Graeme Richard Martin

Characterization of a 5‐HT1B receptor on CHO cells: functional responses in the absence of radioligand binding

1 Chinese hamster ovary (CHO) cells have been reported to be devoid of 5‐HT receptors and have frequently been used as hosts for the expression of cloned 5‐HT receptors. Unexpectedly, 5‐HT was found to induce profound inhibition of forskolin‐stimulated cyclic AMP production in these cells and the aim of this study was to classify the 5‐HT receptor involved. 1 In CHO(dhfr‐) cells 5‐HT was a potent agonist and caused 80–100% inhibition of forskolin stimulated cyclic AMP production. A study using several 5‐HT1 receptor agonists revealed the following potencies (p[A50]): RU24969 (9.09±0.17) > 5‐carboxamidotryptamine (8.86±0.20) > 5‐HT (8.07±0.05) > CP‐93,129 (7.74±0.10) > sumatriptan (5.93±0.04). All five agonists achieved a similar maximum effect. Irreversible receptor alkylation studies yielded a pKA estimate of 7.04±0.34 for 5‐HT. 3 The 5‐HT1A/1B antagonist, (±)‐cyanopindolol (4–100 nM), caused parallel rightward shifts of the 5‐HT concentration‐effect curve with no change in asymptote. Schild analysis yielded a pKB estimate of 8.69±0.09 (Schild slope 1.13±0.10). (±)‐Cyanopindolol actually behaved as a partial agonist with an intrinsic activity of 0.2‐0.5 and a p[A50] of 8.55. 4 5‐HT (0.01–10 μm) also elicited a concentration‐dependent increase in intracellular [Ca2+] in CHO(dhfr‐) cells thus demonstrating that dual coupling is not a phenomenon restricted to systems in which there is overexpression of transfected receptors. 5 This agonist and antagonist profile is consistent with the presence of a 5‐HT1B receptor. 8‐OH‐DPAT (1 μm) and renzapride (3 μm) were without effect on forskolin‐stimulated cyclic AMP production and ketanserin (0.3 μm) did not antagonize the inhibition produced by 5‐HT, thus excluding the involvement of 5‐HT1A, 5‐HT4, and 5‐HT2 receptors. 6 The possibility that expression of a 5‐HT1B receptor was associated with the dhfr‐ mutation was excluded since RU24969, 5‐HT and CP‐93,129 were also potent agonists in unmutated, CHO‐K1 cells: p[A50] 9.03±0.03, 8.34±0.05, 7.69±0.07 respectively, and (±)‐cyanopindolol (0.1 μm) shifted the 5‐HT curve to the right and yielded a pA2 estimate of 8.70±0.06. 7 Little or no specific binding of [3H]‐5‐HT (0.1–200 nM) or of the high affinity ligand [125I]‐iodocyanopindolol (0.01–3 nM) to CHO(dhfr‐) cell membranes could be detected. 5‐HT also failed to elicit any increase in the binding of [35S]‐GTPγS to CHO membranes. 8 In conclusion, cultured CHO cells express 5‐HT1B receptors which are negatively coupled to adenylyl cyclase and positively coupled to increases in intracellular calcium. The absence of radioligand binding was unexpected in view of the high potency of 5‐HT and the partial agonist activity of the normally ‘silent’ competitive antagonist, (±)‐cyanopindolol. This implies very efficient receptor‐effector coupling of a low density of 5‐HT1B receptors. Clearly, the absence of detectable radioligand binding cannot be assumed to mean the absence of receptors capable of eliciting a significant functional response.

