Administration Of Pertussis Toxin Research Articles

Reversal of pertussis toxin-induced thermal allodynia by muscarinic cholinergic agonists in mice

The intrathecal administration of pertussis toxin (PTX) not only blocks the antinociceptive effects of the muscarinic cholinergic receptor agonist oxotremorine administered systemically, but also produces a long-lasting thermal allodynia in mice. The purpose of the present studies was to determine both the antinociceptive effects in normal mice and the antiallodynic effects in PTX-treated mice of systemically administered muscarinic cholinergic receptor agonists and cholinesterase inhibitors. In normal mice, antinociceptive effects were tested using a 55°C water-bath tail-flick test. In mice treated 7 days previously with PTX (0.3 μg i.t.), antiallodynic effects were tested using a 45°C water-bath tail-flick test. The nonselective high-efficacy muscarinic agonists oxotremorine, H-TZTP (3-(1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine oxalate), and methylthio[2.2.1], (exo (+)3-(3-methylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.1]heptane oxalate), as well as vedaclidine, a mixed M 2/M 4 muscarinic receptor partial agonist and M 1/M 3/M 5 muscarinic receptor antagonist, the nonselective partial agonists RS86 and pilocarpine, and the cholinesterase inhibitors physostigmine and tacrine all produced dose-related antinociception. Oxotremorine, H-TZTP and methylthio[2.2.1] produced dose-related reversals of PTX-induced thermal allodynia whereas vedaclidine produced a partial reversal and RS86 and pilocarpine, as well as physostigmine and tacrine, failed to reverse the allodynia. The present results provide further evidence that decrements in PTX-sensitive G i/o-protein functioning may be involved in initiating and/or maintaining some persistent or neuropathic pain states. Moreover, the present results suggest that muscarinic receptor agonists such as vedaclidine may be useful in the treatment of persistent pain states that are due at least in part to dysfunction of inhibitory second messenger systems.

Pharmacologic reversal of pertussis toxin-induced thermal allodynia in mice

We have previously demonstrated that the intrathecal administration of pertussis toxin produces a long-lasting thermal allodynia in mice. The purpose of the present studies was to compare the antinociceptive and the antiallodynic effects of drugs that are commonly used in treating neuropathic allodynia in untreated mice and in mice which had been administered vehicle or pertussis toxin intrathecally 7 days previously. In untreated mice, morphine, fentanyl, clonidine, oxymetazoline, desipramine and lidocaine, but not MK801, produced dose-related antinociception when tested using a 55°C water tail-flick test. However, 7 days after the intrathecal injection of pertussis toxin, which induced a condition of thermal allodynia when tested using a 45°C water bath, the full opioid and the full α 2-adrenergic receptor agonists fentanyl and clonidine, but not the partial opioid nor the partial α 2-adrenergic receptor agonists morphine and oxymetazoline, reversed the pertussis toxin-induced thermal allodynia. Moreover, lidocaine, desipramine, carbamazepine and MK801 failed to reverse the pertussis toxin-induced thermal allodynia. The present results suggest that decrements in G i/G o-protein function may be involved in initiating and/or maintaining some neuropathic pain states. Moreover, the results of the present study suggest that the use of full, but not partial, opioid or α 2-agonists may be useful in the treatment of thermal allodynic pain states which may be due at least in part to inhibitory second messenger system dysfunction. Further, the underlying biochemistry of the apparent allodynic pain state induced by intrathecal administration of pertussis toxin warrants further investigation.

Activation of I(2)-imidazoline receptors enhances supraspinal morphine analgesia in mice: a model to detect agonist and antagonist activities at these receptors.

