Prejunctional Receptors Research Articles

Serotonergic Modulation of Neurovascular Transmission: A Focus on Prejunctional 5-HT Receptors/Mechanisms.

5-Hydroxytryptamine (5-HT), or serotonin, plays a crucial role as a neuromodulator and/or neurotransmitter of several nervous system functions. Its actions are complex, and depend on multiple factors, including the type of effector or receptor activated. Briefly, 5-HT can activate: (i) metabotropic (G-protein-coupled) receptors to promote inhibition (5-HT1, 5-HT5) or activation (5-HT4, 5-HT6, 5-HT7) of adenylate cyclase, as well as activation (5-HT2) of phospholipase C; and (ii) ionotropic receptor (5-HT3), a ligand-gated Na+/K+ channel. Regarding blood pressure regulation (and beyond the intricacy of central 5-HT effects), this monoamine also exerts direct postjunctional (on vascular smooth muscle and endothelium) or indirect prejunctional (on autonomic and sensory perivascular nerves) effects. At the prejunctional level, 5-HT can facilitate or preclude the release of autonomic (e.g., noradrenaline and acetylcholine) or sensory (e.g., calcitonin gene-related peptide) neurotransmitters facilitating hypertensive or hypotensive effects. Hence, we cannot formulate a specific impact of 5-HT on blood pressure level, since an increase or decrease in neurotransmitter release would be favoured, depending on the type of prejunctional receptor involved. This review summarizes and discusses the current knowledge on the prejunctional mechanisms involved in blood pressure regulation by 5-HT and its impact on some vascular-related diseases.

Functional Characterization of the Prejunctional Receptors Mediating the Inhibition by Ergotamine of the Rat Perivascular Sensory Peptidergic Drive

Calcitonin gene-related peptide (α-CGRP) released from perivascular sensory nerves induces decreases in diastolic blood pressure (DBP). Experimentally, this can be shown by spinal thoracic (T9-T12) electrical stimulation of these afferent fibers. Because ergotamine inhibits these neurogenic vascular responses and displays affinity for monoaminergic receptors that inhibit neurotransmitter release, we investigated whether this ergotamine-induced inhibition results from activation of serotonin 5-HT1B/1D, dopamine D2-like, and α2-adrenergic receptors. Wistar rats were pithed and, under autonomic ganglion blockade, received intravenous infusions of methoxamine followed by ergotamine (0.1-3.1 μg kg-1 min-1). Thoracic T9-T12 electrical stimulation or an intravenous bolus of α-CGRP resulted in decreases in DBP. Ergotamine inhibited the electrically induced, but not α-CGRP-induced, responses. The vasodilator sensory inhibition by 3.1 μg of ergotamine kg-1 min-1 was resistant to simultaneous blockade of 5-HT1B/1D, D2-like, and α2-adrenergic receptors upon addition of antagonists GR127935, haloperidol, and rauwolscine. Moreover, the inhibition by 0.31 μg of ergotamine kg-1 min-1 was unaltered by GR127935 and haloperidol, partly blocked by GR127935 and rauwolscine or rauwolscine and haloperidol, and abolished by GR127935, haloperidol, and rauwolscine. These findings imply that prejunctional 5-HT1B/1D, D2-like, and α2-adrenergic receptors mediate the sensory inhibition induced by 0.31 μg of ergotamine kg-1 min-1, whereas larger doses may involve other receptors. Thus, ergotamine's ability to inhibit the perivascular sensory peptidergic drive may result in facilitation of its systemic vasoconstrictor properties.

Regulatory Effects of 5-Hydroxytryptamine Receptors on Voiding Function.

