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C D Richards

Publications and source records attributed to C D Richards.

At least 73 records · Page 4Linked to original sources
PubMed1993

Excitatory and inhibitory synaptic mechanisms in anaesthesia.

To understand the cellular and molecular basis of the anaesthetic state, it is important to remember that, in the intact CNS, synapses operate within elaborate nerve networks. From the data presented above, it is evident that block of impulse conduction in presynaptic fibres does not explain the effects of most anesthetics on synaptic activity. This is not surprising since some anaesthetics, the barbiturates in particular, may both depress excitation and enhance inhibition. General anaesthetics modulate the activity of presynaptic voltage-gated calcium channels and this appears to be sufficient to account for the reduction in transmitter secretion they produce. Transmitter operated ion channels in the postsynaptic membrane are modulated by smaller concentrations of anaesthetics than are required to modulate the presynaptic voltage-gated calcium channels. For this reason, transmitter operated channels appear to represent a major target site for anaesthetics. Finally, there are subtle effects of anaesthetics on the patterns of impulse propagation in nerve axons and on action potential generation in the cell body which result from modulation of membrane excitability. The overall effect of an anaesthetic agent depends on summation of events occurring at the many individual synapses and neurones that make up the network. The effects of anaesthetics on different neuronal pathways may therefore depend on the nature of the receptors and ion channels of the cells that comprise the network. The anaesthetic state may be the result of all these actions, but the characteristics of the state may differ somewhat from agent to agent.

Anesthesia, General↗
PubMed1992

Recombinant oncostatin M stimulates the production of acute phase proteins in HepG2 cells and rat primary hepatocytes in vitro.

Acute inflammation is characterized by increased liver output of acute phase proteins (APP). Several cytokines including IL-6, leukemia inhibitory factor, and IL-11 are capable of stimulating APP synthesis by hepatocytes and hepatoma cells. We have tested the activity of a separate and unique cytokine oncostatin M (OM) and have found potent APP-inducing activity of human recombinant OM on hepatocytes. OM acted in a dose-dependent fashion (ED50 5 to 10 ng/ml) in stimulating APP synthesis in human HepG2 cells, rat H35 cells, and primary rat hepatocyte cultures, but not human Hep3B cells. Human OM induced equivalent to or greater responses than IL-6 in HepG2 cells, however, it was less effective than human IL-6 in stimulating rat cells. Northern analysis showed that OM stimulated mRNA levels of haptoglobin and alpha 1-antichymotrypsin in HepG2 cells. OM induced CAT activity in HepG2 cells transfected with CAT constructs containing IL-6-responsive elements, suggesting that OM induces transcription of native proteins through mechanisms involving IL-6-responsive element-like sequences in gene promoters. OM was also shown to act additively with IL-6 or leukemia inhibitory factor and synergistically with glucocorticoid or IL-1 in the induction of specific APP. These results suggest that OM plays a role as a mediator of APP synthesis in inflammatory responses.

Acute-Phase Proteins↗
PubMed1992

The action of anaesthetics on stimulus-secretion coupling and synaptic activity.

1. Anaesthetics are known to depress excitatory synaptic transmission and the mechanism of this inhibition has been investigated using bovine adrenal chromaffin cells as an experimental model. 2. These cells are homologous with post-ganglionic sympathetic neurons and have well characterized receptor and secretory mechanisms. They are amenable both to the direct measurement of evoked secretion with its associated ion fluxes, and to electrophysiological investigation using the patch clamp technique. 3. These approaches have been used to study the influence of anaesthetics on pre- and post-synaptic mechanisms involved in stimulus-secretion coupling. 4. A variety of agents inhibited secretion evoked by direct depolarization, and this was shown to be due to a reduction in calcium influx. 5. Direct inhibition of voltage-gated calcium currents was confirmed by whole-cell patch clamp measurements. 6. In addition, anaesthetics powerfully modulated nicotinic receptor mediated events: carbachol-evoked secretion was more sensitive to anaesthetics than that stimulated by high potassium. 7. The mechanism of anaesthetic action on the nAChR was examined in more detail with patch-clamp experiments. 8. These showed that anaesthetics reduced the probability of channels being in the open state, largely as a result of reduction in mean channel open time. 9. The data are discussed in relation to excitatory synaptic transmission.

