Priming-like effect and successful desensitization after anaphylactic shock by latex sublingual immunotherapy.

The present report deals with some unusual events observed restarting allergy vaccine in a case of anaphylactic shock which occurred giving the maximum scheduled dose (25 drops) of rush sublingual immunotherapy (SLIT) to latex. Restarting SLIT by usual (not rush) scheme we observed long-lasting fall of reactivity threshold. The maximum tolerable dose was reduced to 2 drops, a dose fivefold smaller than the one well tolerated during previous rush phase (10 drops). We have excluded a possible nocebo effect and proved that the reduced tolerance was real by double blind placebo-controlled challenge test. We have considered this effect in some ways similar to priming effect. SLIT was continued with two drops every day. After about twenty months we could demostrate a significant reduction of skin reactivity by end-point technique and an improved response to the controlled exposition to latex by use-tests. In the same time the tolerance to vaccine was improved to three drops. The better safety profile allowed us to restart and continue with SLIT also in the reported case of anaphlicatic shock by latex vaccine and, after about two years, to induce valuable hyposensitization. Latex SLIT is confirmed as a safe and effective method.

Interaction between phenytoin and valproic acid: plasma protein binding and metabolic effects.

The effect of sodium valproate (400 mg three times daily) on the disposition kinetics of intravenous phenytoin (250 mg) was investigated in seven normal subjects. After valproate, the free (unbound) fraction of phenytoin in serum rose from 9.6 +/- 0.9% (SD) to 15.6 +/- 1.4% on average (p < 0.001). The effect was associated with an increase in systemic clearance and apparent volume of distribution of total drug. There was a strong positive correlation between percent increment in each of these parameters and percent increment in unbound drug in serum. Free phenytoin concentration in serum and phenytoin concentration in saliva increased during valproate administration. As a result, both the clearance and the apparent volume of distribution of free drug were reduced. There was an increase in the renal excretion of unchanged phenytoin during valproate administration, but the effect was too small to have an appreciable influence on the overall clearance of the drug. There were no consistent changes in the excretion of the major metabolite 5, p-hydroxyphenyl, 5-phenyl, hydantoin (pHPPH), in the urine. These results suggest that valproic acid may have two separate and opposing effects on phenytoin disposition: (1) displacing phenytoin from plasma protein binding sites, thereby enhancing the systemic clearance of total drug, and (2) inhibiting phenytoin metabolism, thereby increasing the concentration of free drug in the serum.

DNA methylases separated through the HeLa cell cycle methodology show allosteric properties.

Two DNA methylases (DNAmets) can be separated through the cell cycle. The first appears as a minor peak in G1, the second as a major peak in S. Both enzymes protect from HpaII a plasmid (H31), constructed with the pBR322 vector (4.3 kbp) and the inverted A gamma fragment of the human globin gene (3.5 kbp), inserted at its HindIII site (the vector carries several HpaII sites, the insert only one HpaII site). DNAmets G1 and S show distinct Km values and different kinetics vs the ionic strength of the medium, while their Michaelis-Menten and Lineweaver-Burk plots are sigmoidal and hyperbolical curves, respectively. This is the first suggestion about the allosteric nature of the eukaryotic DNAmet system.

Plasma protein binding of drugs in pregnancy.

The degree of binding to plasma proteins is an important determinant of drug disposition and response. Normal human pregnancy is associated with concentration of plasma proteins, free fatty acids and possibly other endogenous substances interfering with drug binding. The possibility of an associated change in plasma binding capacity therefore needs to be taken into consideration. Experimental studies conducted mostly in vitro have shown that the plasma protein binding of many (but not all) drugs is decreased during pregnancy, particularly during the last trimester. This phenomenon should be taken into account when interpreting serum concentrations of total (free + protein-bound) drug in clinical practice. Notable examples of drugs whose unbound fraction increases during pregnancy include diazepam, valproic acid, phenytoin, phenobarbitone, salicylic acid, pethidine, lignocaine, dexamethasone, sulphafurazole and propranolol. For many drugs, important differences have been demonstrated in the degree of protein binding between maternal and cord plasma. In some cases, this may provide an explanation for the finding of marked differences in total drug concentration between maternal and fetal plasma at the time of delivery.

