CCK-8-stimulated insulin secretion in vivo is mediated by CCKA receptors.

The effects of the cholecystokinin A (CCKA) receptor antagonist, L-364,718, and the CCKB receptor antagonist, L-365,260, on CCK-8-stimulated insulin secretion were studied in vivo in the mouse. It was found that CCK-8-stimulated insulin secretion was suppressed by L-364,718 at a low dose level (0.078 mumol/kg). In contrast, L-365,260 caused a partial inhibition of CCK-8-stimulated insulin release only at the high dose level (24 mumol/kg). It is concluded that the CCK-8-stimulated insulin release in vivo is mediated by CCK receptors of the CCKA subtype.     

Central effects of nicotinamide and inosine which are not mediated through benzodiazepine receptors.

The actions of nicotinamide and inosine were investigated on rat cerebellar Purkinje cells using ionophoretic and extracellular recording techniques. Ionophoretic application of nicotinamide or inosine showed that they were potent inhibitors of Purkinje cell firing. This inhibition differed from that induced by benzodiazepines in that it was not reversed by the GABA antagonists bicuculline methiodide and picrotoxin. RO 15-1788, the specific benzodiazepine antagonist, did not reverse the effects of nicotinamide. Chlordiazepoxide has been shown to increase significantly social interaction between pairs of male rats and this increase can be reversed by RO 15-1788, 20 mg kg-1 i.p. Nicotinamide also caused a small increase in social interaction but this effect was not reversed by the benzodiazepine antagonist. Inosine did not increase social interaction. [3H]-flunitrazepam binding studies showed that nicotinamide and inosine have only low affinities for the benzodiazepine binding site. These results suggest that while nicotinamide may exert some neuronal depressant and anxiolytic activity, its site of action appears not to be associated with the benzodiazepine receptor site. Similarly, inosine exerts a neuronal depressant effect dissimilar from that of benzodiazepines.     

The analgesic effect of clonixine is not mediated by 5-HT3 subtype receptors

• 1.1. The analgesic effect of clonixinate of L-lysine (Clx) in the nociceptive C-fiber reflex in rat and in the writhing test in mice is reported.
• 2.2. Clx was administered by three routes, i.v., i.t. and i.c.v., inducing a dose-dependent antinociception.
• 3.3. The antinociceptive effect of Clx was 40–45% with respect to the control integration values in the nociceptive C-fiber reflex method.
• 4.4. The writhing test yielded ED50 values (mg/kg) of 12.0 ± 1.3 (i.p.), 1.8 ± 0.2 (i.t.) and 0.9 ± 0.1 (i.c.v.) for Clx administration.
• 5.5. Ondansetron was not able to antagonize the antinociception response of Clx in the algesiometric tests used.
• 6.6. Chlorophenilbiguanide did not produce any significative change in the analgesic effect of Clx in the nociceptive C-fiber reflex method.
• 7.7. It is suggested that the mechanism of action of the central analgesia of Clx is not mediated by 5-HT₃ subtype receptors.

     

Kindling induced by pentylenetetrazole in rats is not directly associated with changes in the expression of NMDA or benzodiazepine receptors

Repeated injections of a subconvulsant dose of pentylenetetrazole (PTZ, 30 mg/kg IP three times weekly for 13 injections) in Wistar and hooded Lister rats resulted in kindled seizures, the extent of which varied between strains. Wistar rats achieved stage 4 of clonic-tonic seizures, whereas hooded Lister rats only reached stage 2 of convulsive waves axially through the body. Rats were killed 10 days after their final injection, and radioligand binding was used to measure the expression of NMDA receptors in cortex and hippocampus using [3H]MK-801 and [3H]L-689,560, the latter binding specifically to the NR1 subunit. [3H]Ro 15-1788 measured expression of GABA(A)-benzodiazepine binding sites containing alpha1, alpha2, alpha3, or alpha5 subunits. Specific analysis of GABA(A) receptors containing the alpha5 subunit, which are preferentially localized in the hippocampus, was assessed with [3H]L-655,708. In the cortex, there was no effect of strain or treatment on the K(D) or B(max) of any of the ligands. Similarly, there was no effect of strain or treatment on hippocampal [3H]L-689,560 or [3H]Ro 15-1788 binding. However, in the hippocampus there was a significant, albeit modest, effect of treatment on the B(max) of [3H]MK-801 binding and the B(max) and K(D) of [3H]L-655,708 binding, i.e., PTZ-treated rats had fewer [3H]MK-801 and [3H]L-655,708 binding sites (NMDA and alpha5-containing GABA(A) receptors, respectively), but, these reductions were significant only in the relatively seizure-insensitive hooded Lister strain. This suggests that the increased susceptibility to kindling in Wistar rats is not directly related to alterations in the expression of NMDA or GABA(A) receptors.     

