Identification and characterisation of heterogeneous somatostatin binding sites in rat distal colonic mucosa

We have previously shown that the somatostatin (SRIF) sst₂ receptor-selective peptide, BIM-23027, is a potent antisecretory agent in rat isolated distal colonic mucosa (RDCM) and in radioligand binding studies in RDCM membranes, it only maximally inhibited approximately 40% of [¹²⁵I]-Tyr¹¹-SRIF-14 binding (McKeen ES, Feniuk W, Humphrey PPA (1995) Naunyn-Schmiedeberg's Arch Pharmacol 352:402–411). The aim of this study was to characterise the BIM-23027-sensitive and -insensitive SRIF binding sites in more detail and to compare their properties with those of the recombinant sst₂ receptor stably expressed in mouse fibroblast (Ltk^–) cells.SRIF-14, SRIF-28, CGP-23996 and D Trp⁸-SRIF-14 abolished [¹²⁵I]-Tyr¹¹-SRIF-14 binding (pIC₅₀ values, 8.7–9.7) but the competition curves had Hill slopes which were less than unity. Octreotide and L-362,855 inhibited binding over a wide concentration range (0.1 nM-1 M) and inhibition of binding was incomplete at the highest concentration studied. BIM-23056 (PIC₅₀ <6.5) was a weak inhibitor of [¹²⁵]-Tyr¹¹-SRIF-14 binding. GTPS decreased [¹²⁵I]-Tyr¹¹-SRIF-14 binding by 40%. Further binding experiments with [¹²⁵I]-Tyr¹¹-SRIF-14 were carried out in RDCM in the continuous presence of BIM-23027 (1 M). Under these conditions, seglitide had no effect on [¹²⁵I]-Tyr¹¹-SRIF-14 binding at concentrations up to 10 M, whilst SRIF-14 and SRIF-28 abolished specific [¹²⁵I]-Tyr¹¹-SRIF-14 binding in a manner which was consistent with the ligand binding to two sites. SRIF-14 and SRIF-28 displayed high affinity (pIC₅₀ values of 9.8 and 9.3 respectively) for approximately 70% of these binding sites and low affinity (pIC₅₀ values of 7.8 and 7.3) for the remaining sites. Octreotide, L-362,855 and BIM-23056 were weak inhibitors of [¹²⁵I]-Tyr¹¹-SRIF-14 binding (PIC₅₀ <6.5). [¹²⁵I]-BIM-23027 labelled a single population of SRIF binding sites in RDCM membranes and mouse fibroblast (Ltk^–) cells stably expressing the human recombinant sst₂ receptor. There was a significant correlation between the affinitestimates of a range of SRIF analogues at inhibiting [¹²⁵I]-BIM-23027 binding in RDCM membranes and binding to the recombinant sst₂ receptor in Ltk^– cells, suggesting that the sites labelled by [¹²⁵I]-BIM-23027 in RDCM are similar to the sst₂ receptor. GTPS (100 M) decreased [¹²⁵I]-BIM-23027 binding in RDCM by 60%.The results from these studies demonstrate that [¹²⁵I]-Tyr¹¹-SRIF-14 labels a heterogeneous population of high affinity SRIF binding sites in RDCM membranes. The majority of these sites are insensitive to GTPS and display negligible affinity for the cyclic hexapeptides, BIM-23027 and seglitide. The remaining high affinity binding sites can be selectively labelled with [¹²⁵I]-BIM-23027, are sensitive to GTPS and show similar characteristics to the recombinant sst₂ receptor which appears to mediate the antisecretory effects of SRIF in the mucosa (McKeen ES, Feniuk W, Humphrey PPA (1995) Naunyn-Schmiedeberg's Arch Pharmacol 352:402–411).     