This work investigates the receptor acted upon by imidazoline compounds in the modulation of morphine analgesia. The effects of highly selective imidazoline ligands on the supraspinal antinociception induced by morphine in mice were determined. 2. Intracerebroventricular (i.c.v.) or subcutaneous (s.c.) administration of ligands selective for the I(2)-imidazoline receptor, 2-BFI, LSL 60101, LSL 61122 and aganodine, and the non selective ligand agmatine, increased morphine antinociception in a dose-dependent manner. Neither moxonidine, a mixed I(1)-imidazoline and alpha(2)-adrenoceptor agonist, RX821002, a potent alpha(2)-adrenoceptor antagonist that displays low affinity at I(2)-imidazoline receptors, nor the selective non-imidazoline alpha(2)-adrenoceptor antagonist RS-15385-197, modified the analgesic responses to morphine. 3. Administration of pertussis toxin (0.25 microg per mouse, i.c.v.) 6 days before the analgesic test blocked the ability of the I(2)-imidazoline ligands to potentiate morphine antinociception. 4. The increased effect of morphine induced by I(2)-imidazoline ligands (agonists) was completely reversed by idazoxan and BU 224. Identical results were obtained with IBI, which alkylates I(2)-imidazoline binding sites. Thus, both agonist and antagonist properties of imidazoline ligands at the I(2)-imidazoline receptors were observed. 5. Pre-treatment (30 min) with deprenyl, an irreversible inhibitor of monoamine oxidase B (IMAO-B), produced an increase of morphine antinociception. Clorgyline, an irreversible IMAO-A, given 30 min before morphine did not alter the effect of the opioid. At longer intervals (24 h) a single dose of either clorgyline or deprenyl reduced the density of I(2)-imidazoline receptors and prevented the I(2)-mediated potentiation of morphine analgesia. 6. These results demonstrate functional interaction between I(2)-imidazoline and opioid receptors. The involvement of G(i)-G(o) transducer proteins in this modulatory effect is also suggested.

Analysis of experimental autoimmune encephalomyelitis induced in F344 rats by pertussis toxin administration

To elucidate the factor(s) accelerating the autoimmune disease processes, we induced two types of experimental autoimmune encephalomyelitis (EAE), severe and very mild, in F344 rats by immunization with myelin basic protein (MBP) plus pertussis toxin (PT) (PT+) or with MBP alone (PT-) and compared the differences between the two. Immunohistochemical examinations showed that although the nature of inflammation was essentially the same between the two groups, the proportion of Vβ8.2 + T cells in the CNS lesion of PT (+) rats was larger than that of PT (-) rats. Cytokine analysis by competitive PCR revealed that IL-10 mRNA in the lymphoid organ was significantly suppressed in the PT(+) group, whereas levels of IFN-γ,TNF-α and TGF-β mRNA were insignificantly different after PT administration. In addition, T cells taken from PT (+) rats proliferated well in response to MBP, while those from PT (-) rats showed a marginal response to the same antigen. However, this finding does not indicate the switching of non-encephalitogenic to encephalitogenic T cells upon PT administration because PT (-) rats contained encephalitogenic T cells and/or their precursor cells as revealed by adoptive transfer experiments. Taken together, these findings suggest that suppression of IL-10 by PT administration is the major factor contributing to the exacerbation of EAE in PT(+) rats.

The role of potassium channels in antihistamine analgesia

The effect of the administration of pertussis toxin as well as modulators of different subtypes of K + channels on the antinociception induced by the H 1-antihistamines pyrilamine, diphenhydramine and promethazine was evaluated in the mouse hot plate test. Pretreatment with pertussis toxin (0.25 μg/mouse i.c.v.) prevented pyrilamine, diphenhydramine and promethazine antinociception. The K ATP channel openers minoxidil and pinacidil potentiated the antinociception produced by the H 1-antihistamines whereas the K ATP channel blocker gliquidone prevented the anti H 1-induced analgesia. The Ca 2+-gated K + channel blocker apamin antagonized pyrilamine, diphenhydramine and promethazine analgesia. Pretreatment with an antisense oligonucleotide (aODN) to mKv1.1, a voltage-gated K + channel, at the dose of 3.0 nmol/single i.c.v. injection, never modified the antinociception induced by the H 1-antihistamines in comparison with degenerate oligonucleotide (dODN)-treated mice. At the highest effective doses, none of the drugs used modified animals’ gross behaviour nor impaired motor coordination, as revealed by the rota rod test. The present data demonstrate that both K ATP and Ca 2+-gated K + channels, contrary to voltage-gated K + channel Kv1.1, represent an important step in the transduction mechanism underlying central antinociception induced by H 1-antihistamines.