A growing body of evidence suggests that 5-hydroxytryptamine (5-HT; serotonin) has both physiological and pathological functions in the lower urinary tract. A wide variety of 5-HT receptor subtypes are variably expressed in different organs, both peripheral and central. On urinary bladder smooth muscle, 5-HT1A, 5-HT2, 5-HT3, and 5-HT7 subtypes could function as postjunctional receptors. Postjunctional 5-HT2 receptors induce detrusor contraction of the bladder body. 5-HT1A is suggested to have a similar effect to 5-HT2, while 5-HT3 might suppress detrusor contraction evoked by direct muscle stimulation. Postjunctional 5-HT7 is reported to induce relaxation of the bladder neck, which might be required for efficient voiding. 5-HT1A, 5-HT2A, 5-HT2C, 5-HT3, 5-HT4, and 5-HT7 subtypes also could act as prejunctional receptors in autonomic excitatory nerve terminals. 5-HT2A, 5-HT2C, 5-HT3, 5-HT4, and 5-HT7 subtypes facilitate the neurogenic contraction of the detrusor by enhancing cholinergic or purinergic transmission, whereas 5-HT1A receptors might inhibit the release of acetylcholine in the detrusor. Furthermore, 5-HT1D could be involved in the suppression of ATP release from the urothelium, aiding visceral sensation of the urinary bladder. In the central pathways controlling the micturition reflex, 5-HT1A, 5-HT2A, and 5-HT7 are involved in regulation of bladder and urethral sphincter activities. Their functions, especially that of 5-HT1A, vary in a species- and site (spinal or supraspinal)- dependent manner. In addition to urinary bladder, 5-HT could be involved in prostate contraction and cell proliferation. Evidence indicates that 5-HT receptor subtypes may be novel therapeutic targets for lower urinary tract symptoms. Grants-in-Aid for Scientific Research (C) (KAKENHI 23590707, 24590722, and 26460694) from the Japan Society for the Promotion of Science.

794 Downregulation of prejunctional CB1 and CB2 cannabinoid receptors and TRPV1 channels is involved in the impaired neurogenic contraction of bladder in insulin-resistant obese Zucker rat

794 Downregulation of prejunctional CB1 and CB2 cannabinoid receptors and TRPV1 channels is involved in the impaired neurogenic contraction of bladder in insulin-resistant obese Zucker rat

PTH-receptors regulate norepinephrine release in human heart and kidney

Recent data suggests that chronic renal failure and hyperparathyroidism are associated with sympathetic overactivity. Since peptide hormones are known to modulate norepinephrine (NE) release by activating prejunctional receptors, this study investigates whether parathyroid hormone fragment (1–34) (hPTH(1–34)) increases neuronal NE release in human heart and kidney. Using specific PTH-receptor agonists and antagonists, this study furthermore highlights functional differences between PTH1 and PTH2 receptors.Human atrial and renal tissues were incubated with [3H]-NE and superfused. Three electrical stimulations (5Hz, 1min) induced a stable [3H]-NE release which was taken as an index of endogenous NE release. RT-PCR with specific primers for PTH1- and PTH2-receptor was performed in heart and kidney.hPTH(1–34) (0.01–0.1μmol/L) and a stable analog of its second messenger cAMP (8-bromo-cAMP) increased [3H]-NE release in human atria. This facilitatory effect of PTH was also observed in human renal cortex. The PTH1-receptor antagonist (D-Trp12, Tyr34)-pTH-(7–34) (0.5μmol/L) abolished the effect of hPTH(1–34). This data was verified using isolated perfused mouse kidneys. Tuberoinfundibular peptide of 39 residues (TIP-39) (0.1nmol/L–0.1μmol/L) decreased [3H]-NE release in atria.PTH1- and PTH2-receptor expressions were demonstrated in human heart and kidney. Moreover, a splice variant of the PTH2-receptor was detected in human kidney.In conclusion, PTH is able to facilitate NE release in human atria and renal cortex by activation of PTH1-receptors. The highly increased PTH levels that can be observed in chronic renal failure might be one contributor for the elevated sympathetic nerve activity and the associated cardiovascular mortality in patients with end stage renal disease.

Neuropeptide Y overflow and metabolism in skeletal muscle arterioles

The purpose of this study was to characterize neuropeptide Y (NPY) overflow and metabolism from isolated skeletal muscle arterioles of female rats. Gastrocnemius first-order arterioles were removed from young (2 months), young adult (6 months) and middle-aged (12 months) F344 female rats. Arterioles were isolated, cannulated and pressurized in a microvessel bath with field stimulation electrodes. NPY overflow from isolated arterioles was assessed at 0 s and 30 s post-field stimulation. Dipeptidyl peptidase IV (DPPIV) activity was quantified via fluorometric assay of whole vessel homogenate. In young adult and middle-aged rats, NPY overflow increased 0 s and 30 s following field stimulation. In young adult rats, DPPIV inhibition resulted in an increase in NPY overflow at 30 s, while middle-aged rats had no increase in NPY overflow with DPPIV inhibition (P <0.05). DPPIV activity was influenced by factors such as age, vessel type, and endothelium (P <0.05). The present data suggest that DPPIV plays a significant role in modulating the actions of NPY in arterioles of young adult females; however, this role appears to diminish with age.

