Andrew G. Ramage

Evidence that the putative 5-HT1A receptor agonists, 8-OH-DPAT and ipsapirone, have a central hypotensive action that differs from that of clonidine in anaesthetised cats.

Thoracic preganglionic sympathetic nerve activity, blood pressure, heart rate and femoral arterial conductance were recorded in anaesthetised, paralysed cats. Cumulative dose-response curves were constructed for 8-OH-DPAT, ipsapirone and clonidine. All three drugs caused dose-related falls in blood pressure which were associated with minimal changes in femoral arterial conductance. However, 8-OH-DPAT and ipsapirone differed from clonidine in that their hypotensive action was associated with moderate sympathoinhibition and a profound bradycardia, whereas clonidine caused profound sympathoinhibition and, as it did not increase central vagal tone, only a moderate bradycardia. 8-OH-DPAT also caused sympathoinhibition in bi-vagotomised cats and decreased carotid sinus nerve activity along with blood pressure. As 8-OH-DPAT and ipsapirone bind selectively to central 5-HT1A receptors it is concluded that central stimulation of these receptors causes sympathoinhibition and an increase in vagal tone, whereas stimulation of central alpha 2-adrenoceptors causes only sympathoinhibition. In addition, the present data suggest a peripheral vasodilator mechanism may also contribute to the hypotensive effects of 8-OH-DPAT and ipsapirone in the cat. The nature and relative importance of this remains to be established.

The role of central 5‐hydroxytryptamine (5‐HT, serotonin) receptors in the control of micturition

At present the most investigated 5‐HT receptor that has been shown to play a role in the control of micturition is the 5‐HT1A receptor followed by 5‐HT7, 5‐HT2 and 5‐HT3 receptors. Most experiments focus on the control these receptors have on the parasympathetic outflow to the bladder and the somatic outflow to the external urethral sphincter (EUS) in the rat. Furthermore, 5‐HT1A and 5‐HT7 receptors have been identified as having an excitatory physiological role in the control of bladder function. 5‐HT1A receptors act, at least in the rat, at both a spinal (probably a heteroreceptor) and supraspinal (probably an autoreceptor) level, while 5‐HT7 receptors only act at a supraspinal level. Additionally, in the rat, 5‐HT administered at a spinal or supraspinal site has an excitatory action, although earlier experiments have shown that activating 5‐HT‐containing brain areas causes inhibition of the bladder. Recent experiments have also indicated that blockade of the 5‐HT1A receptor pathway shows rapid tolerance. However, no data exist for the development of tolerance for the 5‐HT7 receptor pathway. Neither receptor seems to play a role in the control of the urethra. Regarding 5‐HT2 receptors, activation of this receptor subtype inhibits micturition, and this inhibitory action may occur at a spinal, supraspinal or both levels. Although no physiological role for 5‐HT2C receptors can yet be identified, 5‐HT2C receptors have been implicated in the proposed supraspinal tonically active 5‐HT1A autoreceptor (negative feedback) pathway. This proposition reconciles the data that central 5‐HT‐containing pathways are inhibitory to micturition, while 5‐HT1A receptors, although inhibitory to adenylyl cyclase, have an excitatory function. This is because activation of 5‐HT1A autoreceptors reduces the release of 5‐HT thus reducing the activation of the 5‐HT2C receptors, which are inhibitory in the control of micturition (disinhibition). Furthermore, 5‐HT2A receptors in the rat and 5‐HT2C receptors in the guinea pig cause activation of the EUS. In this respect, 5‐ht5A receptors have also been identified in Onufs nucleus, the site of somatic motoneurones controlling this sphincter. In the cat there is very little evidence to indicate that 5‐HT receptors are involved in micturition except under pathological conditions in which activation of 5‐HT1A receptors causes inhibition of micturition. Interestingly, under such conditions 5‐HT1A receptors cause excitation of the EUS. Nevertheless, spinal 5HT3 receptors have been implicated in the physiological control of micturition in the cat, but not yet in the rat. Overall, the data support the view that 5‐HT receptors are important in the control of micturition. However, many more studies are required to fully understand these roles and why there are such species differences.

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.

In vivo effects of 5-hydroxytryptamine receptor activation on rat nucleus tractus solitarius neurones excited by vagal C-fibre afferents

The effects of ionophoretically applied 5-hydroxytryptamine (5-HT) and 5-HT receptor agonists were studied on rat nucleus tractus solitarius (NTS) neurones receiving unmyelinated vagal afferent input. 5-HT excited 15 of 34 neurones (44%), inhibited 10 (29%) and had no effect on nine. 8-Hydroxy-2-(di-N-propylamino)tetralin HBr (8-OH-DPAT) excited 23 of 53 neurones (43%), inhibited 24 (45%) and had no effect on six neurones and (+/-)-2,5-dimethoxy-4-iodoamphetamine HCl activated 18 of 37 neurones (49%), inhibited nine (24%) and had no effect on 10. These results demonstrate that activation of 5-HT1A and 5-HT2 receptors can excite or inhibit populations of NTS neurones. Phenylbiguanide, however, excited 20 of 23 neurones (87%), inhibited only one (4%) and had no effect on two indicating that 5-HT3 receptor activation has an excitatory action. NTS neurones receiving cardiac vagal afferent input were more likely to be excited by 5-HT (five of five, 100%) or 8-OH-DPAT (four of five. 80%) than the population as a whole. In conclusion, the data demonstrate that 5-HT1A, 5-HT2, and 5-HT3 receptor subtypes are functionally present on NTS neurones receiving excitatory vagal afferent input. Further, the subpopulation of NTS neurones receiving input from cardiac afferents are excited by 5-HT, possibly by an action on 5-HT1A or 5-HT3 receptors.

