskolin, in the presence or absence of acetylcholine, inhibited the plateau phase and reduced phasic [Ca2+]cyt and contractile responses. 4. Vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP), had similar effects to isoprenaline and forskolin. 5. Increases in the plateau phase of slow waves and the associated increases in [Ca2+]cyt and tension caused by direct activation of voltage-dependent Ca2+ channels by Bay K 8644 (0.1 microM) were also reduced by forskolin. 6. Isoprenaline and forskolin induced negative chronotropic effects, but VIP increased frequency. 7. At a given level of [Ca2+]cyt, contractions were greater under control conditions than in the presence of isoprenaline, VIP and CGRP, suggesting that part of the inhibition produced by these agents may be due to decreased Ca2+ sensitivity of the contractile apparatus. 8. Experiments performed on alpha-toxin-permeabilized muscles confirmed that cyclic AMP-dependent effects involve reduced Ca2+ sensitivity of the contractile apparatus. Addition of cyclic AMP (3-300 microM) caused a reduction in Ca(2+)-induced contraction at a constant level of Ca2+ (pCa 5.5). 9. These results suggest that increased cyclic AMP and probably subsequent activation of protein kinase A: (i) decrease [Ca2+]cyt and contraction by an inhibition of Ca2+ influx during slow waves, and (ii) decrease the sensitivity of the contractile apparatus to [Ca2+]cyt. The membrane effects might occur directly by inhibition of Ca2+ channels or indirectly by increasing the open probability of K+ channels which would tend to cause premature repolarization of slow waves.

Animals↗
PubMed1992

Calcium oscillations in freshly dispersed and cultured interstitial cells from canine colon.

Spontaneous oscillations in intracellular Ca2+ concentration ([Ca2+]i) and membrane potential were used to monitor rhythmicity in freshly dispersed and cultured interstitial cells (IC) from the canine colon. The frequency of oscillations and responses to a number of channel blockers, agonists, ionic substitutions, and temperature were similar in freshly dispersed and cultured cells. An increase in the amplitude of Ca2+ oscillations after 3-6 days in culture and an increase in the rate of decline of [Ca2+]i in cultured IC were two differences noted between freshly dispersed and cultured cells. The frequency and amplitude of oscillations were a function of extracellular Ca2+ concentrations, and oscillations were abolished when the transmembrane flux of Ca2+ was reduced by nicardipine, La3+, or removal of Ca2+ from the extracellular medium. Oscillations persisted in the presence of ryanodine and ouabain. Lowered temperatures or a reduction in the concentration of Ca2+ in the medium reduced the frequency of spontaneous oscillations. Carbachol and substance P caused a transient increase in [Ca2+]i. Substance P then abolished spontaneous events. ATP and calcitonin gene-related peptide increased the frequency of spontaneous activity. Vasoactive intestinal peptide caused a temporary delay in spontaneous oscillations when added to the medium. Results indicate that freshly dispersed and cultured IC may be useful in studies of the mechanisms of rhythmicity in the gastrointestinal system.

Adenosine Triphosphate↗
PubMed1992

Regulation of calcium current by voltage and cytoplasmic calcium in canine gastric smooth muscle.

The regulation of Ca2+ current by intracellular Ca2+ was studied in isolated myocytes from the circular layer of canine gastric antrum. Ca2+ current was measured with the whole cell patch-clamp technique, and changes in cytoplasmic Ca2+ ([Ca2+]i) were simultaneously measured with indo-1 fluorescence. Ca2+ currents were activated by depolarization and inactivated despite maintained depolarization. Ca2+ current inactivation was fit with a double exponential function. Using Ba2+ or Na+ as charge carriers removed the fast component of inactivation, whereas enhanced intracellular buffering of Ca2+ did not remove the fast component. Ca2+ currents were associated with a rise in [Ca2+]i. The decrease in [Ca2+]i following repolarization was exponential, and during the relaxation of [Ca2+]i, Ca2+ current was inactivated. The inward current recovered with a similar time course as the decrease in [Ca2+]i, suggesting that [Ca2+]i regulates the basal availability of Ca2+ channels. These data support the hypothesis that, although [Ca2+]i may influence the resting level of inactivation, it is the "submembrane" compartment of [Ca2+]i that regulates the development of inactivation.

Animals↗
PubMed1992

Negative-feedback regulation of excitation-contraction coupling in gastric smooth muscle.