Pertussis toxin treatment differentially affects cholinergic and dopaminergic receptor stimulation of midbrain dopaminergic neurons

Extracellular single-unit recordings and iontophoresis were used to compare the effect of a single administration of pertussis toxin (PTX, 1 μg), into midbrain dopamine (DA) nuclei (A9 and A10 regions), on the muscarinic, NMDA and DA receptor responses of midbrain DA cells in the anesthetized rat. Iontophoretic applications of DA, or apomorphine (50 μg/kg, i.v.), markedly reduced the firing of DA cells in control rats. In PTX-treated animals, these inhibitory responses were totally abolished, indicating that, in both DA nuclei, the inhibitory DA receptors are coupled to Gi/o proteins. In parallel, there was a significant decrease in the number of active DA cells per track which returned to baseline 5 weeks after the treatment. Applications of the muscarinic agonist oxotremorine M (OXO M) or of NMDA produced a potent increase in the firing of DA cells in control rats. DA neurons treated with PTX were still responsive to OXO M, although their sensitivity to the agonist was significantly reduced by 40%. In contrast, NMDA-induced activation remained unchanged, indicating that PTX did not non-selectively dampen all excitatory responses. Applications of cell-permeable cAMP derivatives did not change the basal firing of DA neurons. On the other hand, the phospholipase C inhibitors neomycin and ET-18-OCH 3 (200 μg, i.c.v.), reduced significantly the activation of DA cells induced by OXO M. These data suggest that muscarinic activation of DA cells involves an M1-like receptor, possibly coupled to Gq /11 proteins, but also the participation of a PTX substrate.

KSR-1 Binds to G-protein βγ Subunits and Inhibits βγ-induced Mitogen-activated Protein Kinase Activation

The protein kinase KSR-1 is a recently identified participant in the Ras signaling pathway. The subcellular localization of KSR-1 is variable. In serum-deprived cultured cells, KSR-1 is primarily found in the cytoplasm; in serum-stimulated cells, a significant portion of KSR-1 is found at the plasma membrane. To identify the mechanism that mediates KSR-1 translocation, we performed a yeast two-hybrid screen. Three clones that interacted with KSR-1 were found to encode the full-length gamma10 subunit of heterotrimeric G-proteins. KSR-1 also interacted with gamma2 and gamma3 in a two-hybrid assay. Deletion analysis demonstrated that the isolated CA3 domain of KSR-1, which contains a cysteine-rich zinc finger-like domain, interacted with gamma subunits. Coimmunoprecipitation experiments demonstrated that KSR-1 bound to beta1 gamma3 subunits when all three were transfected into cultured cells. Lysophosphatidic acid treatment of cells induced KSR-1 translocation to the plasma membrane from the cytoplasm that was blocked by administration of pertussis toxin but not by dominant-negative Ras. Finally, transfection of wild-type KSR-1 inhibited beta1 gamma3-induced mitogen-activated protein kinase activation in cultured cells. These results demonstrate that KSR-1 translocation to the plasma membrane is mediated, at least in part, by an interaction with beta gamma and that this interaction may modulate mitogen-activated protein kinase signaling.

Role of potassium channels in the antinociception induced by agonists of alpha2-adrenoceptors.

1. The effect of the administration of pertussis toxin (PTX) as well as modulators of different subtypes of K+ channels on the antinociception induced by clonidine and guanabenz was evaluated in the mouse hot plate test. 2. Pretreatment with pertussis toxin (0.25 microg per mouse i.c.v.) 7 days before the hot-plate test, prevented the antinociception induced by both clonidine (0.08-0.2 mg kg(-1), s.c.) and guanabenz (0.1-0.5 mg kg(-1), s.c.). 3. The administration of the K(ATP) channel openers minoxidil (10 microg per mouse, i.c.v.), pinacidil (25 microg per mouse, i.c.v.) and diazoxide (100 mg kg(-1), p.o.) potentiated the antinociception produced by clonidine and guanabenz whereas the K(ATP) channel blocker gliquidone (6 microg per mouse, i.c.v.) prevented the alpha2 adrenoceptor agonist-induced analgesia. 4. Pretreatment with an antisense oligonucleotide (aODN) to mKv1.1, a voltage-gated K+ channel, at the dose of 2.0 nmol per single i.c.v. injection, prevented the antinociception induced by both clonidine and guanabenz in comparison with degenerate oligonucleotide (dODN)-treated mice. 5. The administration of the Ca2+-gated K+ channel blocker apamin (0.5-2.0 ng per mouse, i.c.v.) never modified clonidine and guanabenz analgesia. 6. At the highest effective doses, none of the drugs used modified animals' gross behaviour nor impaired motor coordination, as revealed by the rota-rod test. 7. The present data demonstrate that both K(ATP) and mKv1.1 K+ channels represent an important step in the transduction mechanism underlying central antinociception induced by activation of alpha2 adrenoceptors.