Interpretation of drug levels in acute and chronic disease states.

Serum drug concentration monitoring can be an invaluable aid to patient management, particularly in certain pathological conditions when individualisation of dosage is particularly critical. To be clinically useful, however, drug levels must be interpreted in the context of all factors that could influence the correlation between the concentration of the drug in plasma and the intensity of action. Several such factors may be operating in acute and chronic disease states. For example, a number of pathological conditions are associated with marked changes in the fraction of free, pharmacologically active drug in plasma and this will result in disruption of the normal relationship between total serum drug level and effect, as seen for phenytoin in uraemia. An altered response to a given serum drug level in disease states may also be caused by changes in tissue distribution, by abnormal accumulation of pharmacologically active metabolites in plasma or by changes in end-organ responsiveness. The latter are best illustrated by the altered sensitivity to digoxin in patients with various conditions, including hypokalaemia and thyroid disease. In addition to the factors listed above, consideration should also be given to potential interactions with concomitantly used drugs and to the possibility of analytical errors, especially in view of the evidence that the performance of otherwise reliable drug assays may be grossly impaired in certain diseases (e.g. uraemia), due to abnormal plasma composition and/or accumulation of interfering metabolites. In view of these complexities, a correct interpretation of serum drug levels requires a good knowledge of clinical pharmacology and a close collaboration between physician and laboratory. In any case, serum drug concentrations, like other laboratory tests, are not a substitute for careful patient observation, and any decision about drug treatment should be primarily based upon evaluation of the clinical state and, whenever possible, direct measurement of drug effects.

The effect of catecholamines and sympathetic stimulation on the release of acetylcholine from the guinea-pig colon.

1. In isolated guinea-pig terminal colon, the effect of sympathetic stimulation on contraction and acetylcholine release elicited by pelvic and transmural stimulation was investigated.2. Sympathetic stimulation reduced the nerve-mediated contractile responses more than those produced by added acetylcholine.3. Sympathetic stimulation also reduced the acetylcholine released during pelvic and transmural stimulation at low frequency. The inhibitory effect on acetylcholine released from resting colons is concealed by the simultaneous release of acetylcholine in considerable amounts from stimulated periarterial nerves which probably contain parasympathetic fibres.4. The inhibitory effect of endogenous and exogenous catecholamines prevails when cholinergic neurones fire at low rates. It was confirmed that adrenaline is more active than noradrenaline.5. The release of acetylcholine from unstimulated colons was for the most part maintained by nerve-conducted activity, because tetrodotoxin was able to reduce it to about one-tenth.6. It is suggested that the sympathetic control of gastrointestinal tone and motility is exerted through two different routes: inhibition of intramural cholinergic plexuses and direct relaxation of smooth muscle cells.7. The possible site and mechanism of action of catecholamines on intramural cholinergic structures is briefly discussed.

A pharmacological analysis of the peristaltic reflex in the isolated colon of the guinea-pig or cat.

1. The peristaltic reflex in the colon was elicited by a localized intraluminal stimulus. The contractile response of the longitudinal coat, which consists of two phases, begins before the start of propulsion. Although the contractions of the longitudinal and circular musculature are usually associated, they may be independent of each other. In particular, the longitudinal contraction does not seem to be necessary for propulsion.2. Both the longitudinal reflex contraction and the segmental responses of the circular muscle to distension, namely a contraction above and a relaxation below the bolus, are abolished by tetrodotoxin and ganglion blocking agents.3. In the guinea-pig, longitudinal and circular reflex contractions are usually resistant to antimuscarine, antihistamine and antitryptamine drugs but in the cat they are abolished by antimuscarine drugs. In both species, however, atropine and hyoscine can impair propulsion by blocking selectively the descending inhibition. In the cat, it is possible to find doses which abolish the descending inhibition without affecting the contractile responses of the longitudinal and circular muscle.4. Sympathetic denervation and pretreatment with reserpine do not affect the propulsive activity. The maintenance of the descending inhibition in denervated organs suggests that the inhibitory neurones to the circular muscle are not adrenergic.5. On the basis of the effects of drugs, the possible nervous mechanism subserving the polarity of propulsion has been examined. Such a mechanism seems to require an inhibitory pathway involving muscarinic receptors at some point.6. Pelvic nerve stimulation facilitates propulsive activity. The effect of transmural stimulation is different at low and at high frequencies of stimulation. The inhibitory effect of sympathetic stimulation on the reflex responses seems to be due mainly to an action on intrinsic nervous structures.

