Robert Frew

Differential Effects of Ketamine Isomers on Neuronal and Extraneuronal Catecholamine Uptake Mechanisms

Contractile responses of isolated rabbit aortic strips to epinephrine and norepinephrine were potentiated in a dose-related manner by (+) ketamine but not by (-) ketamine (1.1 X 10(-5) M - 3.7 X 10(-4) M). Potentiation was blocked completely by pretreatment with the extraneuronal uptake inhibitor cortisol (83-138 microM) but was unaffected by the neuronal uptake inhibitor cocaine (29 microM). Responses of the rat anococcygeus muscle to these catecholamines were potentiated by both isomers, with (+) ketamine being more potent than its optical antipode. These effects were blocked completely in tissues from 6-hydroxydopamine sympathectomized animals. Results suggest that inhibition of extraneuronal uptake of catecholamines by racemic ketamine is due solely to an action of the (+) isomer, whereas both isomers appear capable of inhibiting neuronal uptake.

Pharmacological evidence for an ω-conotoxin, dihydropyridine-insensitive neuronal Ca2+ channel

Inactivation of N-type voltage-sensitive Ca2+ channels (VSCC) with ω-conotoxin (ω-CgTx) in tissue obtained from chicken brain produces a concentration dependent (0.01–0.1 μM) inhibition of K−-stimulated Ca2+ influx (ΔK+) the rise in [Ca2+]1 and acetylcholine (ACh) release. In identical preparations from rat brain, Ca2+ influx and the rise in [Ca2+]1 were only marginally affected by much higher (1–10 μM) concentrations of ω-CgTx. The release of ACh, however, was inhibited to the same degree with similar amourts of ω-CgTx as those used in chicken brain. An L-type VSCC inhibitor failed to affect any of these parameters alone, or to augment the effect of ω-CgTx. The results suggest that almost all the VSCC in chicken brain are of the N type and that these channels regulate neurotransmitter release. In rat brain, on the other hand, Ca2+ channels resistant to N- or L-type blockers account for almost 75% of the measurable Ca2+ influx and rise in [Ca2+]1. The conspicuous dissociation between the regulation of Ca2+ influx and ACh release demonstrated in rat brain by using ω-CgTx, suggest that neurotransmitter release is governed by only a small proportion of strategically located N-type, ω-CgTx sensitive. VSCC in the presynaptic terminal.

Stimulation of Ca2+ influx through ATP receptors on rat brain synaptosomes: identification of functional P2X7 receptor subtypes

ATP receptors of the P2X class have previously been identified on autonomic nerve endings and on a limited population of CNS neurons. In the present study P2X receptors on mammalian cortical synaptosomes have been identified by a variety of functional and biochemical studies. In choline buffer ATP analogues caused concentration/time dependent Ca2+ influx. Relative to the effects caused by ATP, benzoylbenzoyl ATP (BzATP) was about seven times more active than ATP while 2‐me‐S‐ATP and ATPγS were much less active. α,β‐me‐ ATP and β,γ‐me‐ATP were virtually inactive. In sucrose buffer, relative to choline buffer, the activity of BzATP was more than doubled while activity in sodium buffer was reduced. Moreover, the P2X antagonists PPADS or Brilliant Blue G both significantly attenuated influx. These observations suggest the presence of P2X receptors on synaptosomes which subserve Ca2+ influx. This activity profile of the ATP analogues and the response to blocking agents are characteristic of responses of P2X7 receptors. Influx was unaffected by the VSCC inhibitors ω‐CTx‐MVIIC and (−) 202 – 791, indicating that ATP induced Ca2+ influx occurred primarily through P2X receptors. P2X7 receptor protein was identified by Western blotting and immunohistochemical staining. Purified preparations were devoid of significant concentrations of GFAP or the microglial marker OX‐42 but contained greatly enriched amounts of syntaxin and SNAP 25. The various pharmacological and biochemical studies were all consistent with the presence of functional P2X7 receptors.