Some new insights into the effects of opioids in phasic and tonic nociceptive tests

In this review we critically appraise the value of some phasic and tonic nociceptive tests as models for differentiating the antinociceptive effects of opioid agonists. Using heat-evoked withdrawal of the hind paw or tail of a rodent, several early studies have assessed the effects of stimulus intensity upon antinociceptive potency of opioid agonists. After intrathecal (i.t.) administration of either morphine or sufentanil, for example, for any incremental change in stimulus intensity, the degree of right shift in the dose-response relationship was greater for morphine than for sufentanil. At first glance, such data appear to provide robust support for the pharmacological model of fractional receptor occupancy (FRO), which, according to the historical tenets of classical receptor theory, describes the relationship between intrinsic efficacy and the total receptor concentration. However, new data which elegantly characterize the relative contribution of small calibre unmyelinated and myelinated nociceptive afferents in mediating thermal-evoked responses challenge the exclusivity of such explanations with origins in classical theory. Within our review we report the results of experiments which provide direct electrophysiological evidence that noxious skin heating at a low rate activates C-polymodal nociceptors, but does not effectively activate A-delta mechanothermal nociceptors. In contrast, a high rate of skin heating activates both nociceptor classes, but produces a more intense activation of A-delta nociceptors that occurs after a shorter onset latency compared with the activation of C-fibre nociceptors. Thus, in direct challenge to the traditional model of FRO, a shift in the dose-response relationship of morphine to the right with a reduction in efficacy, may reflect the limited effectiveness of morphine to attenuate the A-delta-mediated component which assumes increasing dominance at high intensity heating. In our appraisal of other nociceptive models we provide an in-depth characterization of afferent processing in the early neonate rat, in which opioids have been tested in both phasic (tail flick and hot-plate) and tonic (formalin) tests. Afferent processing in this model is typified by several behavioural, anatomical and functional features which, although not pathological, are characteristic of those observed in models of nerve injury using the adult rat. Notably, these features include a lack of segmental inhibition (`disinhibition') and afferent input in large diameter myelinated fibres which make synaptic contacts within superficial laminae of the dorsal horn that in the adult are predominantly nociceptive. Paradoxically, because this paradigm demonstrates increased sensitivity to the antinociceptive effects of opioids it may have special merit as a model of tonic pain. It was recently announced that the i.t. administration of pertussis toxin (PTX) caused hyperalgesia and allodynia that appears similar to the symptoms reported by patients suffering from neuropathic pain. Unlike the effects of other opioids so far tested, buprenorphine-induced antinociception is not blocked in this model. This is an exciting finding and provides new optimism that some opioids, notably buprenorphine, may have a special role in managing some types of neuropathic pain.

Interaction between neuronal nitric oxide synthase and inhibitory G protein activity in heart rate regulation in conscious mice.

Nitric oxide (NO) synthesized within mammalian sinoatrial cells has been shown to participate in cholinergic control of heart rate (HR). However, it is not known whether NO synthesized within neurons plays a role in HR regulation. HR dynamics were measured in 24 wild-type (WT) mice and 24 mice in which the gene for neuronal NO synthase (nNOS) was absent (nNOS-/- mice). Mean HR and HR variability were compared in subsets of these animals at baseline, after parasympathetic blockade with atropine (0.5 mg/kg i.p.), after beta-adrenergic blockade with propranolol (1 mg/kg i.p.), and after combined autonomic blockade. Other animals underwent pressor challenge with phenylephrine (3 mg/kg i.p.) after beta-adrenergic blockade to test for a baroreflex-mediated cardioinhibitory response. The latter experiments were then repeated after inactivation of inhibitory G proteins with pertussis toxin (PTX) (30 microgram/kg i.p.). At baseline, nNOS-/- mice had higher mean HR (711+/-8 vs. 650+/-8 bpm, P = 0.0004) and lower HR variance (424+/-70 vs. 1,112+/-174 bpm2, P = 0.001) compared with WT mice. In nNOS-/- mice, atropine administration led to a much smaller change in mean HR (-2+/-9 vs. 49+/-5 bpm, P = 0.0008) and in HR variance (64+/-24 vs. -903+/-295 bpm2, P = 0.02) than in WT mice. In contrast, propranolol administration and combined autonomic blockade led to similar changes in mean HR between the two groups. After beta-adrenergic blockade, phenylephrine injection elicited a fall in mean HR and rise in HR variance in WT mice that was partially attenuated after treatment with PTX. The response to pressor challenge in nNOS-/- mice before PTX administration was similar to that in WT mice. However, PTX-treated nNOS-/- mice had a dramatically attenuated response to phenylephrine. These findings suggest that the absence of nNOS activity leads to reduced baseline parasympathetic tone, but does not prevent baroreflex-mediated cardioinhibition unless inhibitory G proteins are also inactivated. Thus, neuronally derived NO and cardiac inhibitory G protein activity serve as parallel pathways to mediate autonomic slowing of heart rate in the mouse.

