Structure and mechanism of the RNA dependent RNase Cas13a from Rhodobacter capsulatus.

Cas13a are single-molecule effectors of the Class II, Type VI family of CRISPR-Cas systems that are part of the bacterial and archaeal defense systems. These RNA-guided and RNA-activated RNA endonucleases are characterized by their ability to cleave target RNAs complementary to the crRNA-spacer sequence, as well as bystander RNAs in a sequence-unspecific manner. Due to cleavage of cellular transcripts they induce dormancy in the host cell and thus protect the bacterial population by aborting the infectious cycle of RNA-phages. Here we report the structural and functional characterization of a Cas13a enzyme from the photo-auxotrophic purple bacteria Rhodobacter capsulatus. The X-ray crystal structure of the RcCas13a-crRNA complex reveals its distinct crRNA recognition mode as well as the enzyme in its contracted, pre-activation conformation. Using site-directed mutagenesis in combination with mass spectrometry, we identified key residues responsible for pre-crRNA processing by RcCas13a in its distinct catalytic site, and elucidated the acid-base mediated cleavage reaction mechanism. In addition, RcCas13a cleaves target-RNA as well as bystander-RNAs in Escherichia coli which requires its catalytic active HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activity. Our data provide further insights into the molecular mechanisms and function of this intriguing family of RNA-dependent RNA endonucleases that are already employed as efficient tools for RNA detection and regulation of gene expression.

Comprehensive comparison between azacytidine and decitabine treatment in an acute myeloid leukemia cell line.

Azacytidine (AzaC) and decitabine (AzadC) are cytosine analogs that covalently trap DNA methyltransferases, which place the important epigenetic mark 5-methyl-2'-deoxycytidine by methylating 2'-deoxycytidine (dC) at the C5 position. AzaC and AzadC are used in the clinic as antimetabolites to treat myelodysplastic syndrome and acute myeloid leukemia and are explored against other types of cancer. Although their principal mechanism of action is known, the downstream effects of AzaC and AzadC treatment are not well understood and the cellular prerequisites that determine sensitivity toward AzaC and AzadC remain elusive. Here, we investigated the effects and phenotype of AzaC and AzadC exposure on the acute myeloid leukemia cell line MOLM-13. We found that while AzaC and AzadC share many effects on the cellular level, including decreased global DNA methylation, increased formation of DNA double-strand breaks, transcriptional downregulation of important oncogenes and similar changes on the proteome level, AzaC failed in contrast to AzadC to induce apoptosis efficiently in MOLM-13. The only cellular marker that correlated with this clear phenotypical outcome was the level of hydroxy-methyl-dC, an additional epigenetic mark that is placed by TET enzymes and repressed in cancer cells. Whereas AzadC increased hmdC substantially in MOLM-13, AzaC treatment did not result in any increase at all. This suggests that hmdC levels in cancer cells should be monitored as a response toward AzaC and AzadC and considered as a biomarker to judge whether AzaC or AzadC treatment leads to cell death in leukemic cells.

A substance P antagonist, [D-Pro2, D-Trp7,9]SP, inhibits inflammatory responses in the rabbit eye.

Neurogenic factors released by antidromic nerve stimulation are thought to be in part responsible for the vasodilation and breakdown of the blood-aqueous barrier that follows trauma to the eye. Substance P is one candidate for the mediation of the inflammatory response since it is thought to be a neurotransmitter in sensory afferents and since exogenous substance P is capable of eliciting a response characteristic of inflammation. In rabbits, intravitreal or topical application onto the eye of a specific substance P antagonist, [d-Pro2, D-Trp7,9]SP, inhibited not only the irritant effects of exogenous substance P but also the inflammatory response to a standardized trauma (infrared irradiation of the iris). These observations suggest that substance P, or a related peptide, is a neurogenic mediator of the inflammatory response in the eye.

Evidence for an involvement of substance P, but not cholecystokinin-like peptides, in hexamethonium-resistant intestinal peristalsis.