The role of GABAA receptor function in peristaltic activity of the guinea-pig ileum: a comparative study with bicuculline, SR 95531 and picrotoxinin.

1. The peristaltic activity of the guinea-pig ileum was studied in the absence and in the presence of the blockade of GABAA receptors. 2. Bicuculline (1-30 microM), improved at the highest concentrations the efficiency of peristalsis by enhancing the frequency of propulsive contractions and the amount of fluid ejected per unit of time. 3. Neither SR 95531 (0.3-10 microM), a novel GABAA receptor antagonist, which competitively antagonized 3-aminopropane sulphonic acid induced contractions in myenteric plexus-longitudinal muscle preparations (pA2 value: 6.47), nor picrotoxinin (1-30 microM) modified peristaltic parameters or influenced the potentiating effect of bicuculline on peristaltic activity. 4. In myenteric plexus-longitudinal muscle preparations, bicuculline (1-30 microM) enhanced the amplitude of electrically-induced cholinergic contractions without modifying submaximal contractions to applied acetylcholine. SR 95531 and picrotoxinin had no effect on twitch amplitude. In the presence of each of these compounds, bicuculline retained its potentiating effect. 5. The results obtained with SR 95531 and picrotoxinin question the view that GABAA receptors may exert a critical role in intestinal propulsion by modulating the activity of nerve pathways subserving peristalsis. Bicuculline potentiates the peristaltic activity of the ileum probably via a facilitatory effect on enteric cholinergic transmission that is independent of GABAA receptor blockade.

Effects of desensitization to adenosine 5'-triphosphate and adenosine on non-adrenergic inhibitory responses in the circular muscle of rabbit colon.

An approximate eight fold desensitization of the circular coat of the distal rabbit colon to adenosine 5'-triphosphate (ATP) and adenosine could be achieved by repeatedly exposing the organ to relatively low concentrations (10-100 microM) of these compounds. The desensitization was specific and reversible after prolonged washing. It could be overcome by increasing the concentrations of the purine agonists. Dipyridamole potentiated the non-adrenergic inhibition in response to transmural stimulation but failed to influence the caudad relaxation evoked by radial distension. Desensitization to ATP and adenosine (and to ATP + adenosine simultaneously) did not affect the non-adrenergic inhibition in response to radial distension or transmural stimulation. These results suggest that neither ATP nor adenosine are the final transmitters mediating the non-adrenergic inhibitory responses in the distal colon of the rabbit.

Purine receptors in the guinea-pig internal anal sphincter.

1 In the isolated internal anal sphincter of the guinea-pig, adenosine 5'-triphosphate (ATP) and adenosine induced a concentration-dependent and tetrodotoxin-insensitive relaxation. 2 Pretreatment with theophylline (25-50 microM) had no significant effect on the concentration-response curves obtained with either purine compound. 3 Reactive blue 2 (25-100 microM) shifted the curve to ATP to the right in a dose-dependent fashion leaving that to adenosine unaltered. The antagonism appeared to be non-competitive. 4 Neither reactive blue 2 nor purine receptor occupation by ATP or adenosine altered the electrically-induced non-adrenergic, non-cholinergic inhibitory response. 5 The actions of ATP and adenosine in the guinea-pig internal anal sphincter appear to be mediated by separate receptors. These receptors are not involved in the nerve-mediated relaxation.

An in vitro study of the relationship between GABA receptor function and propulsive motility in the distal colon of the rabbit.