Decreased in vivo binding to brain benzodiazepine receptors during social isolation

Pharmacologic studies have demonstrated that benzodiazepines can modulate the ultrasonic vocalizations (USV) associated with social separation of rat pups. In this study, in vivo receptor autoradiography was used to determine if brain benzodiazepine receptors were functionally less available to bind an exogenous ligand during social separation. The labelled benzodiazepine receptor antagonist ³H-RO 15-1788 was given to 10-day-old rat pups during 25minutes of social separation. In autoradiographic studies of brains from separated pups, the binding of ³H-RO 15-1788 was decreased in neocortex (frontal, motor, and somatosensory), hippocampus, and superior and inferior colliculi. As these same regions showed no alteration of in vitro binding of ³H-RO 15-1788, these in vivo binding decreases do not reflect changes in receptor number. The interpretation of decreased in vivo binding and implications of these results for defining the neural substrates of separation behavior are discussed.     

An action of erythromycin in the intestine that is not mediated via motilin receptors

1. Erythromycin lactobionate caused a concentration-dependent inhibition of nerve-mediated contractions of the longitudinal muscle of the guinea-pig ileum, with a threshold for effect of 10-30 mumol/L. The non-antibiotic derivative of erythromycin ABT-229 had a similar effect, but was approximately 10-fold less potent. At a greater concentration (1 mmol/L), erythromycin also depressed the direct contractile effect of 10 mumol/L carbachol on the muscle. 2. Human/porcine motilin (up to 100 mumol/L) did not reduce the nerve-mediated contractions, although it did contract the muscle (threshold 30 mumol/L). Antagonists of motilin receptors (phe3leu13motilin, up to 1 mumol/L, and GM-109, up to 3 mumol/L) did not reduce responses to erythromycin. 3. Erythromycin contracted the longitudinal muscle of the rabbit duodenum, with a threshold concentration of 0.1 mumol/L and ABT-229 contracted this tissue at a threshold concentration of 0.01 mumol/L. Effects of both agonists were antagonized by the motilin receptor antagonists phe3leu13motilin (0.3 mumol/L) and GM-109 (1 mumol/L). 4. It is concluded that the site(s) at which erythromycin acts in the guinea-pig ileum is not a motilin receptor and that ABT-229 is selective for the motilin receptor in comparison with non-motilin erythromycin sites and is unlikely to act at the latter site in therapeutic doses.     

Benzodiazepine inhibition of adenosine uptake is not prevented by benzodiazepine antagonists

Uptake of [3H]adenosine into rat cerebral cortex synaptosomes was studied. Hexobendine (10(-5) M) and the benzodiazepine agonists diazepam (10(-5) M) and flurazepam (10(-4) M) significantly inhibited this uptake, but only if the compounds were pre-incubated for 10 min in the case of the benzodiazepines. The benzodiazepine antagonists Ro15-1788 (10(-5) M) and CGS 8216 (10(-5) M) failed to reverse the action of benzodiazepine agonists or hexobendine on [3H]adenosine uptake. The results add weight to the view that inhibition of adenosine uptake processes by benzodiazepines do not contribute to their behavioural effects.     

Dopamine-induced relaxation of the guinea-pig isolated jejunum is not mediated through dopamine receptors

The possible involvement of specific dopamine receptors in the relaxing effect of dopamine in the guinea-pig isolated jejunum has been investigated. The relaxing effect of dopamine does not show the tachyphylaxis phenomenon and it is present in preparations from guinea-pigs pretreated with reserpine. These results indicate that dopamine has a direct action. Comparison of the effect of dopamine with those of other dopamine receptor agonists, i.e. apomorphine, bromocriptine and the DA1 selective fenoldopam, were made to calculate potency ratios. Since apomorphine, bromocriptine and fenoldopam were shown to relax the guinea-pig jejunum, partly behaving as indirectly acting agents, comparisons were made on reserpine-pretreated guinea-pigs. It has been found that apomorphine is 2.5 times, fenoldopam 3 times and bromocriptine 20 times more active than dopamine in relaxing the guinea-pig jejunum. The order of potency is different from that found in other dopamine receptors containing tissues. The effects of the dopamine receptor blockers, haloperidol and cis-alpha-flupenthixol and the DA1 selective blocker SCH 23390 on the relaxing effect of dopamine were also studied. The relaxing effect of dopamine was not reduced by haloperidol, cis-alpha-flupenthixol and SCH 23390. It is concluded that specific postjunctional dopamine receptors are not involved in the relaxing action of dopamine. Since dopamine is known to interact with alpha- and beta-adrenoceptors in a variety of tissues, the effects of the alpha-adrenoceptor blocker phentolamine and the beta-adrenoceptor blocker propranolol on the relaxing effect of dopamine were also studied. Noradrenaline has been used to check the responsiveness of the tissue. Phentolamine did not block the responses to dopamine and propranolol was able only to partially reduce responses to dopamine, at concentrations higher than those at which it antagonized noradrenaline. Mechanisms other than dopamine, alpha- or beta-receptor activation should be involved in the relaxing effect of dopamine in the guinea-pig jejunum.     