Not a fine wine: the ATP hypothesis may not get better with age

Not a fine wine: the ATP hypothesis may not get better with age

Neuronal and non-neuronal modulation of sympathetic neurovascular transmission.

Noradrenaline, neuropeptide Y and adenosine triphosphate are co-stored in, and co-released from, sympathetic nerves. Each transmitter modulates its own release as well as the release of one another; thus, anything affecting the release of one of these transmitters has consequences for all. Neurotransmission at the sympathetic neurovascular junction is also modulated by non-sympathetic mediators such as angiotensin II, serotonin, histamine, endothelin and prostaglandins through the activation of specific pre-junctional receptors. In addition, nitric oxide (NO) has been identified as a modulator of sympathetic neuronal activity, both as a physiological antagonist against the vasoconstrictor actions of the sympathetic neurotransmitters, and also by directly affecting transmitter release. Here, we review the modulation of sympathetic neurovascular transmission by neuronal and non-neuronal mediators with an emphasis on the actions of NO. The consequences for co-transmission are also discussed, particularly in light of hypertensive states where NO availability is diminished.

Functional crosstalk of prejunctional receptors on the modulation of noradrenaline release in mesenteric vessels: A differential study of artery and vein

The role of angiotensin II receptors, bradykinin receptors and β-adrenoceptors in the modulation of noradrenaline release and the influence of α 2-autoinhibition in these effects was investigated in the mesenteric artery and vein. Rings of mesenteric vessels of male Wistar rats were labelled with [ 3H]-noradrenaline and the effects of modulators on tritium overflow evoked by 100 pulses at 2 Hz (marked α 2-autoinhibition) and by 20 pulses at 50 Hz or 100 pulses at 2 Hz plus yohimbine (1 μM; reduced α 2-autoinhibition) were evaluated. Angiotensin II and bradykinin enhanced noradrenaline release evoked by 100 pulses at 2 Hz, in a concentration-dependent manner, in both vessels. These effects were attenuated under conditions of reduced α 2-autoinhibition. The attenuation was partially reversed by activation of adenosine A 1 receptors in both vessels and by activation of P2Y receptors in the vein. Isoprenaline and the selective β 2-adrenoceptor agonist formoterol enhanced tritium overflow independently of α 2-autoinhibition in the vein. In the artery, the enhancement by formoterol was only observed under reduced α 2-autoinhibition. Pharmacological characterization of the β-adrenoceptors indicated that in the artery the effect of isoprenaline was mediated by the β 1-subtype under marked α 2-autoinhibition and by the β 2-subtype under reduced α 2-autoinhibition whereas in the vein the effect was independent of α 2-autoinhibition. The results indicate that α 2-autoinhibition is a key determinant of the magnitude of facilitation caused by angiotensin II and bradykinin in both types of mesenteric vessels and regulates the effects mediated by β 1-and β 2-adrenoceptors which co-exist in the artery.

Suppression of mediator secretion in murine neogenic motor synapses with the participation of L-type Ca2+-channels and ryanodine receptors

L-type Ca(2+)-channel blockers, verapamil (5 microM) and nifedipine (10 microM), have increased the quantum composition of endplate potentials (EPP) and the level of induced rhythmic activity of neogenic synapses. L-type Ca(2+)-channel activator BAY 8644 (1 microM) has a decreased mediator secretion level. Nifedipine (10 microM) has not changed the frequency and amplitude of diminutive EPPs in the dormant state or during potassium depolarization. Blocking of the prejunctional ryanodine receptor with ryanodine (10 microM) led to an increase in the single EPP quantum composition that was qualitatively similar to nifedipine and verapamil, but more marked, and also caused the reinforcement of mediator release during the rhythmic EPP salvo. Ryanodine receptor activation with ryanodine (1 microM) resulted in reduction of the quantum composition of single and rhythmic EPPs. This effect was partially prevented with nifedipine (10 microM).