Effects of noradrenaline and somatostatin on basal and stimulated mucosal ion transport in the guinea-pig small intestine

Summary Noradrenaline (NA) and somatostatin (SOM) stimulate intestinal water and ion absorption and are found in mucosal nerve fibres and nerve terminals in submucous ganglia of the guinea-pig small intestine. As the main projection of submucous neurons is to the mucosa, NA and SOM might alter mucosal transport either by a direct effect on the epithelium or indirectly, by affecting submucous neurons. In this study these two possible sites of action of NA and SOM have been investigated in mucosa-submucosa preparations of guinea-pig ileum. In addition, the actions of NA and SOM on the secretory responses caused by stimulation of different populations of submucous neurons have been studied. The stimulants of secretion used were a nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP, 10^–5 M), 5-hydroxytryptamine (5-HT, 10^–7 M) and electrical field stimulation (EFS), which activate cholinergic, noncholinergic and mixed populations of submucous secretomotor neurons, respectively.Segments of intestine were dissected free of external muscle and myenteric plexus and mounted in Ussing chambers. Short-circuit current (I _sc) was measured as an indication of net active ion transport across the tissue. NA (10^–8 M) and SOM (>10^–10 M) each caused a decrease in I _sc, indicating a net increase in ion absorption. The NA response was abolished and the magnitude of the SOM response was reduced to 20% by tetrodotoxin (10^–7 M). DMPP, 5-HT and EFS each stimulated nerves that increased I _sc and each of these responses was significantly diminished by NA and SOM; for both NA and SOM the decrease in the DMPP response was significantly greater than the decrease observed in the response to carbachol (10^–6 M). Phentolamine (10^–6 M) abolished all of the effects of NA but caused no change in the SOM effects. These studies have shown that NA and SOM cause similar changes in net ion transport, that their actions are primarily on submucous secretomotor neurons and that NA and SOM can diminish the responses to stimulation of both cholinergic and noncholinergic submucous neurons.In this tissue it is also known that SOM coexists with NA in noradrenergic nerve terminals in the submucosa. However, when applied together, NA and SOM caused no greater decrement in the carbachol and 5-HT responses than would be predicted by adding the separate effects of NA and SOM. Hence there was no obvious interaction between NA and SOM effects on mucosal transport.     

Pharmacological characterisation of neurokinin receptors mediating anion secretion in rat descending colon mucosa

Summary Substance P(SP), neurokinin A (NKA), neurokinin B (NKB), [Sar⁹, Met (O₂)¹¹]-SP (SMSP), senktide, [Ala⁸]-NKA(4–10) and neuropeptide (NP) all stimulate secretory responses in rat descending colon mucosa under voltage clamp conditions. Secretory responses (measured as short circuit current under voltage clamp conditions) were transient and those evoked by SP, SMSP, NKA and senktide were significantly reduced by pretreating tissues with the chloride channel blocker, diphenylamine carboxylate (DPC). Concentration-response curves showed varying degrees of sensitivity to tetrodotoxin (TTX). Senktide-induced secretion was virtually abolished by TTX, while NP and [Ala⁸]-NKA(4–10) were not significantly altered. Rightward shifts of concentration-response curves were observed for SMSP, NKA and SP in TTX treated preparations compared with controls. NKA response curves in the presence of TTX were further inhibited by MEN10,207 and CP-96,345. GR71251, GR82334 and CP-96,345 all inhibited SMSP secretory responses with pA₂ values of 5.8, 6.5 and 6.9 respectively. In conclusion three types of neurokinin receptor exist in preparations of rat colon mucosa and their relative location within neuronal and epithelial surfaces are discussed. Correspondence to H. M. Cox at the above addressK. G. and S. Y. were project students with H. M. C. in the Department of Pharmacology, University of Cambridge. S. Y. was also a Wellcome Trust Vacation Scholar     

Investigation of the effects of platelet-activating factor (PAF) on ion transport and prostaglandin synthesis in human colonic mucosa in vitro

1. We have investigated the effects of platelet-activating factor (PAF), an endogenous mediator of inflammation, on ion transport and prostaglandin synthesis in the human isolated colon.
2. Application of PAF to the serosal surface of human colonic mucosa induced a marked, concentration-dependent increase in ion transport. Mucosal application was without effect.
3. The secretory response to PAF was significantly inhibited by prior application of a specific PAF receptor antagonist WEB 2170, indicating that the response is dependent on PAF receptor activation.
4. The response to PAF was attenuated by prior application of indomethacin or piroxicam, implicating products of the cyclo-oxygenase pathway in the response.
5. The response to PAF was attenuated by the loop diuretic bumetanide, indicating an involvement of chloride ion secretion in the response.
6. Addition of PAF to the serosal surface induced a significant increase in serosal prostaglandin E₂ (PGE₂), but not 6-oxo-PGF_1α release. There was no effect on mucosal application of PAF.
7. In summary, we have shown that PAF is a potent secretagogue in isolated preparations of human colon and that the response is dependent on a specific PAF receptor, cyclo-oxygenase products and bumetanide-sensitive chloride ion transport.