1. The effects of gamma-aminobutyric acid (GABA), 3-aminopropane sulphonic acid (3-APS) and baclofen on spontaneous, electrically-induced and propulsive motility were investigated in rabbit distal colon. 2. In unstimulated longitudinal (LMPs) and circular muscle strip preparations (CMPs) 3-APS (10-200 microM) and GABA caused a clear-cut relaxation susceptible to desensitization. Baclofen (10-200 microM) caused relaxation in a minority (30%) of preparations. The 3-APS response was sensitive to tetrodotoxin (TTX; 1 microM), SR 95531 (a novel competitive GABAA-receptor antagonist) (10 microM), picrotoxinin (30 microM), and insensitive to hyoscine (1 microM) and to a combination of prazosin (1 microM) and propranolol (1 microM). The baclofen response was antagonized by 5-aminovaleric acid (DAVA, 500 microM), TTX and hyoscine and resistant to GABAA-receptor and adrenoceptor blockade. GABAA-receptors were therefore associated with non-adrenergic non-cholinergic (NANC) inhibitory nerve activation while GABAB-receptors were involved in depression of cholinergic tone of smooth muscle. GABA (10-200 microM) elicited both above mentioned effects. 3. In LMPs, baclofen (10-200 microM) dose-dependently inhibited submaximal responses to both cholinergic and NANC inhibitory nerve stimulation. This effect was resistant to SR 95531 and picrotoxinin and prevented by DAVA and baclofen desensitization. GABA (10-200 microM) mimicked the action of baclofen. GABA inhibitory effects persisted in the presence of GABAA-receptor blockade. 4. In segments of distal colon, GABA and baclofen (1-200 microM), but not 3-APS (1-200 microM), dose-dependently decreased the velocity of propulsion of an intraluminally-distended balloon. This effect was antagonized by DAVA and GABA or baclofen desensitization and resistant to SR 95531 and picrotoxinin. These antagonists per se had no effect on propulsion. In preparations in which propulsion was slowed by hyoscine (1 microM), baclofen caused no consistent further depression of propulsive activity. 5. Our results show that GABAA- and GABAB-receptors are present in rabbit colon. GABAA-receptor stimulation activates NANC inhibitory nerves without apparently affecting propulsion. GABAB-receptors are associated with a reduction of neural (mainly cholinergic) activity subserving muscular tone and peristalsis and appear to be located on both cholinergic and NANC inhibitory nerves. However, the persisting propulsive activity during suppression of GABAA- and GABAB-receptor function suggests that GABA in enteric neurones is not crucial for the neural circuitry subserving colonic peristalsis in this species.

Inhibitory effects of SR 58611A on canine colonic motility: evidence for a role of beta 3-adrenoceptors.

1. In order to clarify whether atypical or beta 3-adrenoceptors can modulate canine colonic motility in vivo, we studied the effects of SR 58611A (a selective agonist for atypical beta-adrenoceptors) alone and after pretreatment with beta-adrenoceptor antagonists on colonic motility in the conscious dog. The gastrocolonic response (postprandial increase in motility) was monitored by means of electrodes and strain-gauge force transducers chronically implanted along the distal colon. In some experiments, heart rate was also measured. The possible role of beta 3-adrenoceptors in mediating the effects of SR 58611A was also tested in vitro in circular muscle strips taken from the canine distal colon. 2. Intravenous infusion of SR 58611A, ritodrine or isoprenaline at doses inducing the same degree of tachycardia inhibited the gastrocolonic response to a different extent, with SR 58611A and ritodrine being more effective than isoprenaline. 3. In a dose-response study, SR 58611A was more potent in inhibiting colonic motility than in inducing tachycardia: the ED35 values for inhibition of colonic motility and induction of tachycardia were 23 and 156 micrograms kg-1, i.v., respectively. 4. The inhibitory effect of SR 58611A 100 micrograms kg-1, i.v., on the gastrocolonic response was reversed by alprenolol (non-selective beta-adrenoceptor antagonist), but resistant to CGP 20712A (beta 1-adrenoceptor antagonist) or ICI 118551 (beta 2-adrenoceptor antagonist). 5. In vitro, SR 58611A concentration-dependently relaxed circular muscle strips, an effect that was competitively antagonized by alprenolol with a pA2 value of 7.1, but resistant to CGP 20712A (100 nM), ICI 118551 (100 nM) or tetrodotoxin (1 microM). 6. The present study provides strong functional evidence for a role of atypical or beta 3-adrenoceptors in the modulation of canine colonic motility both in vivo and in vitro by an inhibitory effect most likely at the smooth muscle level.