Evidence for the involvement of central 5-HT7 receptors in the micturition reflex in anaesthetized female rats.

The effects of the selective 5‐HT7 receptor antagonists SB‐269970 (3‐300 μg kg−1; n=5–6) and SB‐656104 (30 μg kg−1; n=5) administered centrally (i.c.v.) were investigated on the ‘micturition reflex’ in the urethane anaesthetized female rat. In cystometric recordings, SB‐269970 caused significant increases in volume of 58±15 and 138±33% and pressure of 140±46 and 149±60% thresholds at 10 and 30 μg kg−1. These changes were associated with significant decreases in distension‐induced bladder contraction of 62±14 and 60±11%, respectively. However, there was no change in residual volume. At the higher doses, SB‐269970 blocked the micturition reflex. SB‐656104 had similar effects to SB‐269970 but in addition significantly increased the residual volume. SB‐269970 (10 μg kg−1; n=5) given i.v. had no effect on the micturition reflex. SB‐269970 (30 μg kg−1; n=4) given intrathecally (i.t.) had no effect on micturition reflex, although the selective 5‐HT1A receptor antagonist WAY‐100635 given i.t. after SB‐269970 caused a significant increase in the volume threshold. Using an isovolumetric method in which urethral changes were measured, SB‐269970 (30 μg kg−1; n=4; i.c.v.) failed to have any effect on these urethral‐evoked changes although they significantly reduced the amplitude of the bladder contraction. These data demonstrate that 5‐HT7 receptors located supraspinally in the rat are involved in the control of micturition.

Investigation of the role of 5-HT2 receptor subtypes in the control of the bladder and the urethra in the anaesthetized female rat

Micturition is controlled by central 5‐HT‐containing pathways. 5‐HT2 receptors have been implicated in this system especially in control of the urethra, which is a drug target for treating urinary incontinence. This study investigates the role of each of the three subtypes of this receptor with emphasis on sphincter regulation.

Evidence that the novel antihypertensive agent, flesinoxan, causes differential sympathoinhibition and also increases vagal tone by a central action

The effects of flesinoxan were studied on thoracic preganglionic, splanchnic and renal sympathetic nerve activity, carotid sinus nerve activity, blood pressure and heart rate in anaesthetised cats. In some experiments femoral or renal arterial conductance was also recorded. Flesinoxan (3-300 micrograms kg-1) caused a dose-related fall in blood pressure and heart rate and also caused sympathoinhibition. This fall in blood pressure was not associated with changes in femoral arterial conductance but was with a large increase in renal arterial conductance. In this respect flesinoxan had a greater sympathoinhibitory action on the renal nerve compared with the other sympathetic outflows. The bradycardia was unaffected by the 5-HT3 antagonist, MDL 72222, but was reversed by atropine and was abolished in bi-vagotomised cats. Flesinoxan also caused sympathoinhibition in bi-vagotomised cats and decreased carotid sinus nerve activity and blood pressure. It is concluded that flesinoxan acts centrally to cause sympathoinhibition and an increase in vagal tone.

The role of central 5‐HT1A receptors in the control of B‐fibre cardiac and bronchoconstrictor vagal preganglionic neurones in anaesthetized cats

1 Experiments were performed to determine whether 5‐HT1A receptors (a) modulate the activity of cardiac and bronchoconstrictor vagal preganglionic neurones (CVPNs and BVPNs) in the nucleus ambiguus (NA) and (b) are involved in pulmonary C‐fibre afferent‐evoked excitation of CVPNs, by right‐atrial injections of phenylbiguanide (PBG). These experiments were carried out on α‐chloralose‐anaesthetized, artificially ventilated and atenolol (1 mg kg−1)‐pretreated cats. 2 The ionophoretic application of 8‐OH‐DPAT (a selective 5‐HT1A receptor agonist) influenced the activity of 16 of the 19 CVPNs tested. 8‐OH‐DPAT tended to cause inhibition at low currents (40 nA) and excitation at high currents (120 nA). The activity of 15 of these neurones increased in response to the application of 8‐OH‐DPAT. In six of the CVPNs tested, this excitatory action of 8‐OH‐DPAT was attenuated by co‐application of the selective 5‐HT1A receptor antagonist WAY‐100635. 3 The pulmonary C‐fibre afferent‐evoked excitation of eight CVPNs was attenuated by ionophoretic application of WAY‐100635. 4 In three out of four CVPNs, the ionophoretic application of PBG caused excitation. 5 In five out of the nine identified BVPNs that were tested with ionophoretic application of 8‐OH‐DPAT, excitation was observed that was attenuated by WAY‐100635. 6 WAY‐100635 (i.v. or intra‐cisternally) also reversed bradycardia, hypotension and the decrease in phrenic nerve activity evoked by the i.v. application of 8‐OH‐DPAT (42 μg kg−1). 7 In conclusion, the data indicate that 5‐HT1A receptors located in the NA play an important role in the reflex activation of CVPNs and BVPNs, and support the view that overall, these receptors play a fundamental role in the reflex regulation of parasympathetic outflow.