     

Activation of submucosal 5-HT3 receptors elicits a somatostatin-dependent inhibition of ion secretion in rat colon

Background and purpose:

5-Hydroxytryptamine (5-HT) is a key regulator of the gastrointestinal system and we have shown that submucosal neuronal 5-HT₃ receptors exerted a novel inhibitory effect on colonic ion transport. The aim of the present study was to investigate the precise mechanism(s) underlying this inhibitory effect.

Experimental approach:

Mucosa/submucosa or mucosa-only preparations from rat distal colon were mounted in Ussing chambers for measurement of short-circuit current (I_sc) as an indicator of ion secretion. Somatostatin release was determined with radioimmunoassay. Intracellular cAMP content was measured with enzyme-linked immunoadsorbent assay (elisa). Immunohistochemical techniques were used to study the expression of 5-HT₃ receptors, somatostatin and somatostatin receptors in colonic tissue.

Key results:

In rat distal colonic mucosa/submucosa preparations, pretreatment with 5-HT₃ receptor antagonists enhanced 5-HT-induced increases in I_sc. However, in mucosa-only preparations without retained neural elements, pretreatment with 5-HT₃ receptor antagonists inhibited 5-HT-induced ΔI_sc. Pretreatment with a somatostatin-2 (sst₂) receptor antagonist in mucosa/submucosa preparations augmented 5-HT-induced ΔI_sc. Combination of sst₂ and 5-HT₃ receptor antagonists did not cause further enhancement of 5-HT-induced ΔI_sc. Moreover, both sst₂ and 5-HT₃ receptor antagonists enhanced 5-HT-induced increase in intracellular cAMP concentration in the mucosa/submucosa preparations. 5-HT released somatostatin from rat colonic mucosa/submucosa preparations, an effect prevented by pretreatment with 5-HT₃ receptor antagonists. Immunohistochemical staining demonstrated the presence of 5-HT₃ receptors on submucosal somatostatin neurons and of sst₂ receptors on colonic mucosa.

Conclusion and implications:

Activation of neuronal 5-HT₃ receptors in the submucosal plexus of rat colon suppressed 5-HT-induced ion secretion by releasing somatostatin from submucosal neurons.     

Autoradiographic analysis of somatostatin SRIF1 and SRIF2 receptors in the human brain and pituitary

The distribution of somatostatin (SRIF) receptor sites was studied by in vitro receptor autoradiography in the human brain and pituitary using the SRIF₁ (sst₂) receptor selective [¹²⁵I]Tyr³-octreotide, the non-subtype selective [¹²⁵I]LTT-SRIF-28 ([Leu⁸,D-Trp²²,¹²⁵I-Tyr²⁵]SRIF-28) and the SRIF₂-receptor selective [¹²⁵I]CGP 23996 (c[Asu-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) in buffer containing 120 mM Na⁺. SRIF receptor autoradiography was compared with mRNA expression of somatostatin receptors sst_1–5 as studied by in situ hybridisation in human brain. High levels of [¹²⁵I]LTT-SRIF-28 and [¹²⁵I]Tyr³-octreotide recognition sites were found in the deep layers of cerebral cortex and molecular layer of cerebellum of the human brain. The hypothalamus, choroid plexus, most areas of the brainstem and dentate nucleus were associated with low levels of binding. In contrast to [¹²⁵I]LTT-SRIF-28 and [¹²⁵I]Tyr³-octreotide, no difference was observed for [¹²⁵I]CGP 23996 labelling in the various layers of cerebral cortex. The choroid plexus, substantia nigra and molecular layer of the cerebellum presented high densities of [¹²⁵I]CGP 23996 binding sites whereas no binding was observed in the hypothalamus and locus coeruleus using this radioligand. Both lobes of the human pituitary displayed low levels of [¹²⁵I]LTT-SRIF-28 and [¹²⁵I]Tyr³-octreotide binding. By contrast, the anterior lobe of the pituitary displayed very high levels of [¹²⁵I]CGP 23996 labelled sites whereas intermediate levels were found in the posterior lobe. There was a partial overlap between sst₂ receptor mRNA and [¹²⁵I]Tyr³-octreotide binding, although the distribution of the binding sites was much wider than that of receptor mRNA. The same observation was made for sst₁ and/or sst₄ receptor mRNA and [¹²⁵I]CGP 23996 labelled sites. The present data show that SRIF₁ and SRIF₂ receptors are present in the human brain with different distributions, especially in the cerebral cortex and the pituitary. The very similar distribution of sites labelled with [¹²⁵I]LTT-SRIF-28 and [¹²⁵I]Tyr³-octreotide suggests (i) that sst₂ receptors are predominant within the SRIF₁ family in the human brain and (ii) that [¹²⁵I]LTT-SRIF-28 under the conditions used in the present study, does not significantly label SRIF₂ sites. Received: 8 August 1996 / Accepted: 8 November 1996     

