Biomedical subjects
Publications and source records attributed to J Donaldson.
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An investigation was made of a range of agents capable of elevating tissue cyclic AMP levels, or acting as a stable analogue of cyclic AMP, upon carbachol induced inositol phosphate responses in bovine tracheal smooth muscle slices. Whereas the beta 2 adrenoceptor agonist salbutamol (1 microM) and the membrane permeable analogue of cyclic AMP, 8-bromo-cyclic AMP (1 mM) were without effect upon total [3H]inositol phosphate formation induced by carbachol, 3-iso-butyl-1-methylaxanthine (IBMX) (EC50 140 microM), the high Km, cyclic AMP selective phosphodiesterase inhibitor rolipram (EC50 41 microM) and theophylline (EC50 76 microM) all inhibited the inositol phosphate response to low (1 microM) concentrations of carbachol. IBMX (IC50 13 microM), rolipram (IC50 4.6 microM) and theophylline (IC50 180 microM) all relaxed bovine tracheal muscle strips precontracted with methacholine (1 microM). The adenylate cyclase activator forskolin (1 microM), produced a much smaller (10% inhibition) effect upon inositol phosphate formation induced by carbachol. Carbachol (1 microM-1 mM) did not inhibit forskolin induced [3H]cyclic AMP formation. An inhibitor of the cyclic GMP preferring phosphodiesterase isozyme, M&B 22948 (1-100 microM), was without effect upon either carbachol induced inositol phosphate formation or trachealis tone. It is concluded that IBMX, rolipram and theophylline inhibit carbachol stimulated inositol phosphate formation, possibly through a cyclic AMP independent mechanism.
The effects of forskolin (1 microM) and EGTA (5 mM) on indirect cyclic AMP responses in slices of guinea-pig cerebral cortex were examined. Forskolin had little effect on the direct 2-chloroadenosine-stimulated cyclic AMP response. However, it completely abolished the glutamate-induced augmentation of this response. In contrast, forskolin had very little effect on the indirect cyclic AMP responses to noradrenaline, 5-hydroxytryptamine, and histamine. Conversely, rapid removal of extracellular calcium with EGTA 2 min before addition of the indirectly acting agent markedly reduced the augmentation responses produced by these latter agonists, but had little effect on the glutamate augmentation. When EGTA was added once a steady level of cyclic AMP had been achieved with the indirect agents, it was without effect on any of the responses. Thus, calcium appears to have a role in the early, but not the later, stages of the noradrenaline, 5-hydroxytryptamine, and histamine responses. A role for protein kinase C in the glutamate augmentation response was suggested, because forskolin inhibited the augmentation of the 2-chloroadenosine response produced by phorbol esters (which mimic the actions of diacylglycerol in activating protein kinase C). We conclude that there is more than one mechanism by which the augmentation of cyclic AMP responses can occur.
1. The effect of fluoroaluminate complexes (AlCl3 plus NaF) upon smooth muscle tone, [3H]-inositol phosphate accumulation and [3H]-cyclic AMP accumulation has been investigated in slices of bovine tracheal smooth muscle. 2. Fluoroaluminate (10 microM AlCl3 + various concentrations of NaF) elicited concentration-dependent contractions of bovine tracheal smooth muscle strips at concentrations of NaF in the range 1-10 mM. The resultant contractile response was reversed by isoprenaline (50 nM) and was preserved in calcium-free medium. 3. Fluoroaluminate stimulated [3H]-inositol phosphate formation at concentrations of NaF over 1 mM. The response to 20 mM NaF + 10 microM AlCl3 was 164 +/- 29% of the response to 1 mM histamine. Fluoroaluminate also increased the incorporation of [3H]-myo-inositol into membrane phospholipids. 4. Fluoroaluminate produced a small rise in [3H]-cyclic AMP levels (2.1 fold increase over basal with 20 mM NaF). The response to forskolin (1 microM, 8.6 fold over basal) was reduced by fluoroaluminate in a concentration-dependent manner, but still remained significantly (P less than 0.05) elevated over the response to fluoroaluminate alone. 5. The [3H]-inositol phosphate response to fluoroaluminate was inhibited by salbutamol (maximum inhibition 60%, IC50 = 0.08 microM), forskolin (1 microM, 46% inhibition) and isobutylmethylxanthine (1 mM, 73% inhibition). 6. These data suggest that inhibition of agonist-induced inositol phospholipid turnover by cyclic AMP in this tissue can occur at the post-receptor level.
