Gian Mario Frigo

Immunological Adverse Effects of Anticonvulsants: What is their Clinical Relevance?

SummaryLong term administration of anticonvulsants is sometimes associated with impairment of the humoral and/or cellular immune response. Furthermore, certain well known adverse reactions to antiepileptics may have an immunotoxicological origin e.g. lymphadenopathy, pseudolymphoma and systemic lupus erythematosus. However, two important questions remain unresolved. First, the possibility that epilepsy per se might be primarily associated with immune alterations makes it difficult to assess the pathogenetic role of a specific drug, especially in a patient population usually on multiple drug therapy. Secondly, the clinical relevance of some of the observed immunological abnormalities is still highly controversial.This review is intended to give an outline of the present state of knowledge on the effects of anticonvulsants on humoral, cellular and nonspecific immunity, with particular regard to some of the major clinical conditions that have been ascribed to drug-induced immune dysregulation, such as pseudolymphoma and systemic autoimmune diseases. The immunotoxic potential of anticonvulsants appears to be low, and immunological monitoring is not usually required except in patients with known immune defects.

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.

Interleukin-8 production induced by the endozepine triakontatetraneuropeptide in human neutrophils: role of calcium and pharmacological investigation of signal transduction pathways

The endozepine triakontatetraneuropeptide (TTN) induces intracellular calcium ([Ca(2+)](i)) changes and is chemotactic for human neutrophils (PMNs). Because interleukin-8 (IL-8) production is Ca(2+) dependent and can be induced by chemotactic stimuli, we have investigated the ability of TTN to induce IL-8 production in PMNs, as well as the signal transduction mechanisms involved. Our results show that TTN increases IL-8 release and IL-8 mRNA expression in a concentration- and time-dependent fashion, and these effects are prevented by the Ca(2+) chelator BAPTA-AM. TTN-induced [Ca(2+)](i) changes and IL-8 mRNA expression are sensitive to pertussis toxin, to the phospholipase C (PLC) inhibitor U73122 (but not to its inactive analogue U73343) and to the protein kinase C (PKC) inhibitor calphostin C. It is therefore suggested that TTN-induced IL-8 production in human PMNs results from a G protein-operated, PLC-activated [Ca(2+)](i) rise, and PKC contributes to this effect. These findings further support the possible role of TTN in the modulation of the inflammatory processes.

Inhibition of endogenous acetylcholine release by blockade of voltage-dependent calcium channels in enteric neurons of the guinea-pig colon.

Abstract— The effects on acetylcholine release from the guinea‐pig colon of the N‐type calcium channel blocker ω‐conotoxin GVIA (ω‐conotoxin), the L‐type calcium channel blocker nifedipine and the putative blocker of T‐type channels, flunarizine, have been investigated. Endogenous basal acetylcholine release and electrically (1 Hz, 1 ms, 450 mA)‐evoked overflow in the presence of cholinesterase inhibitor were studied. ω‐Conotoxin (1–10 nM) and nifedipine (0·03–3 μm) dose‐dependently inhibited basal and electrically‐evoked acetylcholine release. Maximal inhibition of basal or electrically‐evoked acetylcholine release was about 40% for nifedipine and about 75% for ω‐conotoxin. The potency of nifedipine was inversely related to the external calcium concentration: its EC50 value in low‐calcium medium (0·5 Mm) was as low as 12 Nm. Flunarizine inhibited acetylcholine release only at concentrations higher than 0·2 μm. Our results are consistent with an involvement of N‐ and L‐type calcium channels in the control of the endogenous acetylcholine release from the guinea‐pig colon.

Opioid pathways exert a tonic restraint in the guinea‐pig isolated colon: changes after chronic sympathetic denervation

Abstract— We have studied the effects of naloxone on acetylcholine and noradrenaline release in the guinea‐pig isolated distal colon, and have assessed the effect of naloxone on electrically‐induced contractions of the longitudinal muscle and non‐adrenergic, non‐cholinergic (NANC) relaxations of the circular muscle coat. Naloxone dose‐dependently increased resting and electrically‐evoked acetylcholine release and electrically‐evoked noradrenaline release. Naloxone was more potent in increasing resting acetylcholine release in colonic specimens obtained after chronic sympathetic denervation. Naloxone (1 μm) did not affect electrically‐induced contractions of the longitudinal muscle, while it enhanced NANC relaxations of the circular muscle. The effects observed with naloxone in the present experiments suggest that opioid pathways exert a tonic restraint on neurotransmission in the guinea‐pig colon. After suppression of the adrenergic inhibitory tone, the functional relevance of opioid pathways seems to be increased.

Quantitative analysis of intestinal electrical spike activity by a new computerized method

We developed a computerized method that allows quantitative analysis of intestinal spike activity. This method was tested for a series of measurements in six fasting conscious dogs, fitted with bipolar electrodes chronically implanted along the small intestine. Data were stored on 8-channel tape recordings and digitized before computer processing. Spike detection was accomplished by means of a discriminant function able to differentiate spikes from non-spikes (e.g. artifacts) on the basis of six parameters. Computer analysis allowed accurate spike recognition (probability of incorrect classification less than 6%). Spike activity during fasting was monitored by calculating, during each phase of the migrating myoelectric complex, the following parameters per unit of time (30 s): number of spikes, percentage of spiked slow waves and number of spikes per spiked slow wave. Results were given both in tabular and graphical form. This method provides a research tool for a better quantitation of intestinal spike activity.

Effect of desipramine-induced blockade of neuronal uptake mechanisms on adrenoceptor-mediated responses in the guinea-pig colon.