Phospholipase A2 and mediation of the activation of short-circuit current in the rat colonic mucosa

Summary Melittin (0.5–2 g ml^–1) increased the short-circuit current (I_sc) in mucosa-submucosa and mucosa preparations of the rat colon descendens in a dose-dependent manner. In the preparation with the submucosal plexus, quinacrine and indomethacin completely blocked the effect of melittin, indicating activation of phospholipase A₂ and production of prostaglandins induced by the drug. The melittin response was also partially sensitive to the lipoxygenase inhibitor, nordihydroguaiaretic acid. Complete inhibition by tetrodotoxin and atropine gives evidence for the involvement of cholinergic neurons in the mediation of the response induced by melittin. In contrast, in the preparation without the submucosal plexus the effect of melittin was only partially inhibited by quinacrine, indomethacin, or by neuronal blockers, suggesting direct interactions of melittin with the epithelium in addition.The effect of melittin resembles to the action of bradykinin, which is neuronally mediated and quinacrine-sensitive in the mucosa-submucosa preparation, and quinacrine-resistant and not neuronally mediated in the mucosa preparation. In the mucosa-submucosa preparation, the melittin response is even partially sensitive to the bradykinin receptor antagonist [D-Phe⁷]-bradykinin. The results provide evidence for the presence of a quinacrine-sensitive phospholipase A₂ in the preparation with and that without the submucosa. Send offprint requests to: M. Diener at the above address     

Effect of ramosetron on short-circuit current response in rat colonic mucosa

We investigated the effects of ramosetron (YM060, (−)-(R)-5-[(1-methyl-1H-indol-3-yl)carbonyl]-4,5,6,7-tetrahydro-1H-benzimidazole monohydrochloride) on the short-circuit current (I_sc) responses to 5-HT receptor agonists in the rat distal colon, and compared its potency to that of other 5-HT₃ receptor antagonists. 5-Hydroxytryptamine (5-HT) concentration-dependently increased I_sc. The I_sc response to 5-HT was partially reduced by tetrodotoxin and ramosetron, and strongly inhibited by GR113808 ([[1-[(2-methylsulphonyl)amino]ethyl]-4-piperidin-yl]methyl 1-methyl-1H-indole-3-carboxylate). 2-Methyl-5-HT and 5-methoxytryptamine also increased I_sc. The former response was inhibited by ramosetron, and the latter was abolished by GR113808. Ramosetron, YM114 (KAE-393, (−)-(R)-5-[(1-indolinyl)carbonyl]-4,5,6,7-tetrahydro-1H-benzimidazole monohydrochloride) and granisetron concentration-dependently antagonized the I_sc responses to 2-methyl-5-HT with reduction in the maximal response at higher concentrations. Apparent pA₂ values for these antagonists were 10.40, 10.37 and 8.99, respectively. Ondansetron produced clear rightward shifts of the concentration-response curves to 2-methyl-5-HT, with a pA₂ value of 8.53. These results suggest that 5-HT increases I_sc through the 5-HT₃ and 5-HT₄ receptors, and that ramosetron is a potent and selective 5-HT₃ receptor antagonist in rat colonic mucosa.     