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1. The effect on histamine-stimulated [3H]-inositol phosphate accumulation of a range of agents which increase the accumulation, or mimic the actions, of cyclic AMP has been investigated in bovine tracheal smooth muscle. 2. Salbutamol (1 microM), forskolin (1 microM) and vasoactive intestinal peptide (VIP, 1 microM) inhibited the inositol phosphate response to 0.1 mM histamine and increased the accumulation of [3H]-cyclic AMP in [3H]-adenine-labelled slices of bovine tracheal smooth muscle. The effect on inositol phospholipid hydrolysis was mimicked by the membrane permeant analogues of cyclic AMP, dibutrylcyclic AMP (1 mM) and 8-bromo-cyclic AMP (1 mM). 3. In contrast to salbutamol, which was equally effective at producing the two effects, forskolin produced large increases in [3H]-cyclic AMP accumulation (EC50 = 1.2 microM) at much higher concentrations than those required for inhibition of histamine-stimulated [3H]-inositol phosphate accumulation (EC50 = 0.09 microM). However, significant increases in [3H]-cyclic AMP accumulation, of similar magnitude to those obtained with salbutamol and VIP, were observed over the concentration range appropriate for inhibition of the inositol phosphate response to histamine. 4. In the presence of histamine (0.1 mM), isobutylmethylxanthine (IBMX, 1 mM) and rolipram (0.1 mM) both significantly (P less than 0.05) elevated tissue [3H]-cyclic AMP levels. IBMX, rolipram and (to a lesser extent) SKF 94120 significantly (P less than 0.05) reduced histamine-stimulated [3H]-inositol phosphate accumulation by 81%, 68% and 20%, respectively. M&B 22948 was without a significant effect on either [3H]-cyclic AMP or histamine-induced [3H]-inositol phosphate accumulation. 5. Both rolipram and forskolin reduced the increase in incorporation of [3H]-inositol into membrane phospholipids which followed stimulation with histamine. However, a significant inhibition of [3H]-inositol phosphate accumulation could be demonstrated under conditions in which there was no change in the level of [3H]-inositol incorporation.
1. 2-Chloroadenosine (2CA) causes a maintained rise in adenosine 3':5'-cyclic monophosphate (cyclic AMP) content of guinea-pig cerebral cortical slices which is augmented by addition of histamine. We have investigated the temporal profile of the sensitivity of this response to calcium. 2. Rapid removal of extracellular calcium with EGTA (5 mM) at 2CA (30 microM)-induced steady state caused a slight increase in the cyclic AMP response to 2CA alone and completely abolished the augmentation produced by histamine (0.1 mM) added 20 min later. When EGTA was added only 2 min before histamine, the augmentation was reduced by 72%. 3. The calcium sensitivity of the histamine response was also indicated in studies in which EGTA was added 1 or 3 min after histamine at 2CA-induced steady state. Following addition of EGTA at either of these times, the augmentation was not maintained. 4. When calcium was rapidly removed with EGTA once a steady state level of cyclic AMP had been achieved with histamine, the augmentation response was maintained. This was despite the fact that EGTA had a similar effect on both extracellular free calcium and tissue calcium content when it was applied before or after histamine. 5. The 2CA response was augmented by phorbol esters (which mimic the actions of diacylglycerol) in a calcium-independent manner. 6. These results suggest that calcium is important for the initiation and early stages of the histamine-induced augmentation response. The apparent lack of calcium sensitivity of the response at later stages could mean that calcium is not involved in the maintenance of the response or that the intracellular machinery involved in the augmentation process becomes more sensitive to calcium as the response progresses, such that it becomes able to operate at a much lower level of intracellular calcium. A possible role for diacylglycerol in the maintenance of the response is discussed.