Anti-infarct effect of magnesium is not mediated by adenosine A1 receptors in rat globally ischaemic isolated hearts

1. The aim of present study was to investigate the effects of magnesium (Mg) on cardiac function and infarct size and to compare it effects with those of adenosine. The mechanism of Mg-mediated cardioprotection was explored by combined use of Mg and a selective adenosine A(1) receptor antagonist. 2. Rat isolated hearts were used for Langendorff perfusion. Hearts were either non-preconditioned or preconditioned with Mg (6 mmol/L) or adenosine (1 mmol/L) before 30 min sustained ischaemia followed by 120 min reperfusion. Within each of these protocols, hearts were divided into two groups; one group was exposed to the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 200 nmol/L). Infarct size was measured by the triphenyltetrazolium chloride method. Left ventricular function was assessed by left ventricular developed pressure (LVDP), the product of heart rate x LVDP and coronary flow (CF). 3. The administration of Mg had an anti-infarct effect independent of its effect on postischaemic functional recovery in rats. Both Mg and adenosine equipotently reduced infarct size, but this effect of Mg was not blocked by the simultaneous administration of DPCPX. Cardiac function was improved by both adenosine and Mg and blockade of adenosine A(1) receptors attenuated these effects for both agents. 4. In conclusion, the results of the present study indicate that stimulation of adenosine A(1) receptors is not responsible for the anti-infarct effect of Mg in ischaemic myocardium in rats, but that the Mg-mediated protection of postischaemic functional recovery in rats is mediated by these receptors.     

LY 171555-induced hyperdefensiveness in the mouse does not implicate benzodiazepine receptors

In naive mice the selective D2 agonist LY171555 dose-dependently (0.5–5 mg/kg) induces defensive responses toward non-aggressive conspecifics. In order to investigate possible anxiogenic properties of the D2 agonist, its behavioural effects were compared with those produced by the benzodiazepine receptor inverse agonist methyl--carboline-3-carboxylate(-CCM) in the elevated plus maze and in social interactions with non-aggressive opponents. When tested in the elevated plus maze, mice injected with LY 171555 (0.005–1 mg/kg) showed no decrease either of the number of entries or of the time spent in the open arms. At 5 mg/kg an actual increase of these two measures was observed. By contrast,-CCM (1–3 mg/kg) dose-dependently decreased both the number of entries and the time spent in the open arms without altering locomotion. The effects of-CCM were antagonized by the benzodiazepine receptor antagonist RO 15-1788 (3 mg/kg) showing a selective involvement of benzodiazepine receptors in their modulation. On the other hand,-CCM, (1–3 mg/kg) did not produce significant effects on defensive behaviour of mice interacting with non-aggressive opponents and the defensive responses of mice treated with 1 mg/kg LY 171555 were not prevented by 5 mg/kg chlordiazepoxide. These results show that DA D2-mediated hyperdefensiveness and anxiety modulated by benzodiazepine receptors are unrelated phenomena and suggest that this behavioural response may represent a model of those forms of fear-related reaction that do not respond to benzodiazepine treatment.     

Morphine-induced decrease in mechanical allodynia is mediated by central, but not peripheral, opioid receptors in rats with inflammation

The aim of this study was to investigate the mechanism underlying the effect of morphine on allodynia to complete Freund's adjuvant-induced inflammation in rats. Morphine (5 mg/kg, i.v.) markedly inhibited the mechanical stimulation-induced nociceptive reflex of the gastrocnemius muscle in the inflamed hind-limb, and the inhibition was blocked by naloxone (1 mg/kg). Teased fiber recordings were made from the tibial nerve innervating the inflamed hindpaw. Morphine at the same dose did not affect the spontaneous firing rate of A-type fibers, whereas it markedly decreased the spontaneous firing of C-type fibers. The present data suggested that the central, but not peripheral, plasticity triggered by inflammation-induced facilitation of A(beta) fibers plays an important role in morphine-induced alleviation of allodynia, whereas activation of opioid receptor expression on the peripheral terminals of C fibers may contribute to morphine-induced alleviation of persistent pain of inflammation.     