It has previously been found that, in the presence of naloxone, the ganglionic blocking drug hexamethonium fails to completely block peristaltic motility in the isolated ileum of the guinea-pig. This hexamethonium-resistant peristaltic activity is coordinated by enteric nerves since it is abolished by tetrodotoxin. In the present study the neurotransmitter circuitry of this type of peristalsis was studied by means of specific antagonists. Atropine totally suppressed hexamethonium-resistant peristalsis. This type of peristalsis was also strongly inhibited by the tachykinin antagonist, spantide, if a concentration sufficient to antagonize neuronally located substance P receptors was employed. In contrast, the cholecystokinin antagonist, lorglumide, caused only a slight inhibition of hexamethonium-resistant peristalsis. Both substance P and the cholecystokinin-related peptide, ceruletide, potently stimulated the hexamethonium-resistant type of peristaltic activity. These data indicate that, after blockade of nicotinic acetylcholine receptors, tachykinins mediate neuroneuronal coordination of peristalsis whereas acetylcholine acting via muscarinic receptors may be primarily responsible for neuromuscular transmission. Cholecystokinin-like peptides appear to play a modulator rather than a mediator role in hexamethonium-resistant peristalsis.

The mechanism of action of a substance P antagonist (D-Pro2, D-Trp7,9)-SP.

1 A newly synthesized substance P (SP) analogue, (D-Pro2, D-Trp7,9)-SP, specifically antagonizes the contractile effects of SP on the guinea-pig isolated taenia coli. In addition, previous studies had indicated that the SP analogue per se is capable of contracting this preparation. The results of the present study on the guinea-pig taenia suggest that the smooth muscle contractions produced by the SP analogue are due to histamine release. No contractions were observed following blockade of histamine H1-receptors by mepyramine or following pretreatment with the histamine liberating agent, compound 48/80. 2 Analysis of the inhibition of SP-induced contraction by the analogue suggests that the inhibition is of the competitive type; pA2 was calculated to be 6.1. 3 We conclude that (D-Pro2, D-Trp7,9)-SP is a competitive SP antagonist with histamine-releasing properties.

A tachykinin antagonist inhibits gastric emptying and gastrointestinal transit in the rat.

The effect of a substance P antagonist, [D-Pro2, D-Trp7,9]-substance P (SPA), on gastric emptying and gastrointestinal transit in the rat was studied in order to elucidate a possible physiological role of endogenous substance P and other tachykinins in gastrointestinal motility. SPA was given by intraperitoneal injection concurrently with the intragastric administration of a test meal containing charcoal and 51Cr. Examination 15 min after the test meal showed that SPA (0.13-1.3 mumol kg-1) inhibited gastric emptying and gastrointestinal transit in a dose-dependent manner. The inhibitory effect of SPA on gastric emptying and gastrointestinal transit remained unchanged after pretreatment of rats with mepyramine (8.7 mumol kg-1) plus cimetidine (19.8 mumol kg-1) or with guanethidine (67 mumol kg-1). Since a full examination of SPA as a specific tachykinin antagonist was not possible in vivo, SPA was also tested on circular muscle strips from the rat gastric corpus in vitro. Submaximal contractions in response to bombesin or bethanechol were not reduced by SPA (50 microM), whereas those in response to substance P were inhibited. The results suggest that SPA inhibits gastric emptying and gastrointestinal transit by interfering with the action of tachykinins released from enteric nerves and that endogenous tachykinins are involved in the regulation of gastrointestinal motility.

Biological evaluation of substance P antagonists.

Five undeca- and six C-terminal heptapeptide substance P (SP) analogues were tested for their capacity to block the contractile effect of SP on the guinea-pig isolated taenia coli. They had one feature in common, namely substitutions in positions 7 and 9 in the SP molecule. In the majority of analogues D-tryptophan was used for these substitutions. All analogues tested were found to be competitive antagonists to exogenous SP and to be capable of blocking the electrically induced non-cholinergic, non-adrenergic neuronal contraction of the taenia. Of the undecapeptides, (D-Arg1, D-Pro2, D-Trp7,9, Leu11) SP and (D-Arg1, D-Trp7,9, Leu11) SP (Spantide) had the highest pA2 value, 7.1-7.2, and the lowest IC50 value, 10(-6) M. The pA2 values of the heptapeptides were generally lower. Three of the most potent antagonists were tested for specificity and found to block the smooth muscle contraction induced by SP, physalaemin, eledoisin and bombesin but not that induced by bradykinin, carbachol, 5-hydroxytryptamine, histamine, prostaglandins and vasopressin. The SP antagonists were also tested for spasmogenic effect on the taenia and for their capacity to release histamine from rat isolated peritoneal mast cells. The spasmogenic activity displayed by most of the SP antagonists tested is likely to be related to their ability to release histamine since the contractile response was reduced by mepyramine, a histamine H1-receptor antagonist. (D-Arg1, D-Trp7,9, Leu11) SP was notable for combining a high antagonistic potency with a weak spasmogenic effect (and poor histamine releasing effect).