Inhibitory effects of calcium channel blockers on intestinal motility in the dog.

Calcium channel blockers are now widely used for the treatment of cardiovascular disorders. However, data concerning their effects on intestinal motility in vivo are still rather fragmentary. Therefore, we evaluated the effects of three prototype calcium channel blockers (nifedipine, verapamil and diltiazem) on intestinal motility in five fasting, conscious dogs fitted with electrodes and strain-gauges along the small bowel. The myoelectric data were analyzed by a recently developed and validated computer program which allows accurate monitoring of intestinal spike activity. The mechanical data were analyzed by calculating a motility index. After recording of at least two migrating motor complexes (control), an i.v. infusion of one of the following calcium channel blockers was maintained for 3 h: 0.29 or 0.87 mumol/kg per h nifedipine, 1.02 or 2.04 mumol/kg per h verapamil and 1.11 or 2.22 mumol/kg per h diltiazem. Nifedipine 0.29 mumol/kg per h significantly reduced (P less than 0.05) spike activity and motility index during phases II and III without disrupting migrating motor complex cycling. The higher dose suppressed migrating motor complex cycling and almost completely abolished both spike and mechanical activities. The two doses of verapamil had effects similar to those of the two doses of nifedipine. Both doses of diltiazem significantly reduced (P less than 0.05) spike activity and motility index during phases II and III without disrupting migrating motor complex cycling. We conclude that all the agents tested, apart from their well known cardiovascular effects, also have a profound inhibitory effect on intestinal motility in vivo, the order of potency being nifedipine greater than verapamil greater than diltiazem. The search for more selective calcium channel blockers for the treatment of intestinal motor disorders with minimal cardiovascular effects is warranted.

Hyoscine-resistant peristalsis in guinea-pig ileum.

The effect of hyoscine on the peristaltic activity of the proximal and distal ileum of the guinea-pig was studied. Hyoscine strongly impaired peristalsis as indicated by the elevation of the threshold pressure and by the increased number of incomplete peristalses and blockades. Functional activity of the circular musculature was more markedly impaired. However, particularly in the distal ileum, complete peristalses occurred even after 70 min exposure to hyoscine at a concentration of 10(-6) g/ml. A tenfold increase in hyoscine concentration failed to produce further impairment of peristaltic activity and of the oral reflex contraction. The activity which remained in the presence of hyoscine was blocked by methysergide and by d-tubocurarine. The hypothesis is advanced that once the muscarinic receptors have been blocked, increased radial stretch of the circular coat results in activation of a separate, tetrodotoxin sensitive, excitatory nervous pathway, which is sufficient to maintain a discrete degree of peristaltic activity.

Inhibitory action of capsaicin on cholinergic responses induced by GABAA agonists in the guinea-pig ileum.

Pretreatment of the guinea-pig ileum with capsaicin resulted consistently in depression of the neurogenic cholinergic contractions induced by the GABAA receptor agonists 3-aminopropane sulphonic acid (3-APS) and muscimol. Since capsaicin acts mainly by releasing and depleting substance P from its stores in intestinal nerves, it is likely that substance P plays a role in the response caused by GABAA-mimetic compounds, On the whole, our results suggest that excitatory responses to 3-APS and muscimol result from both direct and indirect activation of intrinsic intestinal cholinergic neurons innervating smooth muscle cells.

A re-appraisal of the mode of action of 5-HT in inhibiting GABA-induced cholinergic contractions in the guinea-pig ileum.

In the guinea-pig ileum, pretreatment with 5-hydroxytryptamine (5-HT) (5 microM) for 4-5 min inhibited both 5-HT- and gamma-aminobutyric acid (GABA)-induced cholinergic contractions without consistently altering those induced by electrical field stimulation. Cisapride (1 micron) antagonized 5-HT-induced cholinergic contractions but left those induced by GABA or twitch responses unchanged. These results indicate that the 5-HT action in inhibiting GABA-induced cholinergic responses may arise at the interneuronal level, thus suggesting that GABA may also indirectly activate cholinergic terminal neurons. These findings rule out the possibility of 5-HT acting as an intermediate transmitter in this type of response.

