J.M. Barnes

Identification and characterisation of 5-hydroxytryptamine 3 recognition sites in human brain tissue.

Abstract: [3H]Zacopride displayed regional saturable specific binding to homogenates of human brain tissues, as defined by the inclusion of BRL43694 [endo‐N‐(9‐methyl‐9‐azabicyclo[3.3.1]non‐3‐yl)‐l‐methylindazole‐3‐carboxamide] in the incubation media. Scatchard analysis of the saturation data obtained from amygdaloid and hippocampal tissues identified the binding as being of high affinity and to a homogeneous population of binding sites (KD= 2.64 ± 0.75 and 2.93 ± 0.41 nmol/L and Bmax= 55 ± 7 and 44 ± 9 fmol/ mg of protein in the amygdala and hippocampus, respectively). 5‐Hydroxytryptamine3 (5‐HT3) receptor agonists and antagonists competed for the [3H]zacopride binding site, competing with up to 40% of total binding with a similar rank order of affinity in both tissues; agents acting on various other neurotransmitter receptors failed to inhibit binding. Kinetic data revealed a fast association that was fully reversible (k+1= 6.61 × 105 and 7.65 × 105/mol/L/s and k‐1= 3.68 × 10−3 and 3.45 × 10−3/s in the amygdala and hippocampus, respectively). It is concluded that [3H]zacopride selectively labels with high affinity 5‐HT3 recognition sites in human amygdala and hippocampus and, if these binding domains represent 5‐HT3 receptors, may provide the opportunity for 5‐HT3 receptor antagonists to modify 5‐HT function in the human brain

Angiotensin II inhibits the release of [3H]acetylcholine from rat entorhinal cortex in vitro

The effects of angiotensin I and II on basal potassium-induced release of [3H]acetylcholine were investigated in slices of rat entorhinal cortex. Potassium (10-25 mM) produced a concentration-dependent increase in the release of [3H]acetylcholine in the presence of extracellular calcium. Angiotensin II (10(-9)-10(-5) M) (but not angiotensin I) reduced the potassium-induced release of [3H]acetylcholine in a concentration-related manner to 60% of control levels, but did not effect basal tritium release. The effect of angiotensin II was antagonised by [1-sarcosine, 8-threonine] angiotensin II, an angiotensin II receptor antagonist, but not by agents acting on alpha- and beta-adrenoceptors, muscarinic, nicotinic, histamine or 5-hydroxytryptamine receptors nor by the angiotensin converting enzyme (ACE) inhibitor SQ 29852. The results indicate that angiotensin II acting via an angiotensin II receptor can inhibit the release of [3H]acetylcholine in slices of the rat entorhinal cortex. It is hypothesised that the ability of ACE inhibitors to facilitate cognitive processes may be related to a reduced availability of angiotensin II.

Angiotensin-converting enzyme inhibition, angiotensin, and cognition.

In these studies, we have investigated possible cognition-enhancing effects of angiotensin-converting enzyme (ACE) inhibition, and putative neurochemical correlates for these actions. In a mouse habituation model, ACE inhibitors improved basal performance and antagonized scopolamine-induced deficits. The performance of aged mice and those with lesions of the nucleus basalis was also improved. ACE inhibition also improved scopolamine-impaired performance of rats in a swim-maze model. Neurochemical studies showed that a low dose (10 micrograms/kg i.p.) of ceranapril caused significant alterations in ex vivo rat brain catecholamine levels in the nucleus accumbens, amygdala, and septum. In further studies, angiotensin II (Ang II) was shown to decrease potassium-stimulated [3H] acetylcholine release from slices of rat entorhinal and human temporal cortex, an effect that could be antagonized by the angiotensin receptor antagonist [1-sar,8-thr]Ang II. It is concluded that ACE inhibition can improve both basal and impaired performance in animal models of learning, and that this improvement may be in part a consequence of the removal by ACE inhibition of an inhibitory tone on central acetylcholine release, and/or an effect on central catecholaminergic function.

Angiotensin II inhibits acetylcholine release from human temporal cortex: implications for cognition.

Angiotensin II was shown to inhibit potassium-stimulated release of [3H]acetylcholine from slices of fresh human temporal cortex, obtained at surgery, and subsequently loaded with [3H]choline for the biochemical analyses. The inhibitory effect of angiotensin II was antagonised by the specific angiotensin II receptor antagonist [1-sarcosine, 8-threonine]-angiotensin II. High affinity binding sites were identified in the human temporal cortex using [125I]angiotensin II, and may provide the functional site of action of angiotensin II to modify [3H]acetylcholine release.

The differential activities of R(+)- and S(−)-zacopride as 5-HT3 receptor antagonists

R(+)- and S(-)-zacopride were assessed as potential 5-HT3 receptor antagonists in behavioural and biochemical tests. The S(-)isomer was more potent than the R(+)isomer to antagonise the hyperactivity induced by the injection of amphetamine or the infusion of dopamine into the nucleus accumbens in the rat. In contrast, the R(+)isomer was more potent to reduce the aversive behaviour of mice to a brightly illuminated environment and in a marmoset human threat test, to facilitate social interaction in rats, to increase performance in a mouse habituation test and prevent a scopolamine-induced impairment, and to antagonise the inhibitory effect of 2-methyl-5-hydroxytryptamine to reduce [3H]acetylcholine release in slices of the rat entorhinal cortex. In binding assays, [3H]S(-)-zacopride and [3H]R(+)-zacopride labelled homogenous populations of high-affinity binding sites in the rat entorhinal cortex, R(+)-zacopride compete for a further 10 to 20% of the binding of [3H]R(+)/S(-)-zacopride or [3H]R(+)-zacopride in excess of that competed for by (S)(-)-zacopride. It is concluded that both isomers of zacopride have potent but different pharmacological activities, with the possibility of different recognition sites to mediate their effects.