Alpha2 adrenoceptors modulate histamine release from sympathetic nerves in the guinea pig vas deferens

In our previous studies, we have identified that histamine (HA) is co-released with noradrenaline (NA) from the sympathetic nerve terminals and acts as a novel sympathetic neurotransmitter. However, the modulation of sympathetic HA release by sympathetic prejunctional receptors is still unknown. Therefore, in this study, we explored the modulation effect of α 2 adrenoceptors on sympathetic HA release and the interaction of α 2 adrenoceptors with HA H 3 receptors in vas deferens isolated from guinea pig. The selective α 2 adrenoceptor agonist brimonidine decreased the HA overflow in a concentration-dependent manner and abolished the contractile responses mediated by sympathetic HA release. Yohimbine competitively blocked the effect of brimonidine on HA release. Similarly, the HA H 3 receptor agonist R-(α)-methylhistamine also decreased HA release, and thioperamide blocked the effect of R-(α)-methylhistamine. When used singly, both yohimbine and thioperamide facilitated HA release. In addition, the inhibitory effect of brimonidine on HA release was stronger in the presence of thioperamide, while it was reduced when HA H 3 receptors were activated by R-(α)-methylhistamine. Our findings indicate for the first time that sympathetic HA release is negatively modulated both by prejunctional α 2 receptors and HA H 3 receptors and that these two prejunctional receptors interact with each other in modulating sympathetic HA release.

Characterization of Prejunctional Muscarinic Receptors: Effects on the Release of VIP and Functional Responses and Receptor Expression in the Ovine Submandibular Gland

In the in vivo experiments on anaesthetized sheep, it was presently examined whether muscarinic receptor antagonists with diverse selectivity affect the release of VIP in response to electrical stimulation of the parasympathetic chorda tympanic nerve differently, and if the changes in the release could be associated to altered secretory and vasodilator responses. The location of the muscarinic receptor subtypes was examined also. In the experiments, blood was collected out of the submandibular venous drainage before and during electrical stimulation of chorda tympani nerve in the absence and presence either of pirenzepine or methoctramine. While metchoctramine increased the output of protein, pirenzepine inhibited flow of saliva and increased protein output, vasodilatation, and VIP output. In morphological examinations, the inhibitory muscarinic M4 receptor occurred interacinarily in the gland. It is concluded that prejunctional muscarinic receptors, most likely of the M4 subtype, exert inhibitory modulation of the parasympathetic release of VIP in the ovine submandibular gland.

Pharmacological Effects of Solifenacin on Human Isolated Urinary Bladder

To investigate the effects of solifenacin on human detrusor smooth muscles, we evaluate the effects of solifenacin on the contractions induced by carbachol, KCl, CaCl₂ and electrical field stimulation (EFS), and the EFS-induced acetylcholine release from detrusor smooth muscle strips by using the muscle bath and microdialysis technique. The effects of solifenacin were also compared with effects of other antimuscarinic agents (atropine, oxybutynin and propiverine). Pretreatment with various antimuscarinic agents caused parallel shifts to the right of the concentration-response curves to carbachol. The pA₂ value of the Schild plots for solifenacin was similar to that for oxybutynin. Atropine did not inhibit the KCl- and CaCl₂-induced contractions, while solifenacin, oxybutynin and propiverine significantly inhibited these contractions. EFS-induced contractions were inhibited by various antimuscarinic drugs in a concentration-dependent manner. In the presence of atropine, solifenacin tended to inhibit the residual atropine-resistant contractions induced by EFS, but it was not significant. However, oxybutynin and propiverine inhibited them under the same conditions. Although pretreatment with atropine and propiverine did not cause significant changes in EFS-induced acetylcholine release, solifenacin and oxybutynin caused significant decreases in acetylcholine release. The present results suggest that solifenacin inhibits contractions of human detrusor smooth muscles mainly by the antimuscarinic action and that the high concentration of solifenacin has Ca²⁺ channel antagonist action. Moreover, solifenacin may block not only postjunctional receptors, but also prejunctional receptors to modulate acetylcholine releases in cholinergic nerve endings in human detrusor smooth muscles. The findings support that muscarinic-receptor-inhibitory actions in human bladder mainly contribute to the usefulness of solifenacin as a therapeutic drug for overactive bladder.