In order to clarify whether adrenoceptors in the guinea-pig distal colon are under sympathetic control, we assessed possible variations in the sensitivity to adrenoceptor agonists after blockade of neuronal catecholamine uptake mechanisms by desipramine (DMI). First, experiments were carried out to investigate the effects of DMI added in the organ bath on propulsion velocity, endogenous and [3H] prelabelled acetylcholine overflow, electrically evoked noradrenaline overflow and longitudinal smooth muscle tone. Secondly, we studied the effects of adrenoceptor agonists on the above parameters in untreated animals and in animals chronically treated with DMI.DMI added in the organ bath at concentrations equal to or higher than 30 nM inhibited all the parameters under study. Thus, when evaluating the effect of DMI on concentration-response curves to adrenoceptor agonists, concentrations which were per se inactive were used. DMI added in the organ bath at concentrations up to 30 nM potentiated the inhibitory effects of exogenous noradrenaline on propulsion velocity and acetylcholine overflow, but it did not affect the concentration-response curve to exogenous noradrenaline on longitudinal smooth muscle tone. Furthermore, 30 nM DMI inhibited propulsion velocity during sympathetic nerve stimulation. In preparations obtained from animals chronically treated with DMI, no significant change of propulsion velocity, endogenous and [3H] prelabelled acetylcholine overflow was found with respect to untreated animals. Nevertheless, in such preparations subsensitivity to isoprenaline (acting mainly on muscular β-adrenoceptors) and clonidine (acting on neuronal α2-adrenoceptors) and supersensitivity to phenylephrine were observed. Electrically evoked noradrenaline overflow was enhanced, in a frequency-dependent way, by yohimbine and inhibited by clonidine.We conclude that in the guinea-pig colon: 1) α- and β-adrenoceptors are under tonic neuronal control, as indicated by the sensitivity changes to α- and β-adrenoceptor agonists after chronic DMI treatment; 2) exogenous NA reaching neuronal, but not muscular adrenoceptors, is affected by neuronal uptake mechanisms; 3) NA released by adrenergic terminals undergoes neuronal uptake and is controlled by α2-autoreceptors.

[3H]Acetylcholine release from the guinea-pig distal colon: comparison with ileal [3H]acetylcholine release and effect of adrenoceptor stimulation

Abstract— To study cholinergic function in the guinea‐pig colon, resting and electrically evoked 3H release after preincubation with [3H] choline has been compared in colonic and ileal myenteric plexus preparations. Fractional spontaneous colonic 3H release was significantly higher than ileal 3H release, while the reverse was true for electrically evoked 3H outflow. Electrically evoked 3H outflow in the colon was linearly related to stimulation frequency (0·2–3 Hz range) and current intensity (300–600 mA range), while 3H outflow per pulse was inversely related to stimulation frequency. Electrically evoked 3H outflow was prevented in Ca2+‐free solution, indicating that it probably mirrored neuronal exocytotic [3H]acetylcholine release. Both noradrenaline and clonidine concentration‐dependently inhibited electrically evoked 3H outflow, Clonidine being more potent but less efficacious than noradrenaline. For both noradrenaline and Clonidine, the potency and efficacy for inhibition of 3H outflow were close to the values previously reported for the inhibition of electrically evoked endogenous acetylcholine output from colonic preparations. In conclusion, these data indicate that 3H release after incubation with [3H]choline is a valid alternative to measurement of endogenous acetylcholine output to study colonic cholinergic neuronal function in the guinea‐pig.

Occurrence of α and β Receptors in the Bile Duct

Summary and conclusion By means of a new method which permits one to evaluate separately the behavior in situ of the choledochus and duodenum of guinea pig, we confirmed our previous assumption concerning the existence in the terminal bile duct of α-receptors whose activation caused a constriction and β-receptors whose activation caused a dilatation. The contracting action of epinephrine was selectively blocked by diben-amine, while the relaxing action of isopren-aline was blocked by pronethalol.

Effect of β-casomorphins on intestinal propulsion in the guinea-pig colon

β‐Casomorphins are a family of opioid peptides originally isolated from β‐casein. In view of a possible physiological significance of these milk‐derived compounds, the effects of bovine β‐casomorphin‐5 (β‐CM‐5), β‐casomorphin‐4 (β‐CM‐4) and D‐Ala2‐β‐casomorphin‐4‐NH2 (D‐Ala2‐β‐CM‐4‐NH2) have been investigated on the peristaltic reflex in the guinea‐pig isolated colon and compared with morphine. β‐CM‐5 and D‐Ala2‐β‐CM‐4‐NH2 each dose‐dependently inhibited the velocity of propulsion of an intraluminal bolus; β‐CM‐4 was ineffective. IC50 values were 0·30, 5·21 and 0·29 μfor morphine, β‐CM‐5 and D‐Ala2‐β‐CM‐4‐NH2, respectively. The potency ratios vs morphine were 0·06 and 0·96 for β‐CM‐5 and D‐Ala2‐β‐CM‐4‐NH2, respectively. Blockade of the peristaltic reflex by β‐CM‐5 or D‐Ala2‐β‐CM‐4‐NH2 was reversed by the opioid antagonist naloxone. D‐Ala2‐β‐CM‐4‐NH2 also dose‐dependently inhibited resting acetylcholine output (IC50 = 5·69 μM; potency ratio vs morphine: 0·63). In conclusion, certain β‐casomorphins inhibit intestinal propulsion and cholinergic neurotransmission in the guinea‐pig colon, probably by acting at opioid receptors.