The effect of the phosphodiesterase (PDE) inhibitor rolipram on the cyclic AMP responses to adenosine, histamine and combinations of these two agonists, was examined in [3H]adenine-labelled slices of guinea-pig cerebral cortex. Constant levels of [3H]-cyclic AMP were achieved within 10 min of agonist addition, both in the presence and absence of rolipram (0.1 mM). Histamine (1 mM) produced an 8-fold increase in [3H]-cyclic AMP (compared with basal) which was increased 7-fold by rolipram. The responses to adenosine (0.1 mM) and adenosine and histamine in combination were larger than that to histamine alone (46-fold or more compared with basal) but the potentiation by rolipram was much smaller (2.5-fold or less). With both agonists the effect of rolipram was dose-dependent, the steady state [3H]-cyclic AMP levels increasing 1-2-fold for a 10-fold increase in rolipram concentration. Removal of the histamine or adenosine stimulus once steady state had been reached resulted in a rapid fall in [3H]-cyclic AMP levels with a half time of less than 5 min. Rolipram (0.1 mM) did not significantly alter the initial rates of fall in [3H]-cyclic AMP levels but increased the time taken for them to return to basal levels. The findings of higher steady state levels of cyclic AMP in the presence of rolipram, together with an almost unaltered rate of cyclic AMP turnover, are consistent with an interaction of rolipram with PDE which is overcome by an increase in cyclic AMP concentration. However, the relatively smaller effects of rolipram on the higher steady levels of cyclic AMP produced by adenosine and the rather shallow dose-dependence of the PDE inhibitor on the responses to both agonists are inconsistent with a simple competitive inhibition of total PDE activity in responding cells. The results can be explained, however, by the involvement of different forms of PDE, with the rolipram-sensitive, calcium-independent form dominating at low cyclic AMP levels and the rolipram-insensitive, calcium-dependent form becoming more important when cyclic AMP levels are higher.
Rift Valley fever (RVF) is a major cause of human morbidity and mortality in endemic areas of sub-Saharan Africa and has the potential to cause epidemic disease in receptive areas world-wide. In this study, a RVF viral isolate from the 1977 Egyptian epidemic (ZH-501) inoculated intravenously into rhesus macaques caused a benign viremic infection in most, but resulted in the hemorrhagic fever syndrome in 20 per cent (3 of 15). Serious disease of this type has not previously been observed in nonhuman primates inoculated with RVF virus and may be a consequence of the viral strain used or the route of inoculation. Severe disease was accompanied by extensive liver necrosis, disseminated intravascular coagulation, and microangiopathic hemolytic anemia. We also attempted to prevent RVF by passive transfer of serum from vaccinated rhesus monkeys (plaque-reduction neutralization test titer 1:2,560). As little as 0.025 ml/kg prevented the development of viremia in naive rhesus monkeys after subcutaneous inoculation of virus. The monkey model should be helpful in understanding the pathogenesis and prevention of human RVF.
Histamine, acting via H1 receptors, augments adenosine-induced cAMP accumulation in slices of guinea pig cerebral cortex by an indirect mechanism that appears to involve an intracellular messenger(s). To better characterize this process, the time course of this augmentation was examined in slices prelabeled with [3H] adenine. When histamine (1 mM) was added after the cAMP level had reached steady state with adenosine (0.1 mM), the cAMP level rose to a new steady level within 10 min (t 1/2, 2-3 min). There was no measurable delay in this response, indicating rapid activation of the augmentation after receptor occupation. Studies using the H1 receptor antagonist mepyramine indicated that the continued presence of the histamine stimulus was required to maintain the augmentation. Addition of mepyramine (10 microM) between 1 and 14 min after histamine caused cAMP levels to fall to a level similar to that obtained previously with adenosine alone, but with a delay of 2-3 min. This gives an upper estimate of the lifetime of any intracellular messenger involved in the augmentation process. To determine whether histamine acts by stimulating synthesis of cAMP or by inhibiting its breakdown, the fall in tissue cAMP content was studied after rapid removal of the adenosine stimulus by addition of adenosine deaminase. The initial fall was significantly faster in slices incubated with 0.1 mM adenosine plus 1 mM histamine than in slices with 0.1 mM adenosine alone, indicating increased synthesis and breakdown of cAMP in the presence of histamine. However, the higher breakdown rate probably reflects stimulation of the degradation process by the higher initial level of cAMP with histamine because, at equivalent levels, cAMP content fell at similar rates in both conditions. This was confirmed in other experiments in which similar steady state cAMP levels were achieved with and without histamine by appropriate choice of adenosine concentrations. It is therefore concluded that the direct effect of histamine is primarily to potentiate cAMP synthesis.