PTX-sensitive G proteins and permissive action of prostacyclin in newborn pig cerebral circulation.

The present study of newborn pig cerebral circulation investigated the role of pertussis toxin (PTX)-sensitive GTP binding proteins in the permissive action of prostacyclin in specific dilator responses. Pial arterioles of anesthetized piglets were observed through closed cranial windows. The piglets were treated topically with PTX and intravenously with indomethacin. The effects of hypercapnia (10% CO2 ventilation) and topical 5,6-epoxyeicosatrienoic acid (5,6-EET) on pial arteriolar diameter were noted before and after the intervention. Samples of the artificial cerebrospinal fluid (aCSF) were collected from beneath the cranial windows for determination of the cAMP concentration. After administration of PTX, indomethacin still abolished pial arteriolar dilation to both hypercapnia and 5, 6-EET and also inhibited the cAMP elevation caused by hypercapnia. The addition of phorbol 12-myristate 13-acetate (PMA), but not iloprost, restored the increase in cAMP and vascular responses to hypercapnia and 5,6-EET. Therefore, in the newborn pig cerebral microvasculature, PTX appears to inhibit a G protein involved in the permissive action of prostacyclin. However, the protein kinase C (PKC) activator PMA appears to act downstream from the block, and, therefore, the permissive action of PMA is not affected by PTX. We suggest that the prostacyclin IP receptor may be coupled to phospholipase C via a PTX-sensitive G protein that normally permits vasodilation to specific stimuli via activation of a PKC, resulting in phosphorylation of a component of the adenylyl cyclase pathway.

Intrathecal pertussis toxin produces hyperalgesia and allodynia in mice

Pertussis toxin (PTX), which causes the ADP-ribosylation and thereby inactivation of G i-proteins, has been employed in analgesia testing to elucidate receptors that are coupled to inhibitory G-proteins, such as the mu-opioid receptor. Consistent with previous findings, the antinociceptive effects of morphine (1–10 μg) as measured by tail-flick latency using a 55°C water bath, were blocked by a single intrathecal injection of 0.5 μg PTX 6 days prior to intrathecal morphine administration. In addition, mice treated intrathecally with 0.5 μg of PTX had significantly shorter baseline tail-flick latencies compared with vehicle treated mice using a 55°C water bath when tested 6 days after PTX or vehicle administration. Morphine-induced antinociception was blocked in a dose-dependent manner by PTX with complete blockade of morphine following a 0.3- μg dose of PTX. Further, mice administered 0.1 μg or 0.3 μg PTX intrathecally had significantly shorter tail-flick latencies compared with vehicle injected mice using a 40, 45 or 50°C water bath when tested 7 days after intrathecal injection. Shorter tail-flick latencies were observed at 45°C as early as 48 h after intrathecal administration of 0.03, 0.1 or 0.3 μg PTX and these shorter tail-flick latencies were observed up to 90 days after intrathecal PTX administration. The intrathecal administration of PTX caused hyperalgesia and allodynia that appears similar to the symptoms reported by patients suffering from neuropathic pain, and suggests that deficiencies in inhibitory systems, as compared with increases in excitatory systems, may play a role in the pathophysiology of at least some central or neuropathic pain states.

In vivo pharmacology of butylthio[2.2.2] (LY297802 / NNC11-1053), an orally acting antinociceptive muscarinic agonist.

Butylthio[2.2.2] (LY297802 / NNC11-1053) is a mixed muscarinic cholinergic receptor agonist/antagonist that produces antinociception in mice and rats. As such, butylthio[2.2.2] may have therapeutic utility in the treatment of pain. Butylthio[2.2.2] was fully efficacious in the mouse grid shock, writhing, tail-flick and hot plate tests with ED50 values ranging from 1.5 to 12.2 mg/kg after oral administration. In contrast, the ED50 values for morphine ranged from 7.3 to 72 mg/kg after oral administration. Scopolamine was a competitive antagonist of the antinociceptive effects of butylthio[2.2.2]. Butylthio[2.2.2] did not produce either salivation or tremor at therapeutic doses; rather, there was a 50- to >100-fold separation between therapeutic doses and doses which produced side-effects. Butylthio[2.2.2] had high affinity for muscarinic receptors, but little if any affinity for other neurotransmitter receptors or uptake sites. In isolated tissues, butylthio[2.2.2] was an agonist with high affinity at M1 receptors in rabbit vas deferens, an antagonist at M2 receptors in guinea pig atria as well as an antagonist at M3 receptors in guinea pig urinary bladder. Although it has been suggested that M1 receptors mediate the antinociceptive effects of muscarinic agonists, M1 efficacy is not a requirement for antinociception, and, in vivo, the antinociceptive effects of muscarinic agonists are blocked by the intrathecal administration of pertussis toxin, indicating the involvement of m2 or m4 receptors. Since butylthio[2.2.2] is an M2 antagonist, antinociception is therefore most likely mediated by m4 receptors. Butylthio[2.2.2] is currently undergoing clinical development as a novel analgesic.