Characterisation of bradykinin B1 and B2 receptors using rat isolated vas deferens

The actions of bradykinin and its metabolite des-Arg(9) bradykinin are mediated through activation of bradykinin B(2) and B(1) receptors, respectively. The aim of the present study was to characterize native bradykinin receptors focusing on induction and desensitization using rat isolated vas deferens. Tissues were mounted in organ baths for isometric recordings and neurogenically mediated contractions were evoked by electrical stimulation. Des-Arg(9) bradykinin enhanced the magnitude of the electrically evoked contractions and this effect (which was sensitive to blockade by the peptide bradykinin B(1) receptor selective antagonist B9858, Lys-Lys-(Hyp(3),Cpg(5),D-Tic(7),Cpg(8))des-Arg(9) bradykinin) was only observed following a pre-incubation period and was greatest following 5 h of pre-incubation. Bradykinin also potentiated neurogenically evoked contractions and this effect was sensitive to blockade by Hoe 140 (D-Arg(Hyp(3),Thi(5),D-Tic(7),Oic(8))bradykinin, a peptide bradykinin B(2) receptor antagonist) and was present without pre-incubation but was increased by pre-incubation and reached maximum at the 5-h incubation time point. Responses to bradykinin were larger than those to des-Arg(9) bradykinin. Bradykinin responses did not show desensitization on repeated agonist stimulation. These data confirm in rat isolated vas deferens bradykinin B(2), but not B(1), receptors are constitutively expressed, that both receptor populations are inducible and B(2) receptors do not exhibit desensitization.     

Indirect effects of bradykinin on ion transport in rat colon descendens: mediated by prostaglandins and enteric neurons

Summary The effect of bradykinin on two preparations of rat colon descendens was examined. In a mucosa-submucosa preparation consisting of the submucosal plexus, the mucosal plexus and the epithelium bradykinin (10^–10-5 × 10^–9 mol · 1^–1) caused an increase in Isc, Gt and Pd which was to more than 70% diminished by TTX. However, in a mucosa preparation consisting of only the mucosal plexus and the epithelium bradykinin caused an increase in Isc, Gt and Pd, which was not affected by TTX. Ten times higher concentrations of bradykinin were needed in the mucosa preparation to reach the same effects as in the mucosasubmucosa preparation. All effects of bradykinin were markedly reduced in the presence of indometacin indicating that they were mediated by prostaglandins in both preparations. The bradykinin effect in the mucosa-submucosa preparation but not in the mucosa preparation was reduced about 50% by atropine. The results suggest that bradykinin activates prostaglandin synthesis. Prostaglandins subsequently stimulate neurons in the submucosal plexus which induce a secretory response on the epithelium partially mediated by a muscarinic receptor. In a high concentration bradykinin due to the induction of prostaglandin synthesis can also activate directly the mucosal epithelium. Send offprint requests to M. Diener     

Localization and pharmacological characterization of somatostatin sst2 sites in the rat cerebellum

Radioligand binding studies were performed in membranes of rat cerebellum using [¹²⁵I]-[Tyr³]octreotide ([¹²⁵I]204-090) to characterize the nature of cerebellar somatostatin receptors. Saturation experiments suggest the presence of a single class of binding sites with high affinity, pK_d = 9.53 ± 0.11, but low receptor density, B _max = 12.7 ± 1.0 fmol/mg protein. The pharmacological profile of [¹²⁵I]204-090 sites in cerebellar membranes was established using a range of ligands known to interact with SSTR-2 (now called sst₂) and other somatostatin (SRIF) receptors. SRIF analogues such as octreotide (SMS 201-995), seglitide (MK 678) and somatuline (BIM 23014) displayed very high affinity for cerebellar [¹²⁵I]204-090 binding sites. The data were compared to results obtained using the same ligand in rat cerebral cortex membranes known to represent sst₂ binding. The pharmacological characteristics of the cerebellar sites were in close correlation with those of the cerebral cortex (r = 0.976, n = 19, p < 0.001) and CHO-cells expressing human recombinant sst₂ receptor (r = 0.977, n = 19, p < 0.001). By contrast, there was very little correlation between cerebellar binding and published affinities for rat sst₅ receptors (r = 0.465), for which octreotide has also high affinity. In vitro autoradiographic studies performed in cerebellar slices using [¹²⁵I]204-090 demonstrated the presence of binding sites in the molecular layer of the rat cerebellum. In situ hybridization studies using sst₂ receptor mRNA selective oligoprobes confirmed the presence of sst₂ receptor mRNA in the rat cerebellum. Together, the present data demonstrate the presence of a low density of SRIF receptors in the molecular layer of the adult rat cerebellum which are best characterized as sst₂. This is the first pharmacological characterization and localization of sst₂ receptors in the adult rat cerebellum.     