Effects of neuraminidase on equine isolated bronchi

It has been demonstrated in mammals that the airway hyper-responsiveness, which accompanies viral infections, is the result of increased reflex bronchoconstriction due to inhibition of muscarinic prejunctional receptors, which belong to M2 subtypes. Multiple mechanisms account for virus-induced M2 receptor dysfunction. Viral neuraminidase may deglycosylate the M2 receptor, decreasing acetylcholine affinity. Equine influenza remains a common viral respiratory disease of horses worldwide, which results in loss to the equine industry, by decreasing performance, convalescence time and loss of peak performance due to chronic sequelae, such as airway hyper-responsiveness. The purpose of this study was to evaluate the effect of neuraminidase on equine isolated bronchi, assessed in equine bronchial smooth muscle rings, derived from five healthy equine male lungs. A pretreatment with vehicle did not modify contraction induced by electrical field stimulation (EFS) studies at each frequency tested. A pretreatment with pilocarpine (1–100 μM) significantly reduced, while methoctramine (1–100 μM) significantly increased contraction induced by EFS. Finally, neuraminidase (0.5 UI) significantly increased contraction induced by EFS. These results suggest that airway hyper-responsiveness that follows a viral influenza infection might be related to a dysfunction of muscarinic prejunctional receptors.

ATP MODULATES THE RELEASE OF NORADRENALINE THROUGH TWO DIFFERENT PREJUNCTIONAL RECEPTORS ON THE ADRENERGIC NERVES OF RAT PROSTATE

1. The effects of adenosine and ATP receptor agonists on the release of endogenous noradrenaline from electrically stimulated (2 Hz, 0.1 msec) rat prostate were examined in order to clarify the pharmacological properties of prejunctional receptors for adenosine and ATP on the adrenergic nerve varicosities in the prostate. Noradrenaline was quantified by HPLC coupled with electrochemical detection techniques. 2. Both adenosine and ATP receptor agonists (1 micromol/L) inhibited noradrenaline release and the relative order of inhibitory effect was N(6)-cyclopentyl-adenosine (CPA) > 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine > adenosine > 2-methylthio-ATP (2mSATP) > AMP > ATP. 3. The adenosine receptor agonist CPA (1 nmol/L-1 micromol/L) and the ATP receptor agonist 2mSATP (100 nmol/L-100 micromol/L) inhibited the stimulation-induced release of noradrenaline in a concentration-dependent manner. The concentrations of CPA and 2mSATP that produced 50% inhibition of noradrenaline release were 9.6 nmol/L and 1.4 micromol/L, respectively. 4. 1,3-Dipropyl-8-cyclopentylxanthine, an adenosine A(1) receptor antagonist, significantly reduced the inhibitory effects of not only CPA, but also 2mSATP. 5. Suramin, an ATP receptor antagonist, significantly reduced the inhibitory effects of 2mSATP, but not those of CPA. 6. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, another ATP receptor antagonist, had no effect on the inhibitory action of either agonist. 7. These results suggest that, in the sympathetic nerve terminals of rat prostate, adenosine and ATP induce inhibition of noradrenaline release via the activation of adenosine A(1) and/or xanthine-sensitive ATP receptors, which play an inhibitory regulatory role in adrenergic neurotransmission in the prostate.

Regulation of Norepinephrine Release from Isolated Bovine Irides by Histamine

In the present study, we investigated the effect of histamine on sympathetic neurotransmission from isolated, superfused bovine irides. We also studied the pharmacology of prejunctional histamine receptors that regulate the release of norepinephrine (NE) from this tissue. The effect of exogenous histamine and various histamine receptor agonists was examined on the release of [(3)H]-norepinephrine ([(3)H]NE) triggered by electrical field stimulation using the Superfusion Method. Histamine receptor agonists caused a concentration-dependent inhibition of field-stimulated [(3)H]NE overflow with the following rank order of potency: imetit > histamine > R-alpha-methylhistamine. In all cases, the inhibitory action of histamine receptor agonists was attenuated at high concentrations of these compounds. The histamine receptor antagonists, clobenpropit (H(3)-antagonist/H(4)-agonist) and thioperamide (H(3)-antagonist) blocked the inhibitory response elicited by R-alpha-methylhistamine and imetit, respectively. Inhibitory effects of R-alpha-methylhistamine and clonidine were not additive suggesting that prejunctional H(3)- and alpha(2)-adrenoceptors coexist at neurotransmitter release sites. We conclude that histamine produces an inhibitory action on sympathetic neurotransmission in the bovine iris, an effect mimicked by selective H(3)-receptor agonists and blocked by H(3)-antagonists.