Effect of Pertussis Toxin Injected Into the Ventral Tegmental Area on Amphetamine-Induced c-Fos in the Nucleus Accumbens

Amphetamine produces an enhanced locomotor stimulatory response in animals injected with pertussis toxin into the ventral tegmental area. This response is dependent on the activation of D1 receptors in the nucleus accumbens. The immediate early gene, c-Fos, has been used as a cellular marker for increases in dopamine neurotransmission in the nucleus accumbens. The purpose of the present study was to determine whether the administration of pertussis toxin into the ventral tegmental area results in an increased ability of amphetamine to induce c-Fos-positive immunoreactivity in the nucleus accumbens. Amphetamine (1 mg/kg and 2 mg/kg IP) produced a greater number of c-Fos-positive cells in the nucleus accumbens of pertussis toxin-treated animals as compared to vehicle-treated controls. However, the increase in Fos immunoreactivity at the higher amphetamine dose was not associated with a corresponding increase in locomotor activity. These data suggest that amphetamine produces an enhanced increase in dopamine neurotransmission in the nucleus accumbens of pertussis toxin-treated animals, resulting in an increased induction of c-Fos or Fos-related antigens.

Delineation of the minimal encephalitogenic epitope within the immunodominant region of myelin oligodendrocyte glycoprotein: diverse V beta gene usage by T cells recognizing the core epitope encephalitogenic for T cell receptor V beta b and T cell receptor V beta a H-2b mice.

The nature of the autoimmune T cell response to myelin oligodendrocyte glycoprotein (MOG), recently recognized as a potential target antigen in multiple sclerosis (MS), has not yet been characterized, in contrast to the T cell reactivity to other potential target antigens in MS such as myelin basic protein and proteolipid protein. Here, we show that the encephalitogenicity of the recombinant Ig-like domain of human MOG is associated, in H-2 b mice, with an immunodominant T cell reactivity against a single region of MOG spanning amino acids 35-55, accounting for the previously reported strong encephalitogenic activity of pMOG 35-55. A single injection of pMOG 35-55 with or without administration of pertussis toxin was sufficient to induce severe clinical experimental autoimmune encephalomyelitis (EAE) in H-2 b mice. Encephalitogenic pMOG 35-55-specific T cell lines derived from C3H.SW (V beta b) mice were diverse in their TCR V beta gene usage (V beta 1, V beta 6, V beta 8 and V beta 15), although V beta 8.2 was most predominantly expressed (48%). However, V beta 8 + T cells may only be part of the encephalitogenic MOG-specific T cell repertoire in H-2 b mice, as demonstrated by the susceptibility of C57L (V beta a) mice to disease induced by pMOG 35-55. Encephalitogenic T cell lines from V beta a mice were also diverse in their TCR V beta gene usage (V beta 1, V beta 2, V beta 6, V beta 14 and V beta 16). Such a heterogeneous TCT V beta gene expression by pMOG 35-55/I-A b-reactive T cells from both V beta a and V beta b H-2 b mice suggested multiple epitopes within pMOG 35-55. Analysis of the pattern of reactivity by pMOG 35-55-reactive T cells to a set of truncated peptides was not commensurate with independent nested epitopes, but revealed a requirement for recognition of a core sequence, YRSPFSRVV (pMOG 40-48). However, optimal stimulation was obtained with longer peptides, with each additional amino acid flanking either the N or the C terminus differentially increasing the stimulatory capacity of pMOG 40-48. Nonetheless, pMOG 40-48 was the minimal encephalitogenic epitope for both V beta a and V beta b mice. Thus, the T cell reactivity against the immunodominant encephalitogenic region of MOG is characterized by a diverse V beta gene usage and a requirement for the same core epitope. This pattern of reactivity may favor epitope-directed, rather than TCR-targeted, approaches to immunospecific therapy for MOG-related autoimmune disease.