Neuronally mediated and direct effects of prostaglandins on ion transport in rat colon descendens

Summary Two preparations of rat colon descendens were used in order to localize the action sites of iloprost and prostaglandin E₂ (PGE₂). One preparation, the mucosa-submucosa preparation contained the submucosal and mucosal plexus whereas for the mucosa preparation in addition the submucosa with the submucosal plexus was removed. Iloprost (10^–6 mol · 1^–1) caused an increase in short-circuit current (Isc), potential difference (Pd) and tissue conductance (Gt) of the mucosa-submucosa preparation reflecting net Cl^– secretion as confirmed by unidirectional ion flux measurements. The Cl^– secretion was due to an increase in J _infsm ^supCl and a decrease in J _infms ^supCl . These effects were completely abolished by addition of 5 × 10^–5 mol · l^–1 atropine. Iloprost had only small and inconsistent effects in the mucosa preparation. In contrast PGE₂ (10^–6 mol · l^–1) increased Isc, Pd and Gt due to Cl^– secretion in both preparations. The Cl^– secretion was caused by an increase in J _infsm ^supCl and a decrease in J _infms ^Cl Only the PGE₂ effect in the mucosa-submucosa preparation but not in the mucosa preparation was inhibited by about 50% by atropine. The results suggest that the prostacyclin derivative iloprost induces a Cl^– secretion only by an activation of submucosal neurons whereas PGE₂ acts both on the epithelium and the submucosal plexus. The neuronal effects of prostaglandins appear to be, at least in part, mediated by muscarinic receptors. Send offprint requests to M. Diener     

Functional role of 5-HT2 receptors in the regulation of sleep and wakefulness in the rat

Recently developed agents specifically acting on different 5-hydroxytryptamine (5-HT) receptor populations were used to analyze the functional role of 5-HT₂ receptor subtypes in the sleep-wakefulness cycle of the rat. The 5-HT₂ receptor antagonist ritanserin injected intraperitoneally (IP) (0.04–2.5 mg/kg) induced an increase in deep slow wave sleep (SWS2) duration at the expense of wakefulness (W), light slow wave sleep (SWS1) and paradoxical sleep (PS). The stimulation of 5-HT₂ receptors by 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) produced a dose-related increase in W and a dose-dependent decrease in both SWS2 and PS. Pretreatment with ritanserin (0.16–2.5 mg/kg) or with cinanserin (2.5–5 mg/kg), another 5-HT₂ receptor antagonist, dose-dependently reversed the W enhancement and the SWS2 deficit produced by DOM, but not the PS deficit. Sleep-wakefulness alterations (increase in W and SWS1 combined with a suppression of SWS2 and PS) observed after IP injection of two putative 5-HT₁ receptor agonists, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) (2.5 mg/kg) and 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969) (0.63 mg/kg), were not modified by ritanserin pretreatment (0.16–2.5 mg/kg). These results further support the hypothesis that the serotonergic system plays an active role in the regulation of the sleep-wakefulness cycle in the rat and that 5-HT₂ receptors are involved in this action. In addition it is suggested that 5-HT₁ receptor subtypes are unlikely to interact with 5-HT₂ receptors in the sleep-wakefulness modulation mediated through 5-HT₂ receptors.     

Role of voltage-gated cation channels and axon reflexes in the release of sensory neuropeptides by capsaicin from isolated rat trachea

In order to reveal the role of axon reflexes and sensory receptors in sensory neuropeptide release in response to capsaicin, liberation of substance P, calcitonin gene-related peptide and somatostatin from isolated rat tracheae was investigated in the presence of voltage-sensitive Na(+) and Ca(2+) channel blocking agents. Neuropeptide release induced by capsaicin (10 nM) remained unchanged in the presence of 25 mM lidocaine, 1 microM tetrodotoxin or the N-type Ca(2+) channel inhibitor, omega-conotoxin GVIA (100-300 nM). Peptide release by 100 pulses of 2 Hz field stimulation was prevented by lidocaine or tetrodotoxin. Omega-agatoxin TK (250 nM) significantly inhibited and Cd(2+) (200 microM) prevented capsaicin-induced neuropeptide release. These results suggest that chemical stimulation-induced neuropeptide release does not involve activation of fast Na(+) channels or N- and P-type voltage-dependent Ca(2+) channels, but contribution of Q-type Ca(2+) channels is possible. Sensory neuropeptides are released by capsaicin from sensory receptors without axon reflexes.     