Anesthetics↗
PubMed1992

The mechanism by which procaine inhibits catecholamine secretion from bovine chromaffin cells.

1. We have investigated the action of procaine on stimulus-secretion coupling in bovine adrenal chromaffin cells. 2. Procaine inhibited the catecholamine secretion evoked by 500 microM carbachol (CCh) with an IC50 of 35 microM and the associated calcium influx (IC50 60 microM). It inhibited the catecholamine secretion evoked by depolarization with high potassium by less than 20% even at the highest concentrations tested (3.2 mM). 3. The secretion evoked by CCh was associated with an increase in sodium influx. This evoked influx was also inhibited by procaine (IC50 80 microM). 4. This selective action of procaine on the CCh-evoked catecholamine secretion was investigated further by patch-clamp techniques. 5. In agreement with the ion flux studies, procaine inhibited the inward current evoked by CCh. Procaine also altered the spectral characteristics of the noise associated with the agonist-induced current by adding an additional high frequency component. The amplitude of this component showed an e-fold increase for a 55 mV membrane hyperpolarization. 6. Data from cell-attached patches showed that increasing concentrations of procaine produced a progressive fall in the mean channel open time and an increase in mean blocked time. This combination led to a decrease in mean burst length. In addition, Popen was reduced by 50 microM procaine. These changes in channel conducting time were sufficient to account for the reduction in inward current. A limited study of the action of procaine on nicotinic channels in outside-out patches gave similar results. 7. The data were considered in relation to various schemes of anaesthetic-channel interactions. The data did not fit the sequential blocking model or the extended channel block model but could be fitted to a modified sequential blocking model in which the rate constant for channel reopening after block was itself subject to modulation by the anaesthetic and the blocked channel could close without passing through the open state.

Animals↗
PubMed1991

Effects of pentobarbitone on the properties of nicotinic channels of chromaffin cells.

We have used the whole cell patch clamp technique to investigate the action of pentobarbitone on the nicotinic channels of bovine chromaffin cells. Application of agonists induced an inward current associated with a large increase in current noise. The noise could be fitted by Lorentzian functions with time constants of 17 +/- 2 ms for 10 microM acetylcholine and 10 +/- 1 ms for 10 microM carbachol. The single channel conductance estimated from the current variance was about 25 pS in each case. Pentobarbitone decreased the time constants in a concentration-dependent fashion, but the unit conductances were unaffected. Single channel events were recorded in chromaffin cells held under voltage clamp. Pentobarbitone did not reduce the amplitude of channel openings or the probability of channel opening but reduced the mean channel open time. This reduction was sufficient to account for the decrease in inward current produced by pentobarbitone.

Acetylcholine↗
PubMed1991

Molecular cloning and sequence of porcine interleukin 6 cDNA and expression of mRNA in synovial fibroblasts in vitro.

Synovial fibroblasts, derived from enzyme digestion of porcine synovial tissue, released interleukin 6 (IL-6) bioactivity in culture and this release was enhanced by IL-1 alpha. The porcine IL-6 was cloned from a cDNA library made from these cells using human IL-6 cDNA as a probe. Clone PIL-6[13-8] was sequenced and coded for 212 amino acids with 62% homology and 42% homology to published sequences of human and mouse (or rat), respectively. The cDNA was used to probe the expression of pig IL-6 at the RNA level in pig synovial fibroblasts in vitro. IL-1 alpha and tumor necrosis factor markedly induced steady state levels of IL-6 at 20 h in serum-free conditions, whereas transforming growth factor-beta (TGF-beta), epidermal growth factor, and 10% fetal calf serum did not. TGF-beta pretreatment dramatically inhibited TNF-induction of the IL-6 mRNA but did not markedly affect induction by IL-1 alpha. However, TGF-beta did reduce IL-6 activity detected in the supernatants of IL-1-induced cells.

Amino Acid Sequence↗
PubMed1991

Porcine collagenase from synovial fibroblasts: cDNA sequence and modulation of expression of RNA in vitro by various cytokines.