ATP shortens atrial action potential duration in the dog: role of adenosine, the vagus nerve, and G protein

The mechanism by which adenosine 5'-triphosphate (ATP) shortens atrial action potential duration was studied in a canine model in vivo. Previous studies have indicated that the negative chronotropic and dromotropic actions of ATP in the canine heart are mediated by a vagal reflex and by adenosine. However, the mechanism of ATP's action on atrial action potential duration remains unknown. The effects of ATP on endocardial monophasic action potential were determined under baseline conditions (control) and after left cervical vagotomy followed by right vagus nerve afferent blockade with capsaicin (1% in olive oil), and subsequent bilateral cervical vagotomy plus propranolol (1.0 mg/kg, i.v.). In addition, the effects of ATP and adenosine were determined 48 h following the administration of pertussis toxin (PTX, 30 micrograms/kg, i.v.). PTX intoxication was verified by monitoring plasma levels of insulin during glucose tolerance tests. ATP (4 and 6 mumol/kg, rapid bolus into right atrium) markedly shortened right atrial action potential duration at 50% repolarization (APD50) from 101 +/- 8 to 22 +/- 6 and from 111 +/- 8 to 14 +/- 2 ms, respectively. Adenosine (equimolar doses given in an identical mode) had a smaller effect, i.e., APD50 of 106 +/- 8 and 109 +/- 6 was shortened to 77 +/- 12 and 76 +/- 12 ms, respectively. Left cervical vagotomy slightly reduced the effect of ATP but not that of adenosine. Blockade of right vagal C fiber afferent traffic using local application of capsaicin to the right cervical vagosympathetic trunk markedly attenuated the effect of ATP, but not that of adenosine. Autonomic blockade (i.e., bilateral cervical vagotomy and propranolol) markedly attenuated the effect of ATP, but not of adenosine; for example, the effect of ATP (6 mumol/kg) was reduced from 86 +/- 2% shortening of APD50 to 24 +/- 5% (p < 0.05), while that of adenosine was 32 +/- 8 and 20 +/- 4% (ns) before and after autonomic blockade, respectively. Treatment with PTX completely abolished the effect of both ATP and adenosine on atrial action potential duration. These data indicate that (i) the effect of ATP on the canine atrial action potential duration is mediated to a large extent by a vagal reflex triggered by the nucleotide and to a lesser extent by adenosine, the product of ATP's enzymatic degradation,(ii) the afferent traffic of this reflex travels mainly via the right vagal C fibers, and (iii) the effects of both vagal and the adenosine components are mediated by PTX-sensitive guanine nucleotide binding proteins (G proteins) coupled to the muscarinic cholinergic receptors and A1 adenosine receptors, respectively.

Effect of pertussis toxin on the response of rat medial prefrontal cortex cells to the iontophoresis of serotonin receptor agonists.

In this study, we examined the response of spontaneously active as well as quiescent cells (L-glutamate-activated) in the rat medial prefrontal cortex (mPFc) to the iontophoresis of 2-methylserotonin (2-Me-5-HT, 5-HT3 receptor agonist), (+/-)-2,5-dimethoxy-(4-iodo-phenyl)-2-aminopropane (DOI, 5-HT2A,2C receptor agonist), 8-hydroxy-N,N-di-propylamino tetralin (8-OH-DPAT, 5-HT1A receptor agonist) and gamma-aminobutyric acid (GABA, a non-selective GABA receptor agonist) after the intracerebral administration of pertussis toxin, an inactivator of the Gi/o protein. This was accomplished using the techniques of extracellular single cell recording and iontophoresis. The administration of pertussis toxin (0.5 microgram, 24 hours before the experiment) into the mPFc did not alter the response of mPFc cells to the iontophoresis of DOI, 2-Me-5HT or GABA compared to saline treated controls. However, the response of mPFc cells to the iontophoresis of 8-OH-DPAT was significantly attenuated in the animals pretreated with pertussis toxin compared to controls. These results suggest that the 5-HT1A but not 5-HT2A,2C or 5-HT3 receptor is coupled to the Gi/o protein.