Cholinergic innervation of the mouse isolated vas deferens

Recently, a population of nerves has been described in the aganglionic mouse vas deferens, in which electrically evoked contractions were insensitive to high concentrations of the adrenergic neurone blocker, bretylium. In this paper, the pharmacology of this nerve-evoked contraction has been examined in more detail. Bretylium (20 microM) revealed, after 5 h exposure, a new residual neurogenic contraction (20 stimuli at 10 Hz) that was tetrodotoxin-sensitive. The muscarinic antagonist, cyclopentolate (0.1 and 1 microM), reduced this residual component and the inhibition was reversed by the acetylcholinesterase inhibitor, neostigmine (1 and 10 microM). Nicotine (30 microM) enhanced the residual component revealed by bretylium, suggesting that there are prejunctional nicotinic receptors (nAchRs) influencing acetylcholine (Ach) release. In the presence of prazosin (0.1 microM), a selective alpha(1)-adrenoceptor antagonist, and alpha,beta-methylene ATP (1 microM), a purinergic agonist that desensitise P2X receptors, neostigmine increased the hump component of contraction and yohimbine (0.3 microM), an alpha(2)-adrenoceptor antagonist, enhanced both components of the electrically evoked stimulation. The contraction was blocked by cyclopentolate (1 microM). In the absence of bretylium, neostigmine alone increased the hump component of contraction in a frequency-dependent manner. This increase was reversed by atropine (1 microM) and cyclopentolate (1 microM) to control levels. However, in control experiments, atropine or cyclopentolate did not detectably influence the delayed neurogenic contraction. Ach (10 microM) induced a contraction in the mouse vas deferens, either when applied alone or in the presence of neostigmine.Thus, it has been demonstrated unequivocally that the mouse vas deferens is innervated by functional cholinergic nerves, whose action is terminated by cholinesterase. Furthermore, Ach release can be enhanced by activation of prejunctional nAchRs presumably located on the cholinergic nerve terminals.

Characterization of Prejunctional Serotonin Receptors Modulating [3H]Acetylcholine Release in the Human Detrusor

Bladder overactivity (OAB) is a chronic and debilitating lower urinary tract (LUT) disorder that affects millions of individuals worldwide. LUT symptoms associated with OAB, such as urgency and urinary incontinence, cause a hygienic and social concern to patients, but their current pharmacological treatment is largely inadequate due to the lack of uroselectivity. Although OAB etiology remains multifactorial and poorly understood, increasing evidence indicates that serotonin [5-hydroxytryptamine (5-HT)] is an endogenous substance involved in the control of micturition at central and peripheral sites. In this study, we demonstrated the presence of three distinct 5-HT receptors localized at parasympathetic nerve terminals of the human bladder by measuring electrically evoked tritiated acetylcholine release in isolated detrusor strips. These prejunctional receptors, involved in both positive and negative feedback mechanisms regulating cholinergic transmission, have been characterized by means of three highly selective 5-HT antagonists for 5-HT(4), 5-HT(7), and 5-HT(1A) receptors, namely GR113808A ([1-[2-[(-methylsulphonyl) amino] ethyl]4-piperinidyl]methyl1-methyl-1H-indole-3-carboxylate succinate), SB269970 [(R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)pyrrolidine-1-sulfonyl)phenol hydrochloride], and WAY100635 [N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl)-cyclohexane-carboxamide trichloride]. Under these conditions, we confirmed the facilitatory role of 5-HT(4) heteroreceptors on acetylcholine release and revealed for the first time the occurrence of 5-HT(7) and 5-HT(1A) heteroreceptors with a facilitatory and an inhibitory action, respectively. Our findings strengthen the novel concept for the use of recently patented selective 5-HT agonists and antagonists for the control of OAB dysfunctions associated with inflammatory conditions, although their therapeutic efficacy needs to be explored in the clinical setting.