Central administration of 5-HT activates 5-HT1A receptors to cause sympathoexcitation and 5-HT2/5-HT1C receptors to release vasopressin in anaesthetized rats

1 The effects of intracerebroventricular injections to the right lateral ventricle (i.c.v.) of 5‐hydroxytryptamine (5‐HT, 40 and 120 nmol kg−1), N,N‐di‐n‐propyl‐5‐carboxamidotryptamine (DP‐5‐CT; 3 nmol kg−1), 5‐carboxamidotryptamine (5‐CT; 3 nmol kg−1), 8‐hydroxy‐2‐(di‐N‐propylamino) tetralin (8‐OH‐DPAT; 3, 40 and 120 nmol kg−1) and 1‐(2,5‐di‐methoxy‐4‐iodophenyl)‐2‐aminopropane (DOI; 40 and 120 nmol kg−1) on renal sym pathetic nerve activity, blood pressure, heart rate and phrenic nerve activity were investigated in normotensive rats anaesthetized with α‐chloralose. 2 5‐HT caused a long lasting pressor response which was associated with an initial bradycardia and renal sympathoinhibition followed by a tachycardia and renal sympathoexcitation. Pretreatment with the 5‐HT2/5‐HT1C receptor antagonists, cinanserin (300 nmol kg−1, i.c.v.) or LY 53857 (300 nmol kg−1, i.c.v.) reversed the initial bradycardia and sympathoinhibition to tachycardia and sympathoexcitation. Combined pretreatment with LY 53857 (300 nmol kg−1, i.c.v.) and the 5‐HT1A antagonist, spiroxatrine (300 nmol kg−1, i.c.v.), blocked the effects of 5‐HT on all the above variables. 3 Pretreatment with the vasopressin V1‐receptor antagonist, β‐mercapto‐β,β‐cyclopentamethylenepropionyl1, O‐Me‐Tyr2, Arg8‐vasopressin [(d(CH2)5Tyr(Me)AVP, 10 μg kg−1, i.v.] did not affect the magnitude but reduced the duration of the pressor response produced by i.c.v. 5‐HT and reversed the initial bradycardia and renal sympathoinhibition to tachycardia and sympathoexcitation. 4 1‐(2,5‐Di‐methoxy‐4‐iodophenyl)‐2‐aminopropane (DOI) caused a pressor effect which was associated with a bradycardia and sympathoinhibition. These effects were blocked by pretreatment with BW501C67 (0.1 mg kg−1, i.v.), a peripherally acting 5‐HT2/5‐HT1C receptor antagonist. However, BW501C67 (0.1 mg kg−1, i.v.) failed to block the effects of i.c.v. 5‐HT. 5 DP‐5‐CT, 5‐CT and 8‐OH‐DPAT (3 nmol kg−1, i.c.v.) caused sympathoexcitation, tachycardia and a rise in blood pressure. Pretreatment with methiothepin (1 mg kg−1, i.v.) or spiroxatrine (300 nmol kg−1, i.c.v.) attenuated the response to i.c.v. DP‐5‐CT. 6 It is concluded that i.c.v. administration of 5‐HT activates 5‐HT1A receptors to cause sympathoexcitation and 5‐HT2 or 5‐HT1C receptors to cause the release of vasopressin.

Inhibition of endothelium‐dependent vasorelaxation by arginine analogues: a pharmacological analysis of agonist and tissue dependence