Decreasing hospitalization rates for older home care patients with symptoms of depression.

OBJECTIVE: To target medically ill older home care patients with symptoms of depression in order to reduce their rate of hospitalization. DESIGN: A case-control study. SETTING: A private, nonprofit home care organization, the Visiting Nurse Association of St. Louis. PARTICIPANTS: Home care patients 65 years of age and older with symptoms of depression who were participants of a Total Quality Management (TQM) intervention (n = 81) were compared with an historical control of home care patients 65 years of age and older with symptoms of depression (n = 69). INTERVENTION: Utilization of TQM principles to develop a plan including: (a) an educational seminar on depression for home care staff involved in the project; (b) letters to physicians introducing the TQM project; (c) use of the Geriatric Depression Scale (GDS) for screening; (d) recommendation to the primary physician of a home social service (SS) consultation for patients with a GDS of 10 to 14; (e) recommendation to the primary physician of three interventions for patients with a GDS > or = 15: home SS consultation + mental health (MH), or gerontological nurse (GN) consultation + antidepressant medication (a pharmacotherapeutic algorithm sent by facsimile to the primary physician upon request). OUTCOME MEASURES: Hospitalization rates of the control group compared with the TQM intervention group, the degree to which part (e) of the plan was implemented, and the effect this had on hospitalization rates. RESULTS: The TQM intervention patients had a higher mean age than the historical control patients but were not different in percent female, percent white race, percent with a caregiver in the home, functional status, and in 15 of 16 diagnostic categories. Overall, the TQM intervention group had a hospitalization rate of 23.5% (19/81) compared with a rate of 40.6% (28/69) for the historical control group (P = .024). For part (e) of the plan (56/81 patients had a GDS > or = 15), 29/56 (52%) received the recommended SS consultation, 50/56 (89%) received the recommended MH or GN consultation, and 32/56 (57%) received antidepressant medication. One type of intervention did not seem to lower hospitalization rates more than another although having received the MH or GN visits approached significance (12/50, 24%; P = .052) when compared with the control group. CONCLUSIONS: Utilization of TQM principles and the development of an intervention such as the one described here can decrease hospitalization rates for medically ill older home care patients with symptoms of depression.

Spantide II, a novel tachykinin antagonist having high potency and low histamine-releasing effect.

Two undecapeptide substance P (SP) analogues, Spantide I and Spantide II, were tested for their capacity to block the contractile effect of SP on the guinea pig isolated taenia coli and the contractile effect of electrical stimulation of the rabbit isolated (and atropinized) iris sphincter, and for their capacity to mobilize histamine from rat isolated peritoneal mast cells. Spantide I and Spantide II have one feature in common, namely D-tryptophan in positions 7 and 9. Spantide I: D-Arg, Pro2, Lys3, Pro4, Gln5, Gln6, D-Trp7, Phe8, D-Trp9, Leu10, Leu11-NH2. Spantide II: D-NicLys1, Pro2, 3-Pal3, Pro4, D-Cl2Phe5, Asn6, D-Trp7, Phe8, D-Trp9, Leu10, Nle11-NH2. Both Spantide I and II were found to be competitive antagonists to SP on the taenia coli and to be capable of blocking the electrically induced non-cholinergic contraction of the iris sphincter. Spantide II had higher pA2 value (taenia coli) than Spantide I, 7.7 versus 7.0, and higher pIC50 value (blockade of tachykinin-mediated neurotransmission in iris sphincter), 6.0 versus 5.1. Both Spantide I and II mobilized histamine from rat peritoneal mast cells but Spantide II was less effective. Spantide I and II were tested for antagonistic specificity. Both blocked contractions of the taenia induced by SP and neurokinin A. In the concentration used, Spantide II in addition blocked the response to neurokinin B. The contractions induced by carbachol, 5-hydroxytryptamine, histamine and prostaglandins (F2 alpha and E1) were not affected; the contractile response to bombesin was inhibited by Spantide I but not by Spantide II.

Neurogenic relaxation mediated by vasoactive intestinal polypeptide (VIP) in the isthmus of the human fallopian tube.