Effects of endothelin on rat renal proximal tubule Na(+)-Pi cotransport and Na+/H+ exchange

Endothelin (ET), a powerful vasoconstrictive peptide, is distributed ubiquitously in various organs, including the vascular endothelium and tubules of the kidney. Although localized more abundantly to the glomerulus and inner medullary collecting duct, ET receptors have been identified in the proximal tubule. The possible effects of ET on proximal tubule transport and the potential role of second messengers in this process have not been described fully. To define the role of ET in proximal tubule transport, renal cortical slices were incubated for 3 min in the presence of various concentrations of ET. Incubation with low concentrations of ET-1 (1 x 10(-9) to 1 x 10(-11) M) within the physiological range stimulated both Na(+)-Pi cotransport and Na+/H+ exchange. Pretreatment with staurosporine (0.6 microM) for 25 min abolished completely the ET-induced effects on Na(+)-Pi cotransport and Na+/H+ exchange. Similarly, preincubation with phorbol ester 12-O-tetradecanoylphorbol-13-acetate (200 nM) also abolished the effects of ET on these transporters. Incubation with ET decreased significantly intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Intravenous administration of pertussis toxin for 2 days prevented the ET-induced decrease in cAMP and abolished the stimulatory effects of ET on Na(+)-Pi cotransport and Na+/H+ exchange. These findings provide indirect evidence that ET participates in the regulation of proximal tubular Pi and bicarbonate homeostasis. These effects of ET are mediated by activation of protein kinase C and cAMP-dependent protein kinase A.

Further Elucidation of a Pertussis Toxin-Sensitive Transmembrane Signaling Mechanism Involved in Central alpha(2)-Adrenoceptor Activation in the Rat.

In adult male Sprague-Dawley rats anesthetized with pentobarbital sodium, we elucidated the molecular consequence of central alpha(2)-adrenoceptor activation. The hypotensive and negative chronotropic and inotropic actions of the alpha(2)-adrenoceptor agonist guanabenz were used as our experimental index. Intracerebroventricular administration of pertussis toxin (2.5 &mgr;g) significantly attenuated the cardiovascular suppressant effects of the aminoguanidine compound (100 &mgr;g/kg i.v.). However, application of N-ethylmaleimide (0.125 or 0.250 &mgr;g), phorbol 12-myristate 13-acetate (1.25 or 2.50 &mgr;g), cholera toxin (1.25 or 2.50 &mgr;g) or forskolin (12.5 or 25.0 &mgr;g) into the lateral cerebral ventricle elicited no appreciable blunting effect on the circulatory depression produced by guanabenz. These results were essentially duplicated when pertussis toxin (0.125 or 0.250 &mgr;g), N-ethylmaleimide (0.0125 or 0.05 &mgr;g), phorbol 12-myristate 13-acetate (0.125 or 0.25 &mgr;g), cholera toxin (0.125 or 0.25 &mgr;g) or forskolin (1.25 or 2.50 &mgr;g) was microinjected bilaterally to the nucleus reticularis gigantocellularis, a medullary site believed to be intimately related to the antihypertensive action of guanabenz. These findings suggest that stimulation of the alpha(2)-adrenoceptors in the medulla oblongata may result in the activation of a pertussis toxin-sensitive GTP-binding regulatory protein. They further suggest that the biologic signals subsequent to this action may not be linked to Gs, Gi or Gp but possibly Go. Copyright 1994 S. Karger AG, Basel

Modulation by pertussis toxin of salbutamol- and arecoline-induced effects in the isolated heart and aorta of the rat

The modulatory effects of pertussis toxin pretreatment on responses mediated via β-adrenoceptors and muscarinic acetylcholine receptors were investigated in isolated rat hearts and aortic rings 4 days after in vivo administration of pertussis toxin. In isolated hearts, pertussis toxin increased heart weight and baseline coronary flow values but did not effect baseline left ventricular pressure values. In unpaced hearts, pertussis toxin inhibited the arecoline-induced cardiac standstill, while in paced hearts, the β 2-adrenoceptor agonist salbutamol produced a dose-dependent vasodilation with similar characteristics in pertussis toxin and control preparations. Pertussis toxin had no effect on myocardial or aortic cyclic nucleotide levels and the myocardial β-adrenoceptor density ( B max) and dissociation constant ( K d). In precontracted aortic rings, pertussis toxin had no effect on the salbutamol or arecoline induced vasorelaxation. In summary, we demonstrated a reduced cholinergic responsiveness in isolated hearts but an intact β 2-adrenoceptor pathway in isolated hearts as well as in isolated aortic rings after pertussis toxin pretreatment. In aortic rings no change in muscarinic acetylcholine receptor responsiveness occurred.