Collagenase is a metalloproteinase that is important in extracellular matrix turnover and is produced by synovial fibroblasts in response to various cytokines and growth factors. Porcine collagenase cDNA was cloned and the sequence shows a 469-amino acid (AA) peptide with high homology to the human and rabbit enzyme (84% and 83.4% respectively). Predicted amino acid sequence from position #99-114 agree well with previously obtained NH2-terminal AA sequence data of purified mature, active pig collagenase. Using the cloned porcine cDNA as a probe in Northern analysis, it was found that IL-1, TNF and EGF enhanced 24-hour steady state mRNA levels while TGF-beta inhibited basal expression of collagenase. When added 10 hours previously, TGF-beta partially inhibited the induction of collagenase by TNF and EGF, but did not affect induction by IL-1.

Amino Acid Sequence↗
PubMed1990

Ketamine and MK801 attenuate paired pulse inhibition in the olfactory bulb of the rat.

We have investigated the effects of the phencyclidine like-compounds ketamine and MK801 on the evoked field potentials of rat olfactory bulb. Low doses of ketamine (3-6 mg/kg) blocked the inhibition of mitral cells by granule cells evoked by stimulation of lateral olfactory tract fibres or by stimulation of olfactory nerve. This blockade was not accompanied by a decrease in granule cell excitation as revealed by field potential recording. MK801 had a similar effect on the inhibition of mitral cells evoked by stimulation of the lateral olfactory tract. As ketamine does not influence the inhibitory action of GABA (Anis et al. 1983) these results suggest that both ketamine and MK801 block inhibition by an action on intrinsic excitatory feed-back circuits in the olfactory bulb.

Action Potentials↗
PubMed1989

A novel method for absolute calibration of intracellular pH indicators.

In this paper we present methods to measure intracellular pH (pHi) with fluorescent indicators. These methods are based on the change in intracellular pH following the addition of weak acids and weak bases to the extracellular medium. The first method requires that the fluorescence of the indicator is proportional to the change in pHi that follows the addition of a weak acid or weak base to the extra-cellular medium. The second is a null method which uses a mixture of weak acid and weak base that does not change the fluorescent signal. This null method can be used in situations in which the fluorescent signal is a monotonic but non-linear function of pH. The first method depends upon four assumptions. (i) That only the uncharged forms of the weak acids and bases cross the surface membrane. (ii) That the pKa is the same inside and outside the cell. (iii) That the buffering power is constant. (iv) That there is no significant pH regulation on the time scale of the change in pHi. The null method only requires the first two assumptions. We have made estimates of pHi in four different cell types and compared the results obtained with these methods with those obtained from other methods of pHi calibration.

Animals↗
PubMed1988

An in vitro investigation of the action of ketamine on excitatory synaptic transmission in the hippocampus of the guinea-pig.

The effect of ketamine on excitatory synaptic transmission was examined in vitro in slices of guinea-pig hippocampus. Ketamine (100-500 microM) depressed the amplitude of the field excitatory post-synaptic potential (e.p.s.p.) and the population spike evoked in the dentate gyrus by stimulation of the perforant path. It did not affect the coupling between the e.p.s.p. and the population spike. Ketamine (0.2 mM) had no consistent depolarizing or hyperpolarizing effect on the resting membrane potential of the granule cells nor did it alter their input resistance. It did, however, reduce the amplitude of the intracellularly recorded e.p.s.p. Ketamine (5-200 microM) reduced the sensitivity of the granule cells to 1-glutamate applied iontophoretically but did not significantly depress the potassium-stimulated efflux of [3H]glutamate from synaptosomes. It is concluded that ketamine depresses excitatory synaptic transmission in the dentate gyrus by depressing chemical transmission probably via a post-synaptic mechanism.

Action Potentials↗
PubMed1988

The action of volatile anaesthetics on stimulus-secretion coupling in bovine adrenal chromaffin cells.