1 Isolated rings of rabbit external jugular vein (RbJV) and rat thoracic aorta (RA) were used to study the effect of the NO synthase inhibitor l‐NG‐nitroarginine methyl ester (l‐NAME) on muscarinic and 5‐hydroxytryptamine (5‐HT) receptor‐stimulated, endothelium‐dependent vascular relaxations. 2 In RbJV relaxations produced by the endothelial 5‐HT receptor agonist α‐methyl‐5‐HT were potently and non‐surmountably inhibited by l‐NAME (10 μm), whereas acetylcholine relaxations in this tissue were unaffected by this concentration of inhibitor. By contrast, acetylcholine relaxations in RA were virtually abolished by 10 μm l‐NAME. In each case an equivalent concentration of d‐NAME was without effect on agonist‐induced relaxations. 3 The different effect of l‐NAME on acetylcholine relaxations in RbJV and RA was not due to muscarinic receptor differences. Affinity estimates for acetylcholine (pKA = 6.12 ± 0.09; 6.09 ± 0.08 respectively) and for 4‐diphenyl‐acetoxy‐N‐methylpiperidine methobromide (4‐DAMP, pKB = 9.01 ± 0.12; 9.24 ±0.16 respectively) indicated that the receptors in both tissues belong to the same M3 class. Tissue differences resulting from the release of a cyclo‐oxygenase product or a glibenclamide‐sensitive K+‐channel‐linked hyperpolarizing factor were also ruled out by selective inhibition of these pathways. 4 When phenoxybenzamine was used to reduce the efficacy of acetylcholine in RbJV so that it behaved as a partial agonist in this tissue, l‐NAME (10 μm) now produced non‐surmountable inhibition of relaxation responses. In untreated tissues the same concentration of l‐NAME also profoundly inhibited responses produced by butyrylcholine and pilocarpine, both of which behave as partial agonists at the M3 receptor in RbJV. 5 A simple model was developed which describes the theoretical behaviour of receptor‐stimulated synthesis and release of NO. The model predicts that competitive inhibition of NO formation results in parallel displacements of the agonist response curve in the case of high efficacy agonist, but right‐shift with concomitant depression of the curve maximum in the case of low efficacy agonists. Simulations based on the model showed reasonable agreement with the experimental data. 6 It is concluded that analogues of l‐arginine demonstrate tissue‐ and agonist‐dependence in terms of their ability to inhibit receptor‐mediated events involving the liberation of NO. This behaviour can reflect differences in agonist efficacy in the receptor systems being studied, a possibility that should be ruled out before apparent resistance to inhibition is taken as evidence for the involvement of heterogeneous endothelium‐derived relaxing factors (EDRFs).

Effect of the thromboxane A2-mimetic U46619 on 5-HT1-like and 5-HT2 receptor-mediated contraction of the rabbit isolated femoral artery.

1 The influence of the thromboxane A2‐mimetic U46619 (11α,9α‐epoxymethano PGH2) on 5‐hydroxytryptamine (5‐HT)‐induced contractions of the rabbit isolated femoral artery has been examined. 2 In the absence of U46619, 5‐HT responses were mediated predominantly by 5‐HT2‐receptors as judged by potent, surmountable antagonism by the selective 5‐HT2 receptor antagonists, spiperone and ketanserin. Both antagonists unmasked a population of 5‐HT1‐like receptors which accounted for approximately 10–15% of the 5‐HT maximum response. 3 In the presence of U46619 (3–10 nm), 5‐HT‐induced contractions were largely resistant to blockade by 5‐HT2 receptor antagonists since 5‐HT1‐like receptor‐mediated contraction now accounted for approximately 60% of the 5‐HT maximum response. 4 These results show that activation of thromboxane A2 receptors in a tissue possessing both 5‐HT2 and 5‐HT1‐like receptors can convert 5‐HT‐induced contraction from one mediated predominantly by 5‐HT2 receptors to one which is mediated predominantly by 5‐HT1‐like receptors.

Evidence that 5-HT1D receptors mediate inhibition of sympathetic ganglionic transmission in anaesthetized cats.

In anaesthetized cats, 5‐carboxamidotryptamine (5‐CT) or 5‐hydroxytryptamine (5‐HT) (0.3–300 μg kg−1, i.v.) inhibited the postganglionic compound action potential evoked by preganglionic electrical stimulation (0.5 Hz) with a similar potency in the stellate and splanchnic ganglia. In the 5‐HT experiments transmission thorough the inferior mesenteric ganglia was also recorded. The maximal inhibitory effect of 5‐HT was greater on the stellate and splanchnic ganglia (60 ± 4 and 52 ± 5%) than on the inferior mesenteric (15 ± 2%). The effects of 5‐HT were unaffected by pretreatment with antagonists (1 mg kg−1;i.v.) for 5‐HT2 (BW501C67), 5‐HT1A (WAY‐100635) and 5‐HT3 receptors (ondansetron). However, responses to both 5‐HT and 5‐CT were attenuated significantly by GR127935 (1 mg kg−1) except the responses to 5‐HT at the inferior mesenteric ganglia. These results are consistent with the involvement of 5‐HT1D receptors mediating inhibition of sympathetic ganglionic transmission in vivo.