The smooth musculature of the Fallopian tube is important for normal ovum transport, fertilization and implantation. Little is known about the factors controlling the motor activity of the isthmic sphincter. Studies were performed on smooth muscle preparations from the human tube in vitro. Electrical field stimulation of the nerves in the isthmic region reduced the motor activity, particularly in the circular muscle. The response was unaffected by adrenergic and cholinergic antagonists, but blocked by tetrodotoxin, suggesting a neural involvement. Vasoactive intestinal polypeptide (VIP) was considered a likely candidate for the neural mediation of this response in view of the high density of VIP-containing nerve fibres in this region, and in view of the fact that exogenous VIP causes a marked reduction of the tubal motor activity. To test whether VIP might be the endogenous mediator of this effect, nerve stimulation was carried out in the presence of large amounts of exogenous VIP in order to occupy all VIP receptors; the motor inhibitory action of VIP was counteracted by vasopressin. Under these conditions, nerve stimulation failed to reduce isthmic motor activity. This was not due to vasopressin since reduction occurred in the presence of this peptide alone. The results suggest that VIP is responsible for the neurogenic inhibition of motor activity in the isthmus region of the human Fallopian tube.

Immunohistochemical localization of substance P, vasoactive intestinal polypeptide and gastrin-releasing peptide in vas deferens and seminal vesicle, and the effect of these and eight other neuropeptides on resting tension and neurally evoked contractile activity.

Immunohistochemical studies of the vas deferens and seminal vesicle of mouse, guinea-pig, and rabbit showed the presence of nerve fibres containing vasoactive intestinal polypeptide (VIP), substance P (SP), and gastrin-releasing peptide (GRP) supplying the smooth muscle layers as well as blood vessels. The nerve supply was better developed in the seminal vesicle than in the vas deferens. The motor activity of the vas deferens and seminal vesicle of the guinea-pig was studied in vitro. The vas deferens responded to transmural electrical stimulation with a twitch followed by a slow contraction. The twitch was blocked by guanethidine and tetrodotoxin, but not by atropine, propranolol, phenoxybenzamine, or fluphenazine. The slow contraction exhibited features of an alpha-receptor-mediated response. SP, physalaemin and eledoisin contracted the smooth muscle and also potentiated the twitch response to electrical nerve stimulation in a concentration-dependent manner. The SP blocking agent, (D-Pro2,D-Trp7,9)-SP, affected neither the resting tension nor the response to electrical stimulation. It is therefore suggested that the SP fibres act mainly prejunctionally. VIP, Leu-enkephalin, cholecystokinin octapeptide (CCK-8), angiotensin II, vasopressin, neurotensin, bombesin, and GRP had no effect on either the resting tension or the response to electrical nerve stimulation. The seminal vesicle responded to electrical stimulation with a contraction which was unimpaired by atropine, propranolol, phenoxybenzamine, and guanethidine, but abolished by tetrodotoxin. Hence, this contraction is mediated by a non-adrenergic, non-cholinergic neurotransmitter. Bombesin, GRP, SP, physalaemin and eledoisin contracted the smooth muscle and potentiated the response to electrical stimulation. VIP, Leu-enkephalin, CCK-8, angiotensin II, vasopressin, and neurotensin had no effect on the resting tension or on the response to transmural electrical stimulation. The SP antagonist abolished the contraction elicited by SP but did not influence the response to nerve stimulation. The results suggest that the SP and GRP nerves may have prejunctional and facilitating postjunctional effects in the seminal vesicle.

Blockade of sensory nerve mediated contraction of the rabbit iris sphincter by a series of novel tachykinin antagonists.

Electrical stimulation of the isolated rabbit iris sphincter muscle in the presence of atropine gives rise to a contraction that can be blocked by tachykinin antagonists. The ability of a series of novel tachykinin antagonists to inhibit the contractile effect of SP on the guinea-pig taenia coli and to suppress the electrically evoked contraction of the atropinized rabbit iris sphincter was tested. Several of the novel antagonists were found to be more potent in terms of pA2 and pIC50 values than the two previously described analogs, [D-Pro2, D-Trp7,9]SP9(1-11) and [D-Arg1, D-Trp7,9, Leu11]SP-(1-11) (Spantide). Apart from D-Trp in positions 7 and 9 the characteristic features of the potent novel antagonists were D-Cl2Phe (or D-Cys(Bzl] in position 5, Asn in position 6 and Nle in position 11. In addition Pal in position 3 seemed to offer an enhanced potency.

