C. H. V. Hoyle

Colocalization of nitric oxide synthase and NADPH-diaphorase in the myenteric plexus of the rat gut

The pattern of distribution and colocalization of nitric oxide-synthase (NOS) and NADPH-diaphorase in the myenteric plexus of whole-mount preparations of the antrum, duodenum, ileum, caecum, proximal colon and distal colon of the rat were investigated using immunohistochemical and histochemical staining techniques. Almost all the myenteric neurons that were NOS-positive in all regions of the gut examined were also stained for NADPH-diaphorase. However, in the stomach, duodenum and ileum, only a few of the NOS-positive nerve fibres in the tertiary and secondary plexuses and circular muscle layer were also stained for NADPH-diaphorase, whereas in the caecum and distal colon almost all the NOS-positive nerve fibres were also stained for NADPH-diaphorase. The results in the present study are consistent with the view that nitric oxide (NO) has a mediating role in gastrointestinal neurotransmission.

Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in a subpopulation of intrinsic neurones of the guinea-pig heart

This is the first report of the presence of nitric oxide synthase (NOS) immunoreactivity and NADPH-diaphorase (NADPH-d) activity in a subpopulation of the intrinsic neurones that innervate the heart. A cytochemical technique to detect NADPH-d and antisera raised against purified rat cerebellar NOS were employed to examine the expression of these enzymes by cells in a dissociated cell culture preparation from newborn guinea-pig atria and interatrial septum. Comparison of the results obtained by these two techniques and double-labelling experiments indicate that a subpopulation of intracardiac neurones contain both NADPH-d and NOS. These results indicate that some intracardiac neurones are capable of synthesizing nitric oxide. This raises the possibility that nitric oxide plays a role in the neural control of the heart.

Enkephalins modulate inhibitory neuromuscular transmission in circular muscle of human colon via delta‐opioid receptors.

1. A sucrose‐gap technique was used to investigate the neuromodulatory actions of enkephalins on non‐adrenergic, non‐cholinergic inhibitory junction potentials (IJPs) in the circular muscle of the human large intestine. 2. The native enkephalins, [Leu5]enkephalin (LENK) and [Met5]enkephalin (MENK) caused a concentration‐dependent reduction in amplitude of IJPs without a significant effect on the smooth muscle membrane. 3. The actions of LENK and MENK were mimicked by the delta‐selective opioid receptor agonists [D‐Pen2, D‐Pen5]enkephalin (DPDPE) and [D‐Ala2, D‐Leu5]enkephalin (DADLE). 4. The actions of LENK, MENK and DPDPE were antagonized to similar extents by the delta‐selective opioid receptor antagonist ICI 174,864. 5. The mu‐selective opioid receptor agonist [D‐Ala2, Me Phe, Gly‐ol5]enkephalin was approximately 100‐fold less potent than any of the native or synthetic enkephalins at reducing the amplitude of the IJP. Dynorphin A and beta‐endorphin both had very weak activity. 6. Responses to all of the agonists were inhibited by naloxone. The degree of antagonism of DPDPE or DADLE by naloxone (1 microM) was the same as that of LENK or MENK. 7. Neither MENK nor LENK affected hyperpolarization of the smooth muscle membrane induced by ATP or 5‐hydroxytryptamine. Vasoactive intestinal polypeptide (1 pM‐1 microM) did not produce any observable responses and this lack of reactivity was not affected by the enkephalins. 8. It is concluded that in the circular muscle of the human colon, LENK and MENK can act on prejunctional delta‐opioid receptors to produce inhibition of non‐adrenergic, non‐cholinergic inhibitory neuromuscular transmission. Possible physiological significance of this prejunctional receptor is discussed.

Acute paw oedema formation induced by ATP: Re-evaluation of the mechanisms involved

ATP-induced inflammation was investigated using subplantar injection in the mouse hind paw. The order of efficacy of purinoceptor agonists for inducing paw oedema (30 nmol per paw) was ATP=α,β-methylene ATP=2-methylthio ATP > adenosine > UTP > ADP > AMP. Diadenosine polyphosphates effectively induced paw oedema formation with an order of efficacy of: P1, P4-di(adenosine-5′)tetraphosphate =P1,P5-di(adenosine-5′)-pentaphosphate= P1,P6-di(adenosine-5′) hexaphosphate ≫ ATP=P1,P3-di(adenosine-5′)triphosphate > P1,P2-di(adenosine-5′)pyrophosphate. Systemic administration of P2-purinoceptor antagonists (30–100 μmol/kg), suramin, 4,4′-diisothiocyanatostilbene-2,2′-disulphonate, pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid and cibacron blue, reduced the intensity of ATP-induced oedema. At 30 μmol/kg 8-(p-sulfophenyl)theophylline (non-selective adenosine receptor antagonist), 3,7-dimethyl-1,1-propargyl-xanthine (adenosine A2 receptor antagonist), triprolidine (histamine H1 receptor antagonist), ranitidine (histamine H2 receptor antagonist) and ketanserin (5-hydroxytryptamine 5-HT2 receptor antagonist), but neither 8-cyclopentyl-1,3-dipropylxanthine (adenosine A1 receptor antagonist), nor indomethacin (cyclooxygenase inhibitor) inhibited the ATP-induced swelling. Topical (100 nmol per paw), but not systemic (100 μmol/kg) administration of NG-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor) reduced the intensity of the ATP-induced paw oedema. These results show that ATP can induce an inflammatory oedematous reaction and contribute to our understanding of the underlying mechanisms.