The role of phosphatidylinositol (PI) turnover in excitation-contraction coupling was investigated in canine antral smooth muscle. Acetylcholine (ACh; 0.1-1 microM) transiently increased tissue levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and increased the amplitudes of the plateau phase of slow waves and associated Ca2+ transients and phasic contractions. ACh also increased basal concentrations of cytosolic Ca2+ ([Ca2+]c), but these changes were not associated with an increase in resting tension. ATP (0.3 mM) had similar effects on Ins(1,4,5)P3 levels, basal [Ca2+]c, and resting tension. However, in contrast to the effects of ACh, ATP transiently reduced the amplitude of the plateau phase of slow waves and reduced the amplitudes of associated Ca2+ transients and phasic contractions. We investigated the possibility that two products of PI turnover, diacylglycerol (DAG) and Ins(1,4,5)P3, might provide negative feedback to regulate Ca2+ entry during slow waves. 1) DAG is known to activate protein kinase C (PKC). Activation of PKC by phorbol 12,13-dibutyrate (PDBu, 0.5 microM) reduced the amplitude of the plateau phase of slow waves and corresponding Ca2+ transients and phasic contractions. Assay of PKC showed that ACh, ATP, and PDBu stimulated enzyme activity. 2) Ins(1,4,5)P3 is known to increase [Ca2+]c by release of Ca2+ from internal stores. Basal [Ca2+]c was also increased by elevated external K+, ionomycin, thapsigargin, or caffeine. Each of these compounds reduced the amplitude and duration of slow waves. Results suggest that products of PI turnover may provide negative-feedback control of Ca2+ influx during slow waves, tending to reduce the amplitude of phasic contractile activity in gastric muscles. Differences in responses to ACh and ATP can be explained by a G protein-dependent mechanism in which ACh suppresses the voltage dependence of Ca(2+)-activated K+ channels.

Acetylcholine↗
PubMed1991

Time-dependent changes in Ca2+ sensitivity during phasic contraction of canine antral smooth muscle.

1. Relationships between cytosolic Ca2+ concentration ([Ca2+]cyt), myosin light chain (MLC) phosphorylation and muscle tension were examined in circular smooth muscle of canine gastric antrum. 2. Electrical slow waves induced a transient increase in [Ca2+]cyt and muscle tension. [Ca2+]cyt increased before the initiation of contraction and reached a maximum before the peak of the phasic contractions. Following the first Ca2+ transient, a second rise in [Ca2+]cyt was often observed. The second Ca2+ transient was of similar magnitude to the first, but only in some cases was this increase in [Ca2+]cyt associated with a second phase of contraction. Relaxation occurred more rapidly than the restoration of resting levels of [Ca2+]cyt. 3. Acetylcholine (ACh; 3 x 10(-7) M) increased the amplitude of Ca2+ transients, caused MLC phosphorylation and increased the force of contraction. The decay of contraction and MLC dephosphorylation preceded that of [Ca2+]cyt. 4. Increasing external K+ (to 25-40 mM) caused a sustained increase in [Ca2+]cyt, but little change in resting tension. This suggests that the Ca2+ sensitivity decreased as [Ca2+]cyt increased. Increasing K+ to 59.5 mM further increased the level of [Ca2+]cyt, induced MLC phosphorylation and caused a transient contraction. When normal levels of K+ were restored, the rates of MLC dephosphorylation and relaxation exceeded the rate of decay in [Ca2+]cyt. 5. Removal of external Ca2+ in depolarized muscles decreased [Ca2+]cyt below the resting level without affecting resting tension. Readmission of Ca2+ to depolarized muscles caused force to develop at [Ca2+]cyt levels below the original resting level, suggesting that Ca2+ sensitivity was increased when the resting level of [Ca2+]cyt was decreased. 6. The phosphatase inhibitor, calyculin-A (10(-6) M), induced tonic contraction and MLC phosphorylation without an increase in [Ca2+]cyt. During these contractures, electrical activity caused transient increases in [Ca2+]cyt and phasic contractions which were superimposed upon the Ca(2+)-independent contracture. In the presence of calyculin-A, relaxation occurred in two phases. The initial, rapid phase of relaxation was not significantly affected by calyculin-A, but the slow phase was significantly decreased. 7. These results suggest that the relationship between [Ca2+]cyt, MLC phosphorylation and contraction changes as a function of [Ca2+]cyt in canine antral muscles. This may be due to a Ca(2+)-and time-dependent phosphatase that regulates the level of myosin phosphorylation.

Acetylcholine↗
PubMed1991

Relationship between calcium current and cytosolic calcium in canine gastric smooth muscle cells.