Increased potency of antagonists of substance P having asparagine in position 6.

The general structure of antagonists of substance P (SP) which was found with the development of Spantide and analogs based on Spantide served for further refinement. The antagonistic potency was tested in vitro on guinea pig ileum and taenia coli. It was unexpectedly found that introduction of Asn6 gave rise to a considerable increase in potency. The exchange of Gln6 for Asn6 entails the shortening of the side chain by one CH2 unit and seems slight for steric advantages and potency increase. The analog [D-Arg1,D-Cl2Phe5,Asn6,D-Trp7,9,Nle11]SP had pA2 values of 7.4 (ileum) and 8.0 (taenia coli). We then used this sequence as a new lead to introduce new changes, which were made in positions 1, 3, 5, 7 and 9. It was found that Arg1 is important, but Lys3 can be exchanged. The Pal3 derivative had pA2 values of 8.1 and 8.0 and the Nle3 counterpart had 7.7 and 7.4 D-Cl2Phe is an effective substituent in position 5. D-Trp in positions 7 and 9 were superior to other alternatives.

Effects of 4-aminopyridine on mechanical activity and noradrenaline release in the rat portal vein in vitro.

4-Aminopyridine (4-AP) increased the spontaneous mechanical activity of the isolated rat portal vein. Since denervation and adrenergic receptor blockade failed to prevent this effect of 4-AP it is suggested that the drug enhances the electrical excitability of the muscle membrane. 4-AP significantly increased the response of the muscle to electrical nerve stimulation in most experiments but had little effect on the response to applied noradrenaline (NA). Both spontaneous and evoked release of 3H-activity, following preincubation in 3H-noradrenaline, were increased in the presence of 4-AP (10(-3) M). The present results with 4-AP can be explained by its known ability to block the transient potassium conductance which accompanies the action potential in excitable tissues.

Effects of 4-aminopyridine on insulin secretion and plasma glucose levels in intact and adrenalectomized-chemically sympathectomized mice.

The effects of 4-aminopyridine (4-AP) on basal and glucose-induced insulin secretion and on plasma glucose concentrations were investigated in vivo in intact mice and in mice subjected to surgical adrenalectomy plus chemical sympathectomy induced by 6-hydroxydopamine. In normal intact mice, an i.v. injection of 4-AP, 26 micro mol/kg, induced an elevation of plasma glucose concentrations from 6.4 +/- 2 to 10.0 +/- 0.3 mmol/1 (P less than 0.001) seen after 30 min. Thereafter the plasma glucose concentration gradually returned to the normal level. Despite this marked elevation of plasma glucose levels, no change in plasma insulin concentrations was seen. In intact normal mice, the insulin secretion induced by a half-maximal dose of glucose was partially inhibited by 4-AP, 0.26 micro mol/kg. No further inhibition was observed after a larger dose, 26 micro mol/kg. In adrenalectomized-chemically sympathectomized mice, 4-AP, 26 micro mol/kg, did not affect plasma glucose concentrations or plasma levels of insulin. In these animals, 4-AP potentiated glucose-induced insulin secretion by approximately 65% at the two dose levels studied. This potentiation of glucose-induced insulin secretion was not affected by muscarinic receptor blockade. In summary, 4-AP increased plasma glucose levels in normal intact mice, but had no effect on plasma glucose levels in adrenalectomized-chemically sympathectomized mice. In addition, 4-AP inhibited glucose-induced insulin secretion in normal intact mice, but potentiated glucose-induced insulin secretion in adrenalectomized-chemically sympathectomized mice. It is concluded that the effect of 4-AP on insulin secretion and plasma glucose levels in normal mice is exerted indirectly, through stimulation of the sympatho-adrenal system. Further, from the results obtained in animals deprived of their sympatho-adrenal system it is suggested that the drug also has the capability to stimulate insulin secretion by acting directly on the insulin secreting cells.

Gastrins and cholecystokinins release acetylcholine but not substance P from neurons in the guinea-pig taenia coli.