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The effect of 1,4-dithiothreitol (DTT) on histamine H1-receptor agonist affinity and efficacy has been investigated in longitudinal muscle strips of guinea-pig ileum. Exposure of ileal smooth muscle to DTT significantly increased the maximal responses to the partial agonists SKF71473 and DE-2PEA, indicative of an increase in agonist efficacy. This effect was paralleled by a small decrease in EC50 values. In contrast, DTT produced a parallel displacement of the concentration-response curve to the full agonist histamine in the same muscle strips. Studies in which phenoxybenzamine and benzilylcholine mustard were used to reduce the maximum response to histamine suggested that DTT altered both agonist affinity and efficacy. The affinity constant for histamine, calculated by the method of Furchgott & Bursztyn (1967), increased by 2.7 fold in the presence of DTT. Furthermore, agonist efficacy also appeared to increase in the presence of DTT since the maximum response to histamine following phenoxybenzamine treatment increased on application of DTT. [3H]-mepyramine binding studies confirmed that DTT increased agonist affinity. DTT produced a significant parallel shift to the left of the displacement curves for histamine, 2-methylhistamine, 2-pyridylethylamine and 2-thiazolylethylamine. The results of this study therefore suggest that DTT potentiates H1-receptor-mediated contractile activity in guinea-pig ileum by increasing both agonist efficacy and affinity.
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The effect of the disulphide bond reducing agent 1,4-dithiothreitol (DTT) on the binding characteristics of the H1-selective ligand [3H]mepyramine has been examined in homogenates of guinea-pig cerebral cortex and cerebellum. DTT was found to be without effect on antagonist binding. This was evident from studies using different concentrations of [3H]mepyramine (0.5-10.0 nM), which showed no change in either the equilibrium dissociation constant (KD) or specific binding site capacity (Bmax). Furthermore, the IC50 values and slope parameters determined from the inhibition of the binding of 1 nM [3H]mepyramine by non-radioactive mepyramine were similarly insensitive to DTT in both cerebral cortex and cerebellum. In contrast, DTT shifted the inhibition curve for histamine to lower agonist concentrations and reduced the Hill coefficient in these two tissues. Analysis of these inhibition curves as double hyperbolae revealed two binding sites in the presence of DTT and only one low affinity site in the absence of DTT. Similar changes in the location (IC50) and slope (Hill coefficient) parameters were obtained with the H1-selective agonist 2-thiazolylethylamine in cerebellum and with 2-methylhistamine in both brain regions. The results of this study show that DTT affects agonist but not antagonist binding in guinea-pig cerebellum and cerebral cortex, and suggest that DTT stabilises a proportion of the agonist binding sites in a high affinity state.
The effect of histamine and the H1-selective agonist, 2-pyridylethylamine, on the accumulation of inositol monophosphate (InsP), inositol bisphosphate (InsP2) and inositol trisphosphate (InsP3) has been examined in lithium-treated slices of guinea-pig cerebellum and ileal smooth muscle. Following 45 min incubation, histamine produced a large accumulation of [3H]InsP and a smaller accumulation of [3H]InsP2 and [3H]InsP3 in both tissues. In cerebellar slices all three responses to histamine were potently and competitively inhibited by the selective H1-receptor antagonist, mepyramine. In contrast, incubation of ileal slices with mepyramine (0.1 microM) produced only a small reduction (circa 20%) in the maximal accumulation elicited by histamine of each [3H]inositol phosphate with no significant effect on the EC50 or Hill coefficient. However, when 2-pyridylethylamine, instead of histamine, was used to stimulate inositol phospholipid hydrolysis in ileal smooth muscle, the agonist-induced responses appeared to be competitively antagonised by mepyramine. The results presented indicate that there is an apparent dissociation between histamine-induced InsP3 accumulation and H1-receptor-mediated contractile activity in ileal smooth muscle and suggest that agonist-induced inositol phospholipid hydrolysis in this tissue may be involved in other cellular events separate from those involving calcium.