5-HT1B receptors mediate potent contractile responses to 5-HT in rat caudal artery.

5‐Hydroxytryptamine (5‐HT) evoked potent contractile responses in phenoxybenzamine‐treated ring segments of rat caudal artery, partially contracted with U46619. Responses were mimicked by 5‐HT1‐selective agonists with the potency order: RU24969 > 5‐carboxamidotryptamine > 5‐HT = CP 93,129 > > sumatriptan. 8‐Hydroxy‐N,N‐dipropylaminotetralin was virtually inactive. Responses were unaffected by spiperone (0.1 μm) and mesulergine (1.0 μm), but were antagonized competitively by (±)‐cyanopindolol affording agonist‐independent pKB estimates of 8.4 to 8.9. The pharmacological profile of this receptor is consistent with that of the 5‐HT1B subtype. Since the 5‐HT1B receptor is the rodent homologue of the 5‐HT1Dβ subtype, it might be anticipated that 5‐HT1Dβ receptors will be found to mediate vasoconstrictor responses in non‐rodent species.

A comparative study of the prostanoid receptor profile of 9α11β‐prostaglandin F2 and prostaglandin D2

1 The aim of this study was to determine the receptor profile of 9α11β‐prostaglandin F2 (PGF2) and compare it with that of its parent, prostaglandin D2 (PGD2). The experiments were designed to overcome the problems associated with the presence of multiple prostanoid receptor sub‐types in most tissues; the lack of selective antagonists for each receptor means that conclusions regarding efficacy at FP and EP2 receptors must remain provisional. 2 At DP receptors in human platelets and rabbit jugular vein, PGD2 was a full agonist, p[A50] 7.02 ± 0.09 and 6.60 ± 0.12 respectively. 9α11β‐PGF2 was approximately 30–60 fold less potent than PGD2. 3 9α11β‐PGF2 was a full agonist in the FP receptor containing preparation, cat iris sphincter (p[A50] 7.35 ± 0.09) comparable in potency to PGD2 (p[A50] 7.15 ± 0.19). Likewise the two prostanoids showed similar potency at the TP receptor in guinea‐pig aorta (9α11β‐PGF2 p[A50] 6.00 ± 0.07; PGD2 6.24 ± 0.08). 4 9α11β‐PGF2 and PGD2 had efficacy but low potency at EP1 receptors (guinea‐pig oesophageal muscularis mucosa) and demonstrated 2000–3000 fold lower potency than PGE2 (p[A50] 8.35 ± 0.09). Similarly, in the EP2 receptor‐containing preparation, cat trachea, 9α11β‐PGF2 was 3500 fold less potent and PGD2 700 fold less potent than PGE2 (p[A50] 8.06 ± 0.26). 5 9α11β‐PGF2 and PGD2 (10 μm) were without affinity at the IP receptors on human platelets and had no agonist action in the EP3 receptor containing preparation, guinea‐pig vas deferens. 6 9α11β‐PGF2 is a major metabolite of PGD2 in vivo and this conversion clearly represents an inactivation step since 9α11β‐PGF2 is of considerably lower potency than PGD2 at DP receptors. However, it is of similar potency to PGD2 at TP, FP, EP1 and EP2 receptors and it may, therefore, contribute to the biological effects which follow PGD2 administration or endogenous synthesis; its actions at these receptors are likely to be similar to those of PGD2 itself.