Gastrins and cholecystokinins contract the isolated taenia coli of the guinea-pig. Porcine CCK-39 produced the greatest contractile response and human gastrin-17 I and -34 the weakest. Pentagastrin had the highest affinity to the receptors and non-sulphated CCK-8 the lowest. The contractions produced by the CCK peptides were reduced by the neuronal blocker tetrodotoxin and by the muscarinic blocker atropine but not by the substance P antagonist [D-Pro2,D-Trp7,9]SP. It is concluded that gastrin/CCK peptides act directly on smooth muscle cells and that in addition these peptides, notably sulphated forms of CCK, are capable of exciting cholinergic neurons (but not SP neurons) to cause smooth muscle contraction.

Bradykinin contracts the pupillary sphincter and evokes ocular inflammation through release of neuronal substance P.

Bradykinin contracts the isolated rabbit sphincter pupillae muscle. The contraction produced by 10(-8) M bradykinin was resistant to atropine but not to tetrodotoxin, suggesting a non-cholinergic nervous mechanism. The contraction was blocked by specific substance P (SP) antagonists, suggesting the involvement of SP. The SP antagonists tested were [D-Pro2,D-Trp7,9]SP-(1-11) and [Arg5,D-Trp7,9]SP-(5-11). The bradykinin-induced contraction exhibited marked tachyphylaxis in contrast to that induced by SP. It appears that the tachyphylaxis reflects the depletion of a bradykinin-sensitive neuronal pool of SP. Injection of bradykinin into the vitreous chamber of the rabbit eye caused miosis and disruption of the blood-aqueous barrier (manifested as aqueous flare). A second administration of bradykinin a few hours after the first injection evoked a reduced response; the response to SP upon repeated administration was unchanged. Atropine was without effect on the response to bradykinin whereas tetrodotoxin and the SP antagonists reduced the response. The results suggest that bradykinin causes miosis and aqueous flare at least partly through local release of neuronal SP.

Antagonism of the paralysis produced by botulinum toxin in the rat. The effects of tetraethylammonium, guanidine and 4-aminopyridine.

The injection of botulinum toxin type A into the hind-leg of adult rats causes complete paralysis of the leg lasting for several weeks. In the extensor digitorum longus (EDL) muscle transmitter release is reduced to a level of less than 1% of normal. Tetraethylammonium (TEA) and guanidine in concentrations of about 3 mM restore, in EDL muslces in vitro, neuromuscular transmission to about the normal level, provided that the external calcium concentration is 4 mM or higher. 4-Aminopyridine (4-AP) has similar restorative effect but is about 20-30 times more potent. Unlike TEA and guanidine, 4-AP is effective when the ambient calcium concentration is 2 mM; this drug is therefore also active in vivo. The intravenous injection of 4-AP (5 mg/kg body weight) restores neuromuscular transmission from complete paralysis by botulinum toxin to a normal level as shown by the recording of almost normal twitch and tetanic tensions in the EDL muscle. In rats paralysed by a lethal dose of botulinum toxin, the intraperitoneal administration of 4-AP restores general motor activity, the effect lasting 1-2 hours. A study of the effects of these drugs on spontaneous and evoked transmitter release suggests that all three compounds increase the level of free calcium inside the nerve terminals. In botulinum poisoning the transmitter release mechanism appears to be intact, but a reduced sensitivity to calcium has been shown (Cull-Candy et al. 1976), and this could explain why the drugs restore evoked transmitter release in botulinum poisoning.

Hirschsprung's disease: a comparison of the nervous control of ganglionic and aganglionic smooth muscle in vitro.

Specimens from aganglionic (constricted) and ganglionic (dilated) gut were obtained from nine patients with Hirschsprung's disease. Transmural nerve stimulation of ganglionic smooth muscle in vitro evoked an initial relaxation followed by a contraction. This contraction was reduced but not abolished by atropine and it was further reduced by substance P antagonists. Guanethidine did not affect the electrically evoked responses. In aganglionic smooth muscle, an atropine-sensitive contraction but no initial relaxation was registered. Tetrodotoxin abolished all responses to electrical stimulation in both ganglionic and aganglionic specimens. Application of carbachol or substance P produced contraction and the adrenergic agonist isoprenaline or vasoactive intestinal peptide produced relaxation in ganglionic as well as aganglionic specimens. Two other gut neuropeptides, neuropeptide Y and galanin, were without effect. The results do not indicate a different receptor set up in ganglionic v aganglionic gut. The results are compatible with a lack of noncholinergic nonadrenergic inhibitory neurons in the aganglionic gut.