Cholinergic disinhibition in area CA1 of the rat hippocampus is not mediated by receptors located on inhibitory neurons.

We studied the effects of carbamylcholine (carbachol; CCh) on monosynaptic inhibitory postsynaptic potentials (IPSPs) evoked in the presence of the excitatory amino acid receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and D,L-2-amino-5-phosphonovalerate (APV). CCh (30 microM) blocked late afterhyperpolarizations but did not depress GABAA receptor-mediated fast monosynaptic IPSPs or GABAB receptor-mediated late monosynaptic IPSPs. In the presence of CCh the GABAB receptor agonist (+/- )-baclofen (2 microM) reversibly hyperpolarized pyramidal neurons and depressed monosynaptic IPSPs as under control conditions. Phorbol-12,13-diacetate (PDAc; 10 microM) increased fast and depressed late monosynaptic IPSPs, and prevented depression of IPSPs by baclofen. These results suggest that cholinergic disinhibition in area CA1 of the hippocampus results from decreased synaptic excitation of inhibitory neurons.     

Control of spasticity in a multiple sclerosis model is mediated by CB1, not CB2, cannabinoid receptors

BACKGROUND AND PURPOSE: There is increasing evidence to suggest that cannabis can ameliorate muscle-spasticity in multiple sclerosis, as was objectively shown in experimental autoimmune encephalomyelitis models. The purpose of this study was to investigate further the involvement of CB1 and CB2)cannabinoid receptors in the control of experimental spasticity. EXPERIMENTAL APPROACH: Spasticity was induced in wildtype and CB1-deficient mice following the development of relapsing, experimental autoimmune encephalomyelitis. Spastic-hindlimb stiffness was measured by the resistance to flexion against a strain gauge following the administration of CB1 and CB2 agonists. KEY RESULTS: As previously suggested, some CB2-selective agonists (RWJ400065) could inhibit spasticity. Importantly, however, the anti-spastic activity of RWJ400065 and the therapeutic effect of non-selective CB1/CB2 agonists (R(+)WIN55,212-2 and CP55, 940) was lost in spastic, CB1-deficit mice. CONCLUSIONS AND IMPLICATIONS: The CB1 receptor controls spasticity and cross-reactivity to this receptor appears to account for the therapeutic action of some CB2 agonists. As cannabinoid-induced psychoactivity is also mediated by the CB1 receptor, it will be difficult to truly dissociate the therapeutic effects from the well-known, adverse effects of cannabinoids when using cannabis as a medicine. The lack of knowledge on the true diversity of the cannabinoid system coupled with the lack of total specificity of current cannabinoid reagents makes interpretation of in vivo results difficult, if using a purely pharmacological approach. Gene knockout technology provides an important tool in target validation and indicates that the CB1 receptor is the main cannabinoid target for an anti-spastic effect.     

Self-administration of the D1 agonist SKF 82958 is mediated by D1, not D2, receptors

We have previously reported that two D₁ dopamine agonists, SKF 82958 and SKF 77434, are readily self-administered by rats. However, due to the limited selectivities of these agents, it was not possible to attribute their reinforcing effects exclusively to their D₁ actions. To assess the relative involvement of D₁ and D₂ receptors in SKF 82958 reinforcement, rats were pretreated 30 min before self-administration sessions with either the D₁-selective antagonist (+)SCH 23390 or the D₂-selective antagonist raclopride. The D₁ antagonist (+)SCH 23390 (5–20 µg/kg, SC) produced significant, dose-related (compensatory) increases in SKF 82958; in contrast, the D₂ antagonist raclopride (25–400 µg/kg, SC) did not significantly increase SKF 82958 self-administration, although raclopride did increase cocaine self-administration. Compensatory increases in self-administration rates are thought to reflect antagonist-induced reductions in drug reinforcement. Hence, we conclude that SKF 82958 self-administration depends on activation of a D₁-regulated reinforcement substrate.This work was supported by U.S.P.H.S. grants DA-05107, DA-05379, and DA-07747. All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH pub. no. 86-23, revised 1985).     

Reduction of cephalic arteriovenous shunting by ergotamine is not mediated by 5-HT1-like or 5-HT2 receptors.