Depression by morphine of the excitability of intrinsic inhibitory neurons in the guinea-pig colon.

The mechanical responses to morphine were examined in isolated preparations of longitudinal and circular muscle of the guinea-pig colon. In the longitudinal coat, morphine induced a relaxation which was prevented by naloxone, hyoscine and tetrodotoxin. Conversely, in the circular coat morphine caused a contraction which was antagonized by naloxone, mimicked by tetrodotoxin and left unaltered by hyoscine, chlorpheniramine and methysergide. In both muscular layers, morphine depressed (and tetrodotoxin abolished) the non-adrenergic relaxation induced by field stimulation. The action of morphine in the two preparations can thus be explained in terms of inhibition of the tonic excitatory cholinergic or inhibitory non-adrenergic neural control prevailing in the longitudinal and circular muscle respectively.

Mode of action of ATP on propulsive activity in rabbit colon.

ATP induced a concentration-dependent reduction of the velocity of propulsion in isolated segments of rabbit colon as assessed by the aboral displacement of an intraluminal rubber balloon, and delayed the onset of the propulsive wave. ATP depressed both the reflex contraction of the circular coat above the distended balloon and the response of the circular muscle to transmural (cholinergic) stimulation. On the contrary, ATP (up to 200 muM), while causing relaxation of the circular muscle, had no effect on either the muscular contractile response induced by carbachol and histamine or the non-adrenergic inhibitory responses elicited by electrical stimulation and by radial distension of the gut wall. Within the concentration range used (10-200 muM), ATP concentration-depression curves for propulsion and transmural excitatory stimulation were shifted to the right in the presence of theophylline (10 muM). Theophylline, however, had no influence on either the direct inhibitory action of ATP on circular smooth muscle or the non-adrenergic relaxation in response to electrical stimulation. These data are consistent with the concept that at least two populations of purinergic receptors are present in intestinal tissue. Those populations located presynaptically, unlike those located postsynaptically, are blocked by theophylline. Since the contractile machinery does not appear to be affected by ATP concentrations up to 200 muM, the mechanism by which ATP impairs propulsive activity is probably dependent on activation of presynaptic purinergic receptors located on the nervous pathways subserving the wave of contraction, without having any appreciable influence on descending inhibition.

Effects of pentazocine on the intestine and biliary tract of the rabbit in vitro.

Pentazocine impairs peristaltic activity and relaxes longitudinal muscle in the colon and in the ileum. The circular coat is excited in the colon, while in the ileum pentazocine exhibits both excitatory and inhibitory effects depending on the concentration employed. Pentazocine does not exert a spasmogenic effect in the smooth muscle of terminal bile duct but instead reduces the electrically-induced contraction. The effect of pentazocine does not seem to involve endogenous acetylcholine or catecholamine release.

5-HT mediated GABA excitatory responses in the guinea-pig proximal ileum.

GABA (3--100 microM) and 5-HT (0.03--30 microM) caused concentration-dependent transient contractions of the longitudinal muscle of the guinea-pig ileum. The contractile response to GABA was antagonized by hyoscine (2.2 microM). TTX (0.7 microM), bicuculline (3 microM), furosemide (25 microM) and desensitization to GABA itself, while hexamethonium (20 microM) and methysergide (20 microM) were without effect. The contractile response to 5-HT was antagonized by hyoscine (2.2 microM), TTX (0.7 microM) and desensitization to 5-HT itself and was unaffected by bicuculline (10 microM), hexamethonium (20 microM), furosemide (25 microM) and methysergide (20 microM). A desensitization procedure that caused a 84.7-fold increase in the 5-HT EC50 also resulted in a 74.1-fold increase of the GABA EC50. Desensitization to GABA caused a reduction of 5-HT induced response but only in preparations desensitized by high (50 microM) concentrations of GABA. The results indicate that GABA-induced contractions in the guinea-pig ileum are mediated by activation of cholinergic motor neurones. This effect appears to be mediated by interneuronal release of 5-HT rather than by a direct stimulatory action of GABA on the effector neurones.