Selective deletion of the α5 subunit differentially affects somatic–dendritic versus axonally targeted nicotinic ACh receptors in mouse

We have compared the functional properties of nicotinic acetylcholine receptors (nAChRs) within both somatic and presynaptic domains of superior cervical ganglion (SCG) neurones from wild-type (WT) mice with those expressed by SCG neurones from mice with a targeted deletion of the gene for the alpha5-subunit. The functional profile of somatic nAChRs was assayed by direct macroscopic current recording and from measurements of nicotinic agonist-induced calcium transients with fura-2 imaging. The profile of nAChRs at presynaptic sites was assayed by measurement of nicotinic agonist-induced transmitter release (as preloaded [3H]noradrenaline) under conditions of action potential blockade. We have examined the responses to the nicotinic agonists acetylcholine, nicotine, cytisine, dimethylphenylpiperazinium iodide (DMPP) and epibatidine. Macroscopic current and calcium imaging assays revealed several differences in the functional profile of somatic nAChRs in WT SCG neurones compared with those from mice with the alpha5 subunit deleted. Somatic nAChRs in control animals were more potently activated by cytisine as compared to DMPP. In contrast, DMPP was consistently more potent than cytisine in mice lacking the alpha5 nAChR subunit. Differences in the somatic nAChR rank order of potency were most prominent after a least 1 day in vitro. The magnitude of somatic nAChR responses to nicotinic agonists was not substantially different in control mice compared with those of alpha5 subunit-deleted animals. Comparison of presynaptic nAChR-mediated responses in WT versus alpha5 subunit-deleted animals revealed a very different set of changes in the functional profile of prejunctional nAChRs compared with somatic nAChRs. In contrast to somatic nAChRs, the responses of prejunctional receptors were markedly enhanced in alpha5 knockout animals compared with control. Furthermore, all prejunctional receptor responses were most potently activated by DMPP in both control and in alpha5 subunit-deleted mice. Hence, the presence or absence of the alpha5 subunit did not affect the rank order of potency of agonists at preterminal sites but greatly affected the magnitude of presynaptic nAChR-mediated responses. The enhanced efficacy of nicotine at presynaptic receptors was corroborated in an acute atrium preparation from postnatal alpha5 subunit-deleted mice. These results confirm and significantly extend our previous observation that in the sympathetic nervous system, somatic and prejunctional receptors are different and rely on the presence of the alpha5 subunit in a distinct manner.

Different AT1 receptor subtypes at pre- and postjunctional sites: AT1A versus AT1B receptors.

Angiotensin (AT) II is known to enhance responses to electrical field stimulation (EFS) via AT1 receptors located on sympathetic nerve terminals. Differences in potency exist between AT1 receptor antagonists regarding the inhibition of the prejunctional and postjunctional AT1 receptors. It is hypothesized that prejunctional AT1 receptors might belong to the AT1B receptor subtype. Accordingly, the authors investigated whether AT1B receptor inhibition by high concentrations of PD123319 could suppress ATII-augmented noradrenergic transmission (prejunctional) in the rabbit thoracic aorta by means of a noradrenaline spillover model. Additionally, the influence of PD123319 on ATII-enhanced constrictor responses to electrical field stimulation was investigated in the isolated rabbit mesenteric artery. Furthermore, the authors investigated whether PD123319 could influence the constrictor responses (postjunctional) to ATII in both preparations. In the thoracic aorta, ATII (10 nM) caused a significant enhancement of EFS-evoked [3H]-noradrenaline release by a factor of 2.0 +/- 0.1. This reinforcement could be inhibited by PD123319 (0.1, 1, and 10 microM). The constrictor response to ATII was unaffected by PD123319. In the mesenteric artery, ATII (0.5 nM) caused a significant enhancement of constrictor responses to EFS by factors of 2.9 +/- 0.3, 2.3 +/- 0.3, and 1.6 +/- 0.1 at 1, 2, and 4 Hz, respectively. This enhancement could be attenuated by PD123319 (1 and 10 microM). The constrictor response to ATII was unaffected by PD123319. It is concluded that the prejunctional AT1 receptors belong to the AT1B subtype whereas postjunctional AT1 receptors do not.