1. The action of four volatile anaesthetics, ethrane, halothane, isoflurane and methoxyflurane on stimulus-secretion coupling has been studied in isolated bovine adrenal medullary cells. All four agents inhibited the secretion of adrenaline and noradrenaline evoked by 500 microM carbachol at concentrations within the anaesthetic range. Total catecholamine secretion induced by stimulation with 77 mM potassium was also inhibited but at higher concentrations. All four agents inhibited the 45Ca influx evoked by stimulation with 500 microM carbachol and the 45Ca influx in response to K+-depolarization. 2. When total catecholamine secretion in response to potassium or carbachol was modulated by varying extracellular calcium or by adding halothane or methoxyflurane to the incubation medium, the amount of catecholamine secretion for a given Ca2+ entry was the same. 3. The action of methoxyflurane on the relationship between intracellular free Ca and exocytosis was examined using electropermeabilised cells, which were suspended in solutions containing a range of concentrations of ionised calcium between 10(-8) and 10(-4)M. The anaesthetic had no effect on the activation of exocytosis by intracellular free calcium. 4. Halothane and methoxyflurane inhibited the carbachol-induced secretion of catecholamines in a non-competitive manner. 5. Halothane and methoxyflurane inhibited the increase in 22Na influx evoked by carbachol. For halothane and methoxyflurane this inhibition of Na influx appears to be sufficient to account for the inhibition of the evoked catecholamine secretion. 6. We conclude that the volatile anaesthetics ethrane, halothane, isoflurane and methoxyflurane inhibit the secretion of adrenaline and noradrenaline induced by carbachol at concentrations that lie within the range encountered during general anaesthesia. In addition all four also inhibit the secretion of catecholamines induced by depolarization with 77 mM K+ but at much higher concentrations. The decrease in Ca influx caused by methoxyflurane accounts fully for the decrease in secretion in response to depolarization with potassium. Similar actions at synapses within the CNS may underlie the general anaesthetic effects of these agents.

Adrenal Glands↗
PubMed1987

Na+/H+ exchange is the major mechanism of pH regulation in cultured sympathetic neurons: measurements in single cell bodies and neurites using a fluorescent pH indicator.

The regulation of intracellular pH in single cell bodies and in neurites of cultured neurons from rat superior cervical ganglion was studied by continuous monitoring of pH transients using the fluorescent indicator bis(carboxyethyl)carboxy-fluorescein. Intracellular pH was 7.03 +/- 0.05 (n = 8) in bicarbonate-free media at pH 7.4 and was not affected by depolarization with high potassium. Brief exposure to NH4Cl caused rapid cytoplasmic acidification followed by an exponential return of intracellular pH to the resting value. The apparent first order rate constant for recovery from an NH4Cl-induced acid load was 0.2 +/- 0.03 min-1 (37 degrees C) and was similar in media at pH 6.5 or 7.8. Recovery from an acid load was blocked by removal of extracellular Na+ or by amiloride but was not dependent on extracellular Cl- or phosphate or blocked by inhibitors of anion transport, in the presence or absence of bicarbonate. Addition of 5-10 mM bicarbonate at pH 7.4 resulted in a slight alkalinization of the cytoplasm and enhanced complete restoration of pHi after an NH4Cl-induced acid load. Nerve growth factor did not affect intracellular pH of either growing cells deprived of nerve growth factor up to 6 days or of newly isolated neurons left at 4 degrees C for a week before exposure to nerve growth factor. Phorbol 12-myristate 13-acetate had no effect on the pH of cell bodies of growing cells and increased pH of cells deprived of nerve growth factor by less than 0.05 pH units. It is concluded that: pH regulation in cultured sympathetic neurons is largely achieved by Na+/H+ exchange; Bicarbonate may also participate in pH regulation, but not by its exchange with Cl-.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1987

The action of pentobarbitone on stimulus-secretion coupling in adrenal chromaffin cells.