Cranial vascular effects of zolmitriptan, a centrally active 5-HT1B/1D receptor partial agonist for the acute treatment of migraine

The anti-migraine drug zolmitriptan is a novel 5-HT1B/1D receptor partial agonist which, unlike sumatriptan, has been shown to cross the intact blood-brain barrier. In this study we examined whether or not the ability to access the cerebro-vascular intima affects the way in which a centrally-active 5-HT1B/1D receptor agonist influences cranial haemodynamics. The effects of zolmitriptan on carotid arterial blood flow distribution were studied in anaesthetised cats using radiolabelled microspheres. Zolmitriptan (10-1000 microg kg(-1) i.v.) selectively reduced arteriovenous-anastomotic (AVA) conductance producing a maximum decrease of 92.5+/-2.3%. The drug also produced a modest reduction in extra-cerebral conductance (23.9+/-6.5% maximum reduction at 30 microg kg(-1), i.v.), but was without effect on cerebral conductance. Using laser doppler flowmetry in anaesthetised cats, zolmitriptan (1-30 microg kg(-1), i.v.) produced dose-dependent decreases in ear microvascular conductance (15+/-5 to 60+/-6%) which mirrored decreases in carotid arterial conductance (12+/-11 to 61+/-5%). By contrast, zolmitriptan at doses up to 1000 microg kg(-1) was without effect on cerebral microvascular conductance. Although zolmitriptan crosses the blood-brain barrier and can therefore access the cerebro-vascular intima, this study suggests that this property does not adversely affect cerebrovascular function.

Evidence that activation of 5‐HT2 receptors in the forebrain of anaesthetized cats causes sympathoexcitation

1 The aim of the present experiments was to determine whether the effects of lateral ventricular application of 5‐HT on cardiovascular and respiratory variables in anaesthetized cats are mediated by forebrain 5‐HT2 receptors. This was carried out by determining whether the effects of 5‐HT are blocked by the 5‐HT2 antagonist, cinanserin and if they are mimicked by the selective 5‐HT2 agonist, 1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane (DOI) 2 Cats were anaesthetized with a mixture of oc‐chloralose and pentobarbitone sodium, neuromuscularly blocked and artificially ventilated. The following cardiovascular and respiratory variables were recorded: renal, splanchnic and cardiac sympathetic nerve activities, phrenic nerve activity, heart rate, arterial blood pressure, femoral arterial conductance and tracheal pressure. All drugs were administered via the lateral ventricle and the action of these agonists was restricted to forebrain sites by a cannula placed in the Aqueduct of Sylvius 3 Cumulative doses of 5‐HT (10–160 nmol kg−1) and DOI (80–320 nmol kg−1) injected into the lateral ventricle caused significant increases in blood pressure, heart rate, cardiac and splanchnic sympathetic nerve activity and a decrease in femoral arterial conductance. DOI and 5‐HT caused a greater increase in cardiac compared with splanchnic nerve activity and failed to change renal nerve activity 5 ‐HT but not DOI significantly increased the magnitude and the number of phrenic bursts as well as significantly increasing tracheal pressure. The effects of 5‐HT also differed from DOI in that 5‐HT evoked maximal pressor and near maximal sympathoexcitatory effects after the first dose, whereas the pressor and sympathoexcitatory effects of DOI were graded over the complete dose‐range 4 The 5‐HT2 antagonist, cinanserin (265 nmol kg−1, i.c.v.) caused significant falls in blood pressure, heart rate and cardiac nerve activity and an increase in femoral arterial conductance. Splanchnic and renal sympathetic nerve activity, phrenic nerve activity and tracheal pressure were unaffected by cinanserin. After pretreatment with cinanserin all cardiovascular and respiratory effects of 5‐HT were significantly attenuated 5 It is concluded that in the cat, as DOI and 5‐HT have similar effects on the cardiovascular variables recorded and as the effects of 5‐HT are blocked by cinanserin, 5‐HT can act on 5‐HT2 receptors located in the forebrain to cause differential sympathoexcitation and a rise in arterial blood pressure. Further, the sympathoexcitatory effects mediated by 5‐HT2 receptors located in the forebrain differ from those located in the hindbrain in that they mediate increases in cardiac nerve activity and heart rate and also have no effect on renal nerve activity.