We measured free intracellular Ca2+ concentration ([Ca2+]i) and Ca2+ current (ICa) simultaneously in voltage-clamped, indo-1-loaded smooth muscle cells isolated from the circular layer of the canine antrum. Resting [Ca2+]i averaged 144 +/- 20 nM in cells held at -70 mV. Depolarization positive to -50 mV elicited ICa and increased [Ca2+]i. Peak [Ca2+]i occurred between 0 and +10 mV and averaged 372 +/- 48 nM. On repolarization, [Ca2+]i decreased slowly (time constant 2-3 s) and the rate depended on the magnitude of [Ca2+]i. Cells were also voltage clamped with protocols that mimicked the upstroke and plateau phases of slow waves. With simulated plateau potentials of -55 to -45 mV, [Ca2+]i increased transiently as a result of the small transient ICa elicited by the upstroke depolarization. Sustained ICa was of sufficient magnitude with plateau depolarizations positive to -40 mV to cause a secondary rise in [Ca2+]i throughout the plateau phase. These data suggest that at the plateau potential of slow waves in situ, ICa is sufficient to cause a sustained increase in [Ca2+]i. The resulting accumulation of Ca2+ may couple the slow wave plateau to contraction and may increase the open probability of Ca(2+)-activated K channels. The latter may provide the outward current necessary to initiate repolarization.

Animals↗
PubMed1991

Simultaneous measurement of membrane potential, cytosolic Ca2+, and tension in intact smooth muscles.

Microelectrode techniques and the fluorescent Ca2+ indicator indo-1 were used to measure membrane potential, cytosolic Ca2+ ([Ca2+]cyt), and muscle tension simultaneously in canine antral smooth muscles. Responses of muscles from the myenteric and submucosal regions were compared, since electrical activity and excitation-contraction coupling in these regions differ. The upstroke phase of electrical slow waves in both regions induced an increase in [Ca2+]cyt. In myenteric muscles the plateau phase of slow waves often caused either a further rise in [Ca2+]cyt or maintenance of the level reached during the upstroke event. In submucosal muscles, the plateau phase was significantly smaller and did not induce a second phase in the Ca2+ transient. Contractions were related to the amplitudes of Ca2+ transients. Acetylcholine (ACh; 3 x 10(-8)-10(-6) M) increased the amplitude and duration of the plateau phase of slow waves in a concentration-dependent manner. ACh also increased the second phase of Ca2+ transients and contractile responses associated with the plateau potential. In submucosal muscles ACh induced a significant increase in the plateau phase of the slow wave and increased the corresponding phase of Ca2+ transient. Nicardipine (10(-6) M) inhibited plateau phase of slow waves and the associated increases in [Ca2+]cyt and muscle tension. BAY K 8644 (10(-7) M) augmented the plateau potential and increased [Ca2+]cyt and muscle tension. These results suggest that dihydropyridine-sensitive Ca2+ currents participate in the plateau potential. Cholinergic stimulation modulates [Ca2+]cyt and therefore force by regulating the amount of Ca2+ entering cells through these channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Inhibition of electrical slow waves and Ca2+ currents of gastric and colonic smooth muscle by phosphatase inhibitors.

The effects of calyculin A, a phosphatase inhibitor isolated from the marine sponge Discodermia calyx, on the electrical activity of colonic and gastric muscles were studied. Calyculin A reduced the amplitude and duration of slow waves, primarily by inhibiting the plateau component. Okadaic acid, another phosphatase inhibitor, also reduced the amplitude and duration of gastric slow waves. The mechanism of action of calyculin A was investigated by studying its effects on inward currents of isolated gastric and colonic myocytes. Calyculin A reduced the amplitude of the peak and the sustained components of the inward current. Okadaic acid had similar effects. These data suggest that phosphorylation of Ca2+ channels of gastrointestinal smooth muscles may inhibit Ca2+ currents. This mechanism may provide an important means of regulating the currents responsible for excitation-contraction coupling in these muscles.

Animals↗
PubMed1990

Participation of Ca2(+)-activated K+ channels in electrical activity of canine gastric smooth muscle.