Evidence of ω-conotoxin GV1A-sensitive Ca2+ channels in mammalian peripheral nerve terminals

Abstract The existence of ω-conotoxin GV1A (ω-CgTx)-sensitive, voltage-sensitive Ca2+ channels (VSCCs) in mammalian peripheral nerves was investigated in the guinea pig eleum myenteric plexus longitudinal smooth muscle preparation (GPI). ω-CgTx (0.01–1.0 μM) reduced the electrically stimulated GPI twitch height, failed to modify exogenously applied acetylcholine (ACh) contractions, and inhibited Ca2+-dependent KCl-stimulated ACh release as measured by chemiluminescence. The 1,4-dihydropyridine VSCC antagonist (−) 202-791 (0.1–1.0 μM) inhibited the GPI twitch height, reduced contractions to exogenous ACh, but failed to affect ACh release. In the rat aorta, a nerve free preparation, ω-CgTx failed to affect contractions to KCl which were inhibited by (−) 202-791 and potentiated by the VSCC agonist (+) 202-791. The results provide evidence of neuronal N type VSCCs in mammalian peripheral cholinergic nerve terminals.

Characteristics of the relaxant response of adenosine and its analogs in intestinal smooth muscle

Several characteristics of the relaxant response of the isolated longitudinal muscle of the rabbit small intestine in response to the administration of adenosine and related compounds are studied. Following administration of adenosine or ATP the preparation responded with a rapid initial suppression of spontaneous contractile activity followed by a secondary sustained phase of inhibition of lower magnitude. Cumulative application of relaxant doses of adenosine or ATP caused a lesser total response than that obtained by single application of the cumulative dose. Neither procaine, lidocaine or guanethidine antagonized the responses to adenosine or ATP and the responsiveness of muscles obtained from reserpinized animals appeared unchanged. A number of adenosine derivatives and analogs was tested for the ability to relax the muscle. Generally, compounds containing a primary or secondary 6-amino group acted as agonists with the exception of 8-bromoadenosine. Those nucleosides found to be inactive did not modify the responsiveness of the muscle to adenosine. Responses to adenosine and ATP were not appreciably modified by papaverine, imidazole, dipyridamole, 6-(p-nitrobenzylthio)-purine riboside. Antagonism was observed, however, with phentolamine and theophylline. Theophylline at 100 muM inhibited responses to adenosine over a wide dose range; this antagonism was surmountable by high doses of adenosine. 1-Methyl-3-isobutylxanthine did not antagonize adenosine responses. A number of 1,3-alkyl-6-thioxanthines did not modify the adenosine response at doses that did not show any direct action. The results supported the concept of an extracellular receptor site of adenosine and its analogs and the absence of an indirect mechanism of action via nerve stimulation.

Effects of adenosine and related compounds on adenylate cyclase and cyclic AMP levels in smooth muscle.

The hypotheses were tested that the relaxant effect of adenosine and related compounds in the longitudinal muscle of the rabbit small intestine involves interaction with adenylate cyclase and/or the elevation of tissue cAMP levels. Adenylate cyclase was prepared by gentle homogenization of an isolated smooth muscle cell fraction obtained after collagenase digestion of longitudinal muscle strips. A number of analogs and derivatives of adenosine possessing a primary or secondary 6-amino group were found to inhibit the enzyme similarly to adenosine; however, there was no correlation between compounds known to relax the intact tissue and the existence, or the degree of, cyclase inhibition. Isolated muscle strips were exposed to adrenaline, isoprenaline, adenosine or ATP, at doses causing 30-60% relaxation, for 60 sec prior to sampling and analysis of cAMP content. While small increments in cAMP levels were found after administering adrenaline or isoprenaline, no change was found with adenosine in the absence or presence of theophylline of 1-methyl-3-isobutylxanthine. Neither adenylate cyclase inhibition nor changes in cAMP levels appear to be part of the mechanism of the smooth muscle relaxant action of adenosine or ATP.

Relaxation of guinea-pig fundic strip by adenosine, adenosine triphosphate and electrical stimulation: lack of antagonism by theophylline or ATP treatment.

1 Theophylline relaxed isolated strips of guinea‐pig stomach fundus in a dose‐dependent manner; above 50 to 100 μm responses showed no fade for up to 90 min. 2 Relaxant responses to adenosine, adenosine triphosphate (ATP), noradrenaline, and to electric field stimulation of non‐adrenergic inhibitory nerves were not affected in a significant manner in the presence of 50 μm theophylline. 3 In tissues which showed complete fade of initial responses in the continued presence of 50 μm ATP, the effects of stimulation of non‐adrenergic inhibitory nerves remained unaltered, suggesting that the ATP receptor has no function in non‐adrenergic inhibitory transmission in this tissue. 4 These findings are opposite to those of Okwuasaba, Hamilton & Cook (1977), who claimed that 50 μm theophylline almost fully inhibited relaxation induced by adenosine, ATP and nerve stimulation and that ATP‐induced fade also abolished sensitivity to inhibitory nerve stimulation.