Angiotensin II inhibits cortical cholinergic function: implications for cognition.

In the present studies we have shown that angiotensin II (AT II), in a concentration-dependent manner in rat tissue (10−9-10−5 M) or at a single concentration in human tissue (10−6 M), can inhibit potassium-stimulated release of [3H]acetylcholine ([3H]Ach) from slices of rat entorhinal cortex and human temporal cortex preloaded with [3H]choline for the biochemical analyses. The inhibitory effects of AT II (10−6 M) were antagonised by the specific AT II receptor antagonist [1-sarcosine,8-threonine]AT II in a concentration-dependent manner in rat tissue (10−11 −10−8 M) and at the single concentration employed in the human studies (10−7 M). Also demonstrated were other components of the angiotensin system in the human temporal cortex; ACE activity was present (1.03 nmol min−1 mg−1 protein), as were AT II recognition sites (Bmax = 8.6 fmol mg−1 protein). It is hypothesised that the potential cognitive enhancing properties of ACE inhibitors may reflect their action to prevent the formation of AT II and so remove an inhibitory modulator of cholinergic function.

Reserpine, para-chlorophenylalanine and fenfluramine antagonise cisplatin-induced emesis in the ferret

The involvement of 5-hydroxytryptamine (5-HT) with cisplatin-induced emesis in the ferret was investigated using reserpine, para-chlorophenylalanine and fenfluramine. Pretreatment with reserpine (5 mg/kg, 24 hr), fenfluramine (5 mg/kg, 4 days) or para-chlorophenylalanine (100 or 400 mg/kg, 4 days) antagonised cisplatin-induced emesis. All treatments reduced the levels of 5-HT in the area postrema and at other cerebral sites, but whilst this action was relatively selective for small doses of para-chlorophenylalanine [only modest effects on noradrenaline (NA) and no change in the content of dopamine (DA) in the area postrema], other treatments reduced levels of dopamine and noradrenaline. Data are discussed in terms of an involvement of 5-HT/catecholamines in the area postrema with the mediation of emesis induced by cisplatin.

Identification and distribution of 5-HT3 recognition sites within the human brainstem

The present studies demonstrate the presence of specific [3H]GR65630 binding sites within the human brainstem using the techniques of in vitro receptor autoradiography and ligand binding to homogenates. Autoradiography revealed the greatest accumulation of specific binding in the area postrema and subpostrema (AP/ASP). A lower level of specific binding was identified in the nucleus tractus solitarius (excluding area subpostrema). No specific binding was evident in the remainder of the hindbrain at this level. Discrete dissection followed by ligand binding to homogenates revealed that the specific binding of [3H]GR65630 (defined by the presence of 30 microM metoclopramide) was differentially distributed with highest levels in the AP/ASP (112.1 fmol/mg protein) and lower levels in the dorsal vagal complex (nucleus tractus solitarius--excluding the area subpostrema--dorsal motor nucleus of the vagus and hypoglossal nucleus) (DVC) and olivary nucleus (ON) (22.9 and 3.9 fmol/mg, respectively). No specific binding was detectable in the reticular formation (RF) located ventral to the dorsal vagal complex. The specific [3H]GR65630 binding site was pharmacologically similar to the 5-HT3 receptor since the potent and selective 5-HT3 receptor antagonists ICS 205-930 and zacopride (100 nM) and the agonist 5-HT (10 microM) inhibited binding to the same extent as metoclopramide in each of the individual areas (90, 60 and 20% in the AP/ASP, DVC and ON, respectively). The 5-HT1-like and 5-HT2 receptor antagonist methysergide (10 microM) failed to compete for the binding site. 5-HT3 receptor recognition sites within the AP/ASP and the DVC may be functionally involved in the ability of 5-HT3 receptor antagonists to control emesis.

Topographical distribution of 5-HT3 receptor recognition sites in the ferret brain stem

SummaryThe distribution of [3H]zacopride (1.0 nM) to putative 5-HT3 receptor recognition sites in the ferret hindbrain was assessed using autoradiography. Specific binding (defined by the inclusion of granisetron, 1.0 μM) was heterogeneously distributed with highest density within the dorsal vagal complex (area postrema, nucleus tractus solitarius and dorsal motor nucleus of the vagus nerve). Lower densities were detected in the spinal trigeminal nerve complex whilst no other significant specific binding was detected ventral to the dorsal vagal complex. The location of 5-HT3 receptor recognition sites within the dorsal vagal complex may provide sites of action for zacopride and other 5-HT3 receptor antagonists to inhibit the emesis induced by cancer chemotherapeutic agents and x-radiation.

NEUROCHEMICAL CONSEQUENCES FOLLOWING INJECTION OF THE SUBSTANCE P ANALOGUE, DIME-C7, INTO THE RAT VENTRAL TEGMENTAL AREA

The effect on forebrain catecholamine- and indoleamine-related neurochemical levels was investigated following stimulation of the rat ventral tegmental area with the substance P analogue, DiMe-C7. DiMe-C7 (6.0 micrograms) induced a marked hyperactivity in rats with maximal response between 15 and 30 min following the injection. Fifteen min following the DiMe-C7 injection levels of dopamine and/or its metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid) were significantly increased in the nucleus accumbens, amygdala, entorhinal cortex and striatum relative to vehicle-injected animals. Although the increase in dopamine metabolism in the nucleus accumbens is consistent with the behavioural hyperactivity, it is concluded that other forebrain nuclei may also be involved in the mediation of the hyperactivity response.