Mechanistic kinetic modeling generates system-independent P-glycoprotein mediated transport elementary rate constants for inhibition and,in combination with 3D SIM microscopy,elucidates the importance of microvilli morphology on P-glycoprotein mediated efflux activity

Introduction: In vitro transporter kinetics are typically analyzed by steady-state Michaelis-Menten approximations. However, no clear evidence exists that these approximations, applied to multiple transporters in biological membranes, yield system-independent mechanistic parameters needed for reliable in vivo hypothesis generation and testing.

Areas covered: The classical mass action model has been developed for P-glycoprotein (P-gp) mediated transport across confluent polarized cell monolayers. Numerical integration of the mass action equations for transport using a stable global optimization program yields fitted elementary rate constants that are system-independent. The efflux active P-gp was defined by the rate at which P-gp delivers drugs to the apical chamber, since as much as 90% of drugs effluxed by P-gp partition back into nearby microvilli prior to reaching the apical chamber. The efflux active P-gp concentration was 10-fold smaller than the total expressed P-gp for Caco-2 cells, due to their microvilli membrane morphology. The mechanistic insights from this analysis are readily extrapolated to P-gp mediated transport in vivo.

Expert opinion: In vitro system-independent elementary rate constants for transporters are essential for the generation and validation of robust mechanistic PBPK models. Our modeling approach and programs have broad application potential. They can be used for any drug transporter with minor adaptations.     

Simultaneous blockade of 5-HT<Subscript>1A/B</Subscript> receptors and 5-HT transporters results in acute increases in extracellular 5-HT in both rats and guinea pigs: in vivo characterization of the novel 5-HT<Subscript>1A/B</Subscript> receptor antagonist/5-HT transport inhibitor SB-649915-B

Rationale The delay in onset and treatment resistance of subpopulations of depressed patients to conventional serotonin reuptake inhibitors has lead to new drug development strategies to produce agents with improved antidepressant efficacy. Objectives We report the in vivo characterization of the novel 5-HT_1A/1B autoreceptor antagonist/5-HT transporter inhibitor (6-[(1-{2-[(2-methyl-5-quinolinyl)oxy]ethyl}-4-piperidinyl)methyl]-2H-1,4-benzoxazin-3(4H)-one), SB-649915-B. Materials and methods Ex vivo binding was used to ascertain 5-HT_1A receptor and serotonin transporter occupancy. 8-OH-DPAT-induced hyperlocomotion and SKF-99101-induced elevation of seizure threshold were used as markers of central blockade of 5-HT_1A and 5-HT_1B receptors, respectively. In vivo electrophysiology in the rat dorsal raphe and microdialysis in freely moving guinea pigs and rats were used to evaluate the functional outcome of SB-649915-B. Results SB-649915-B (1–10 mg/kg p.o.) produced a dose-related inhibition of 5-HT_1A receptor radioligand binding and inhibited ex vivo [³H]5-HT uptake in both guinea pig and rat cortex. SB-649915-B (0.1–10 mg/kg p.o.) reversed both 8-OH-DPAT-induced hyperlocomotor activity and SKF-99101-induced elevation of seizure threshold in the rat, demonstrating in vivo blockade of both 5-HT_1A and 5-HT_1B receptors, respectively. SB-649915-B (0.1–3 mg/kg i.v.) produced no change in raphe 5-HT neuronal cell firing per se but attenuated the inhibitory effect of 8-OH-DPAT. Acute administration of SB-649915-B resulted in increases (approximately two- to threefold) in extracellular 5-HT in the cortex of rats and the dentate gyrus and cortex of guinea pigs. Conclusions Based on these data, one may speculate that the 5-HT autoreceptor antagonist/5-HT transport inhibitor SB-649915-B will have therapeutic efficacy in the treatment of affective disorders with the potential for a faster onset of action compared to current selective serotonin reuptake inhibitors.