Modulation of Voiding and Storage Reflexes by Activation of α1-Adrenoceptors

Objective: This paper reviews recent studies in animals that examined the effect on lower urinary tract function of α1-adrenoceptor agonists and antagonists. Methods: Bladder reflexes were studied in vivo on anesthetized rats and cats using cystometrographic and electrophysiologic techniques. Neurally-evoked bladder contractions and release of acetylcholine (ACh) were also studied in rat bladder strips in vitro. Results: Administration of the α1-adrenoceptor agonist, phenylephrine (PE) to isolated strips of rat bladder enhanced neurally-evoked bladder contractions and increased basal tone. The former effects of PE were blocked by a selective α1A antagonist and the latter by an α1B antagonist. Activation of α1A receptors by PE enhanced ACh release evoked by electrical field stimulation in bladder strips. PE also enhanced transmission in cat bladder ganglia. PE or noradrenaline act on α- and β-adrenoceptors on urothelial cells to release nitric oxide. It is concluded that facilitatory α1A-adrenoceptors are located prejunctionally in the bladder, whereas α1B adrenoceptors are located postjunctionally. In the central nervous system of the rat and cat facilitatory α1-adrenergic mechanisms can modulate the sympathetic, parasympathetic and somatic outflow to the urinary tract. In addition inhibitory α1 adrenoceptor mechanisms have been detected in the rat spinal cord. Activation of these receptors with PE raises the intravesical pressure threshold for inducing micturition and decreases voiding frequency. Conclusions: α1-adrenoceptors are located at various sites in the bladder and in the neural pathways controlling lower urinary tract function. At most sites these receptors mediate facilitatory responses that enhance smooth muscle activity or facilitate storage or voiding reflexes. However, α1-adrenoceptor inhibitory mechanisms in the rat spinal cord, can also reduce the frequency of voiding reflexes. This effect is possibly mediated by an inhibition in the afferent limb of the micturition reflex pathway.

The role of central 5-HT3 receptors in vagal reflex inputs to neurones in the nucleus tractus solitarius of anaesthetized rats

Brainstem 5‐hydroxytryptamine (5‐HT, serotonin)‐containing neurones modulate cardiovascular reflex responses but the differing roles of the many 5‐HT receptors have not been thoroughly investigated. The present experiments on anaesthetized rats investigated the role of 5‐HT3 receptors in modulating vagal afferent evoked activity of nucleus tractus solitarius (NTS) neurones. Recordings were made from 301 NTS neurones receiving an input at long (> 20 ms) minimum onset latency from stimulation of the vagus nerve. These included 140 neurones excited by activating non‐myelinated cardiopulmonary afferents by right atrial injection of phenylbiguanide (PBG). Ionophoretic application of PBG, a highly selective 5‐HT3 receptor agonist, significantly increased activity (from 2.4 ± 0.4 to 5.5 ± 0.8 spikes s−1) in 96 of 106 neurones tested and in all 17 neurones tested the increase in activity (3.4 ± 1.1 to 7.0 ± 1.9 spikes s−1) was significantly attenuated (3.0 ± 0.9 to 3.8 ± 1.1 spikes s−1) by the selective 5‐HT3 receptor antagonist granisetron. Ionophoretic application of PBG potentiated responses to vagus nerve and cardiopulmonary afferent stimulation, and granisetron significantly attenuated this cardiopulmonary input (20.2 ± 5.7 to 10.6 ± 4.1 spikes burst−1) in 9 of 10 neurones tested. Ionophoretic application of AMPA and NMDA also excited NTS neurones and these excitations could be selectively antagonized by the non‐NMDA and NMDA receptor antagonists DNQX and AP‐5, respectively. At these selective currents, DNQX and AP‐5 also attenuated PBG‐ and cardiopulmonary input‐evoked increases in NTS activity. These data are consistent with the hypothesis that vagal inputs, including non‐myelinated cardiopulmonary inputs to the NTS, utilize a 5‐HT‐containing pathway which activates 5‐HT3 receptors. This excitatory response to 5‐HT3 receptor activation may be partly a direct postsynaptic action but part may also be due to facilitation of the release of glutamate which in turn acts on either non‐NMDA or NMDA receptors to evoke excitation.