Adrenergic, but not cholinergic or purinergic, responses are potentiated in the cecum of diabetic rats

Electrophysiologic properties of smooth muscle strips from the circular muscle of the cecum were compared in streptozocin-treated diabetic (8 wk) and untreated control rats using the sucrose-gap technique. Changes in membrane potential elicited by field stimulation (0.03-32 Hz) and by exogenously applied adenosine triphosphate (10-1000 microM) and noradrenaline (0.03-10 microM) were measured. Nonadrenergic, noncholinergic inhibitory junction potentials were the predominant response to field stimulation. However, in some preparations from both diabetic and control tissues, the inhibitory junction potential was preceded by a small nonadrenergic, noncholinergic excitatory junction potential. In nonatropinized preparations, a cholinergic excitatory junction potential was occasionally elicited; there was no difference between these cholinergic responses in diabetic and control tissues. The inhibitory junction potentials and the hyperpolarization in response to adenosine triphosphate were similar in diabetic and control tissues, although the rate of hyperpolarization of the single inhibitory junction potential was slower in the diabetic tissues. In contrast, exogenous application of noradrenaline revealed significantly greater hyperpolarizing responses in diabetic compared with control tissues. This increase in potency appeared to be due, in part, to an increased sensitivity of alpha-adrenoceptors on smooth muscle. There was no evidence for beta-adrenoceptor activation by noradrenaline. Prejunctional inhibition of the nonadrenergic, noncholinergic neuromuscular transmission by noradrenaline was not affected by streptozocin-induced diabetes. The induced changes in adrenoceptor activity were selective for the postjunctional alpha-adrenoceptors.

Degradation of adenosine by extracellular adenosine deaminase in the rat duodenum

1. Extracellular degradation of adenosine by adenosine deaminase was studied in the rat duodenum using high performance liquid chromatography (HPLC) and pharmacological techniques. 2. Relaxant responses to adenosine (1-10 microM) were potentiated in a concentration-dependent manner by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and deoxycoformycin, both of which are inhibitors of adenosine deaminase. 3. Breakdown of adenosine, determined by HPLC, due to incubation with segments of rat duodenum was inhibited by both EHNA and deoxycoformycin. Cytosolic sources of adenosine deaminase were excluded. 4. Relaxant responses to adenosine were also potentiated by the adenosine transport inhibitor dilazep, which did not inhibit adenosine deaminase activity. 5. The extracellular adenosine deaminase activity (4 units/g tissue) was high compared with activity previously determined in other organs.

Design and pharmacological characterization of selective P2-purinoceptor antagonists

At least five distinct P2-purinoceptor subtypes have been characterized to date, based on the rank order of potency of several ATP analogues: P2X, P2Y, P2U, P2T and P2Z. However, the characterization of P2-purinoceptor subtypes is hampered by an unavailability of potent, highly selective, competitive antagonists. In the search for selective P2-purinoceptor antagonists, the structure-activity relationships for a series of analogues of pyridoxal-5-phosphate and suramin at P2-purinoceptor subtypes were investigated in our laboratories. Two of these compounds were the subject of a more detailed pharmacological characterization: pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) and the symmetrical 3′-urea of 8-(benzamido)naphthalene-1,3,5-trisulfonic acid (NF023). The results demonstrate that the two parent compounds, pyridoxal-5-phosphate and suramin, do not differentiate between P2X- and P2Y-purinoceptors. In contrast, PPADS and NF023 were found to be selective antagonists of P2X-purinoceptor-mediated responses in several smooth muscle preparations. In addition, PPADS and NF023 were shown to displace competitively the binding of [3H]α,β-methylene ATP to rabbit and rat bladder membranes, respectively, which indicates that these two compounds act directly on P2X-receptors. PPADS and NF023 were ineffective at P2U-purinoceptors in rat mesenteric arterial bed. P2T-purinoceptor-mediated platelet aggregation was only affected by PPADS in concentrations higher than 100 μM. Suramin and NF023 were inhibitors of ecto-ATPase activity in the same concentration range needed for P2-purinoceptor antagonism. In contrast, PPADS was only very weakly active in inhibiting ecto-ATPase activity. AT 100 μM, PPADS and NF023 did not interact with α1-adrenoceptors, adenosine A1- and A2-, histamine H1- and muscarinic M1-, M2- and M3-receptors. In conclusion, PPADS and NF023 are specific P2-purinoceptor antagonists showing a high selectivity for the P2X-subtype. These two compounds may prove to be useful starting points in the synthesis of novel, highly potent and selective antagonists at P2-purinoceptor subtypes.