1. The potent, antimigraine drug ergotamine has affinity for both 5-HT1 and 5-HT2 binding sites and constricts arteriovenous anastomoses. Since 5-HT also constricts arteriovenous anastomoses (mainly via 5-HT1-like receptors), this study investigates the involvement of 5-HT receptors in the ergotamine-induced reduction of arteriovenous shunting in the carotid circulation of the cat and pig. 2. In the cat, ergotamine (3, 10 and 30 micrograms kg-1, i.v.) reduced carotid blood flow, predominantly by a reduction in arteriovenous anastomotic blood flow. Pretreatment with ketanserin (0.5 mg kg-1, i.v.) or methiothepin (1 mg kg-1, i.v.) did not antagonize the effects of ergotamine. 3. In the pig, ergotamine (2.5, 5, 10 and 20 micrograms kg-1, i.v.) also reduced carotid blood flow and arteriovenous shunting, which was not affected by pretreatment with methiothepin (1 mg kg-1, i.v.). 4. These results suggest that the reduction by ergotamine in the shunting of carotid arterial blood via cephalic arteriovenous anastomoses is not mediated by 5-HT1-like or 5-HT2 receptors.     

Membrane transduction of oligoarginine in HeLa cells is not mediated by macropinocytosis

The mechanism in which small cationic oligopeptides are able to reach the cytosol of cells is controversial. Macropinocytosis has been recently suggested as a major mechanism for internalization of these peptides. In this report, the involvement of macropinocytosis on cytosolic localization of oligoarginine was quantitatively investigated in HeLa cells. Using a method which allows for the separate measurement of cytosolic versus vesicular oligopeptide, the results show that neither macropinosome nor filopodia formation correlates with cytosolic delivery of oligoarginine. Additionally, unlike macropinocytosis, the cytosolic delivery of oligoarginine was not inhibited by incubation at 16 degrees C, or by treatment with amiloride. Oligoarginine treatment does not contribute to leakage from endocytic vesicles, indicating the lack of endosomolytic properties. Finally, the amount of oligoarginine found in the cytosol was not substantially increased after coincubation with EGF, a known stimulator of macropinocytosis. Taken together, these data indicate that membrane transduction of oligoarginine occurs separately from macropinocytosis in HeLa cells.     

Forearm reactive hyperaemia is not mediated by nitric oxide in healthy volunteers

AIMS: To determine the role of nitric oxide (NO) in forearm reactive hyperaemia in healthy human subjects. METHODS: Ten healthy subjects aged 19-34 years underwent brachial artery cannulation. Forearm circulatory arrest was achieved by means of an upper arm cuff inflated to 200 mmHg for 5 min. The blood flow responses during reactive hyperaemia were measured using venous occlusion plethysmography following a 10 min intra-arterial infusion of 8 micromol min-1 N-monomethyl L-arginine (L-NMMA) and following matching placebo administered in random order. Results were analysed by repeated measures anova and t-tests. RESULTS: L-NMMA resulted in a significant reduction of basal forearm blood flow indicating inhibition of basal NO release (P=0.005). There was no significant difference between the blood flow responses during reactive hyperaemia following L-NMMA and placebo (P=0.97). CONCLUSIONS: Nitric oxide production does not make a significant contribution to the vasodilatation associated with reactive hyperaemia in the human forearm.     

The immobility produced by intermittent swim stress is not mediated by serotonin

Exposure to uncontrollable stressors such as intermittent swim stress (ISS) produces a behavioral syndrome that resembles behavioral depression including immobility in a Forced Swim Test (FST) and escape learning deficits. The results of previous studies suggest that stress causes a temporary sensitization of the brain serotonin (5-HT) system that is necessary and sufficient for producing behavioral depression. If this hypothesis is true in the ISS paradigm, then enhancing or inhibiting 5-HT transmission during stress should exacerbate or block the development of behavioral depression, respectively. The selective 5-HT uptake inhibitor fluoxetine (FLX) was administered prior to ISS or confinement; 24 h later the FST was used to detect behavioral immobility. ISS, but not FLX, significantly increased immobility in the FST. The purported 5-HT uptake enhancer tianeptine (TPT) was administered in place of FLX. Again ISS increased immobility in the FST, but TPT had no effect. These results suggested that 5-HT is not a critical mediator of ISS induced behavioral depression. However, some authors have raised concern that TPT does not act directly on 5-HT. Therefore, the 5-HT synthesis inhibitor, para-chlorophenylaline (PCPA) was administered to deplete central 5-HT before stress. PCPA did not alter immobility in the FST. Finally, a sub-chronic regimen of FLX given after ISS, but before the FST, was without effect on reversing the ISS-induced immobility. Taken together, these experiments indicate that ISS produces a significant behavioral depression manifested as increased immobility but offer no support of the hypothesis that 5-HT is a critical mediator of these effects.