The action of pentobarbitone on stimulus-secretion coupling was studied in bovine isolated adrenal medullary cells. Pentobarbitone inhibited catecholamine release evoked by 500 microM carbachol with half maximal inhibition (IC50) around 50 microM. It also inhibited catecholamine release induced by depolarization with 77 mM potassium (IC50 100 microM). These effects of pentobarbitone were observed with concentrations that lie within the range encountered during general anaesthesia. Evoked secretion required the presence of calcium in the extracellular medium and was associated with an influx of Ca2+ through voltage-sensitive channels. Pentobarbitone inhibited 45Ca influx in response to both carbachol (IC50 50 microM) and K+-depolarization (IC50 150 microM). The action of pentobarbitone on the relationship between intracellular free Ca and exocytosis was examined using electropermeabilised cells which were suspended in solutions containing a range of concentrations of ionised calcium between 10(-8) and 10(-4)M. Catecholamine secretion was measured in the presence of 0, 50, 200 or 500 microM pentobarbitone. The anaesthetic had no effect on the activation of exocytosis by intracellular free calcium. When catecholamine secretion in response to potassium or carbachol was modulated by varying extracellular calcium or by adding pentobarbitone to the incubation medium, the amount of catecholamine secretion for a given Ca2+ entry was the same. Pentobarbitone inhibited the secretion and 45Ca uptake induced by carbachol in a non-competitive manner. The secretion evoked by nicotinic agonists was associated with an increase in 22Na influx. Pentobarbitone inhibited this influx with an IC50 of 100 microM. We concluded that: (a) Pentobarbitone inhibits the catecholamine secretion from bovine adrenal chromaffin cells induced by nicotinic agonists by non-competitive inhibition of the nicotinic receptor. (b) The decrease in Ca influx caused by pentobarbitone accounts fully for the decrease in secretion in response to depolarization with potassium.

Adrenal Cortex↗
PubMed1986

Active suppression of host-versus-graft reaction in pregnant mice. VIII. The uterine decidua-associated suppressor cell is distinct from decidual NK cells.

The fetus resulting from allogeneic mating expresses a variety of antigens that may serve as targets for rejection by the maternal immune system. Accumulation of non-T suppressor cells into the uterine decidua of allopregnant mice may serve to prevent such rejection. It has been previously shown that the suppressor activity in decidua during the second half of murine pregnancy is predominantly associated with a population of small lymphocytes with cytoplasmic granules that lack T-cell markers and inhibit the generation of cytotoxic lymphocytes (CTL) against paternal alloantigens both in vitro and in vivo. Since natural killer cells (NK) also possess cytoplasmic granules and may regulate the murine immune response, we examined the hypothesis that the decidua-associated non-T suppressor cell may represent a regulatory type of NK cell. Similar to NK cells, the decidua-associated suppressor cell expressed FcR for IgG. Unlike NK cells, the decidua-associated suppressor cell proved resistant to treatment with anti-asialo GM1 + C'. Sedimentation velocity examination demonstrated that decidua-associated NK activity was associated with cell population with a modal sedimentation of 4 mm/hr that was larger than the decidua-associated suppressor population. Potent suppressor cell activity was also recovered from the decidua of NK deficient allopregnant bg/bg mice. Therefore, decidua-associated NK cells and suppressor cells represent two distinct populations.

Animals↗
PubMed1986

An analysis of the action of pentobarbitone on the excitatory postsynaptic potentials and membrane properties of neurones in the guinea-pig olfactory cortex.

Intracellular recordings were made from neurones in slices of guinea-pig olfactory cortex maintained in vitro at 37 degrees C. The average membrane potential was 63 +/- 12 mV and the input resistance of these cells was 42 +/- 20 M omega (mean +/- s.d.). Stimulation of the lateral olfactory tract (l.o.t.) generated a transient depolarization in these cells which had the characteristics of an excitatory postsynaptic potential (e.p.s.p.). If the e.p.s.p. was of sufficient amplitude it culminated in an action potential. The e.p.s.p. was potentiated by repetitive stimulation at 10-50 Hz and showed post-tetanic potentiation after a prolonged period of high frequency stimulation (50-100 Hz for 30-60 s). Pentobarbitone (0.1-0.5 mM) depressed the e.p.s.p. reversibly but was without effect on the resting membrane potential, input resistance or time constant of the neurones. Pentobarbitone did not inhibit potentiation of the e.p.s.p. by a preceding conditioning shock. It is concluded that pentobarbitone does not affect the passive membrane properties of neurones in the olfactory cortex. The depressant action of pentobarbitone on synaptic transmission results from a decrease in the amount of transmitter released in response to a nerve impulse, or a decrease in the sensitivity of the postsynaptic membrane to the transmitter or a combination of both effects.

Animals↗