1. The hypothesis that Ca2(+)-activated K+ channels participate in the repolarization of electrical slow waves was tested in isolated cells and intact muscles of the canine gastric antrum. 2. Freshly dispersed cells from the gastric antrum liberally express large conductance channels that were characterized as Ca2(+)-activated K+ channels by several criteria. 3. Mean slope conductance of these channels in symmetrical 140 mM-KCl solutions was 265 +/- 25 pS and reversal potential was 1.3 +/- 3.3 mV. The reversal potential was shifted when K+ was partially replaced with Na+ in a manner consistent with the Nernst equation for the K+ gradient. 4. Open probability was studied in excised patches in solutions containing 10(-7)-10(-6) M-Ca2+ with holding potentials ranging from -100 to +100 mV. Resulting activation curves were fitted by Boltzmann functions. 5. Increasing [Ca2+] from 10(-7) to 10(-6) M shifted the half-maximal activation from +99 to 0 mV. These data suggest that Ca2(+)-activated K+ channels may be activated in the voltage range and [Ca2+]i occurring during the plateau phase of the slow wave. 6. In intact muscles loaded with the photolabile Ca2+ chelator, nitr-5, photo-activated release of Ca2+ during the slow wave cycle produced changes consistent with activation of Ca2(+)-dependent outward currents. 7. The data are consistent with the idea that Ca2+ build-up during electrical slow waves shifts the activation voltage of Ca2(+)-activated K+ channels into the range of the plateau potential. Activation of these channels yields outward current and repolarization. 8. Since the force of contractions depends on slow wave amplitude and duration, regulation of these channels may be important in controlling gastric motility.

Animals↗
PubMed1989

Are relaxation oscillators an appropriate model of gastrointestinal electrical activity?

Mathematical models based on relaxation oscillators have heavily influenced the terminology and experimental designs of investigations in gastrointestinal motility for nearly two decades. Relaxation oscillator equations have been used to stimulate the electrical activities of the esophagus, stomach, small intestine, colon, and rectosigmoid region. It has been suggested that many attributes of gastrointestinal electrical activity cannot be adequately explained by classic "core-conductor" or "cable" models of excitation and conduction. This article critically reviews the relaxation oscillator model and provides an explanation for each of the putative inadequacies of core-conductor theory. Furthermore, we question whether relaxation oscillator equations are able to simulate the waveforms of gastrointestinal slow waves, alterations in waveform in response to drugs or electrical stimulation, patterns of slow-wave activity when stimulated at physiological frequencies, prolonged periods of constant resting membrane potential between gastric slow waves and electrotonic spread into inactive regions. We conclude that the relaxation oscillator equations do not fully describe gastrointestinal electrical activity; excitation and propagation can be modeled by a theory that provides for morphological features, ionic conductances, and other elements included in the cable equations.

Animals↗
PubMed1986

Dynamic responses of electrically coupled systems.

An identified pair of electrically coupled neurons in the buccal ganglion of the freshwater snail Helisoma trivolvis is an experimentally accessible model of electrical synaptic transmission. In this investigation, electrical synaptic transmission is characterized using sinusoidal frequency (Bode) responses computed by Laplace transforms and responses to brief stimuli. The frequency response of the injected neuron shows a 20-dB/decade attenuation and a phase shift from 0 degree at low frequencies to -90 degrees at high frequencies. The response of a coupled cell shows a 40-dB/decade attenuation and a phase shift from 0 degrees at low frequencies to -180 degrees at high frequencies. A simple mathematical model of electrical synaptic transmission is described that displays each of these crucial features of the measured frequency responses. Methods are described to estimate the frequency responses of coupled systems based on presynaptic measurements. The responses of the coupled system to brief pulses of current were computed using the principle of superposition. The electrical properties of coupled systems impose a minimum delay in reaching a peak in all postsynaptic responses. The delays in the postsynaptic responses to brief stimuli are related to the electrical and anatomical parameters of coupled networks.

Animals↗
PubMed1977

Somatotopic projections of mystacial vibrissae on cerebral cortex of cats.

1. Using classical evoked-potential techniques and 100-micron deflections of one vibrissa, at least three loci of potentials were identified in cats' cortices. 2. The relationships of these loci to cortical landmarks and to each other varied sufficiently to prevent specification of homologies between animals. 3. Attempts to differentiate the loci on functional criteria were unsuccessful. The areal extent of each locus appeared similar and there was evidence of a specific somatotopic pattern within each locus; in each locus different vibrissae were represented at different cortical points. 4. Latencies differed slightly between the loci, being shortest at the most rostral site and longest at the caudal site. In all loci, off-center latencies were longer, but this difference was detected only after complete maps of all three loci were produced. 5. The focus of potentials for an individual vibrissa was radically different, depending on the parameter measured (negative peak, positive peak, and latency). The focus of potentials could also be shifted by changing the velocity or direction of the stimulus. 6. The effects of these stimulus parameters on the cortical map suggest that a cortical focus represents more than simply a point on the skin surface.

Animals↗