Abnormalities of innervation of internal anal sphincter in fecal incontinence

Physiological and histological studies have shown that the internal anal sphincter is abnormal in idiopathic fecal incontinence. We have recently demonstrated that the invitro contractile response of the internal anal sphincter to the sympathetic neurotransmitter noradrenaline is decreased in incontinence. In this study we have further defined this reduced sensitivity and provided more information about the intrinsic innervation in both the normal and the incontinent sphincter muscle. Muscle strips from 12 incontinent patients undergoing post anal repair and from 11 controls undergoing rectal excision for low rectal carcinoma were studied. Responses to noradrenaline were recorded initially alone and then in the presence of phentolamine, an α-adrenoceptor blocker. In the presence of phentolamine, noradrenaline caused relaxation: there was no significant difference in the relaxation-response curves and the EC50 was the same in the two groups. These results demonstrate that the previously documented reduced sensitivity to noradrenaline is due to an altered sensitivity of the α-adrenoceptors. Electrical field stimulation produced relaxations in all muscle strips, but only in the controls was the magnitude of the relaxation significantly increased in the presence of phentolamine. This indicates that there is an α-adrenergic excitatory component of the response to electrical field stimulation of the intramural nerves, which was present in tissues from control patients but which was absent in tissues from patients with idiopathic fecal incontinence.

Pre- and postjunctional adrenoceptor types in the circular muscle of the guinea-pig caecum

SummaryThe sucrose gap technique was used to study the effects of applied adrenoceptor agonists on membrane potential and non-adrenergic, non-cholinergic inhibitory junction potentials (IJPs), in the smooth muscle of the circular coat of guinea-pig caecum. Noradrenaline (10 nmol/1–100 μmol/l), phenylephrine (1–100 μmol/l) and isoprenaline (0.1–100 μmol/l) caused hyperpolarisations of the smooth muscle membrane which were rapid in onset and effect. The magnitudes of hyperpolarisations elicited by noradrenaline were significantly reduced by the non-specific α-adrenoceptor antagonist phentolamine (10 gmol/l), theα1-adrenoceptor antagonist prazosin (1 μmol/l) and the β-adrenoceptor antagonist propranolol (5 gmol/1). The α2-antagonist yohimbine (10 gmol/l) did not significantly reduce the magnitude of the hyperpolarisations induced by noradrenaline. Noradrenaline application caused a reduction in UP amplitude, during hyperpolarisation, by up to 50%. The reduction of the UP amplitude elicited by noradrenaline was significantly antagonised by yohimbine and phentolamine, but not by prazosin or propranolol. Clonidine caused a reduction of UP amplitude by up to 20%, but neither phenylephrine nor isoprenaline caused any significant reduction in UP amplitude. It is concluded that the hyperpolarising responses to exogenous noradrenaline in the circular muscle of guinea-pig caecum are mediated by postjunctional α1- and β-adrenoceptors, and that the inhibition of UP amplitude is mediated by prejunctional α2-adrenoceptors.

Effects of vitamin E deficiency on autonomic neuroeffector mechanisms in the rat caecum, vas deferens and urinary bladder

1. Modified sucrose‐gap, standard organ‐bath techniques and transmitter release studies were used to examine neuromuscular transmission in the caecum, vas deferens and urinary bladder in normal rats and in rats maintained for 12 months on a diet free of vitamin E. 2. In the caecum circular muscle, non‐adrenergic, non‐cholinergic inhibitory junction potentials were absent from 48 and 15% of preparations from vitamin E‐deficient and control animals, respectively. Cholinergic excitatory junction potentials were absent from 83 and 8% of vitamin E‐deficient and control preparations, respectively. Responses to applied noradrenaline (0.1‐30 microM), alpha,beta‐methylene ATP (3‐100 microM) and acetylcholine (0.1‐30 microM) were attenuated or absent in vitamin E‐deficient tissues. Responses to applied KCl were similar in both groups. Release of [3H]noradrenaline or endogenous acetylcholine could not be evoked from vitamin E‐deficient tissues. 3. In contrast, in isolated preparations of the vas deferens and urinary bladder, neuromuscular transmission by adrenergic, cholinergic and purinergic components were unaffected by long‐term vitamin E deficiency. 4. In conclusion, vitamin E deficiency causes dysfunction of autonomic neuroeffector mechanisms in the smooth muscle of the rat caecum, at both a pre‐ and postjunctional level. The lesions in autonomic transmission mechanisms brought about by long‐term vitamin E deficiency were found only in the caecum; no changes in sympathetic neuromuscular transmission were observed in the vas deferens, or in parasympathetic neuromuscular transmission in the urinary bladder.