Effect of ω-agatoxin-IVA on autonomic neurotransmission

Abstract ω-Agatoxin-IVA, a peptide from the venom of the funnel-web spider Agelenopsis aperta and a P type Ca 2+ channel inhibitor, was examined for effects on responses to nerve stimulation in isolated autonomic neuroeffector preparations from the rabbit, guinea-pig and rat. Ca 2+ -dependent, tetrodotoxin sensitive, noradrenergic excitatory responses of rabbit pulmonary artery, rat vas deferens, and anococcygeus muscles, and cholinergic guinea-pig myenteric plexus preparations (all highly sensitive to the N type Ca 2+ channel inhibitor ω-conotoxin-GVIA) were unaffected by ω-agatoxin-IVA (100 nM). Similarly, the neurogenic response of rat bladder, which has cholinergic, and non-adrenergic non-cholinergic (NANC) excitatory components, and the NANC inhibitory response of rat jejunum (atropine 0.5 μM- and guanethidine 5.0 μM-treated), which are partially sensitive and insensitive to ω-conotoxin-GVIA, respectively, were unaffected by ω-agatoxin-IVA (100 nM). Neurogenic NANC inhibitory responses of the guinea-pig taenia caecum, and rat anococcygeus muscles (atropine- and guanethidine-treated, and tone raised with prostaglandin F 2α ), were also insensitive to ω-agatoxin-IVA. These results suggest that P type Ca 2+ channels, if present, play an insignificant role in supplying the Ca 2+ necessary for neurotransmitter release in the peripheral autonomic nervous system.

A role for Q type Ca2+ channels in neurotransmission in the rat urinary bladder

1 In isolated bladder strips of the rat, a substantial component (46%) of the Ca2+‐dependent contractile response to electrical field stimulation (5 Hz) was resistant to combined block of both N and P type Ca2+ channels by ω‐conotoxin‐GVIA (300 nM) and ω‐agatoxin‐IVA (100 nM) respectively. 2 The resistant portion (non‐N, non‐P) was sensitive to ω‐conotoxin‐MVIIC (3 μM), which in addition to N and P also blocks Q type channels at this concentration. ω‐Conotoxin‐MVIIC administered alone, inhibited the neurogenic response to the same degree as that observed in the combined presence of ω‐agatoxin‐IVA, ωconotoxin‐GVIA and ω‐conotoxin‐MVIIC. 3 ω‐Agatoxin‐IVA (100 nM), a concentration that fully inhibits P type channels, had a negligible effect on the neurogenic response. Following blockade of N type Ca2+ channels with ω‐conotoxin‐GVIA (300 μM), ω‐agatoxin‐IVA (3 μM) (a concentration well above that used to block P channels, inhibits Q type channels, but spares N type channels), inhibited the residual response to the same degree as ω‐conotoxin‐MVIIC alone. 4 Results suggest that neurotransmission in rat urinary bladder is supported by both N and Q type Ca2+ channels.

Effect of arylazido aminopropionyl ATP (ANAPP3), a putative ATP antagonist, on ATP responses of isolated guinea pig smooth muscle

Abstract Arylazido aminopropionyl ATP (ANAPP 3 ), a photoaffinity analogue of adenosine 5′-triphosphate, photoactivated with visible light (+hv), specifically and irreversibly antagonized ATP contractions of the guinea pig vas deferens. ANAPP 3 (30 μM) antagonized responses to exogenously added ATP in untreated, and in tissues pretreated with indomethacin (2.9 μM) and 6-(2-hydroxy-5-nitrobenzyl)-thio guanosine (10 μM). It was of interest to see if this pharmacological antagonist of ATP could be used to assess the validity of the purinergic nerve hypothesis by allowing a differentiation between, or proof of the identity of, responses to ATP and the non-adrenergic inhibitory transmitter in guinea pig stomach fundus. After photoactivation (+hv) in the organ bath and subsequent washout, ANAPP 3 (30 and 100 μM) failed to antagonize relaxant responses to ATP (1.0 – 1000 μM) in fundic strips. In addition ANAPP 3 failed to antagonize ATP-induced inhibition of the twitch response in electrically stimulated guinea pig ileum longitudinal muscle strips. We conclude that ANAPP 3 does not antagonize all actions of ATP, which may limit its usefulness in assessing the above hypothesis. Results with this compound suggest that ATP excitatory receptors may differ from those mediating relaxation and other ATP actions.