Intestinal peristalsis associated with release of immunoreactive substance P

hkms&ttee Vof. fl, No. 4,pp.913-918,1984

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0304~522~84$3.~iO.~ Pergamon Press Ltd 0 1984IBRO

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INTESTINAL PERISTALSIS ASSOCIATED WITH RELEASE OF IMMUNOREACTIVE SUBSTANCE P J. DONNERER, L. BARTH& P. WOLZFR and F. LIIMBECK Department of Experimental and Clinical Pharmacology, University of Graz, UniversitZtsplatz 4, A-8010 Graz, Austria

Abstract-The release of immunoreactive substance P into the vascular bed of the isolated small intestine of the guinea-pig was investigated. Raising the intraluminal pressure to Smbar for 5 min initiated peristalsis and stimulated the release of substance P. The substance P releasing effect of pressure stimulation was reduced by 46% when hexamethonium (240/rM) was added to the perfusion solution. The ganglion stimulant drug dimethyiphenylpi~ra~nium (32 CM) also stimulated the release of substance P; its effect was completely prevented by hexame~onium (2~~M). Intraarterial infusion of capsaicin (22 PM), a neurotoxin known to act on sensory substance P-containing neurones, stimulated the release of substance P and caused intestinal contractions. The motor effect of capsaicin in the gut can thus be explained by release of substance P from sensory nerve endings in the gut. Systemic pretreatment of the guinea-pigs with capsaicin abolished the release of substance P due to capsaicin, whereas that evoked by elevated intraluminal pressure or dimethylphenylpiperazinium was not reduced. This means that substance P released in the course of peristalsis or by dimethylphenylpiperazinium originates from neurones intrinsic to the intestine. These findings indicate that intestinal peristalsis is associated with the release of substance P from enteric neurones. Substance P is likely to be a neurotransmitter involved in the coordination of the peristaltic reflex.

P (SP)-immunorea~tive neurons have been localized to ail layers of the guinea-pig small intestine,‘0J’*26,4’ The majority of these neurons are intrinsic to the intestine.“*” However, nerve fibres around blood vessels and some fibres in the submucous plexus are of extrinsic, probably sensory, origin, since they disappear after extrinsic denervation of the gutlO.” or after systemic treatment with the sensory neurotoxin capsaicin. Is Electrophysiological*7~33 and pharmacologicals~8~‘2*25~34 observations indicate that SP is released from enteric neurons in response to various stimuli. Several lines of evidence suggest that SP is involved in both neuroneuronal and neuromuscular transmi~ion within the intestinal wall: (i) Seven distinct projections of SP-containing neurons have been identified in the gut.” (ii) SP is capable of exciting both intestinal muscle7~‘2~‘4~40~42 and enteric neurons.2’*** (iii) SP releases acetylcholine2’~4g and stimulates peristalsis via activation of cholinergic neurons.24 Recent research in this laboratory has estabiished that SP plays an important role in the atropineresistant excitatory pathway involved in the peristaltic reflex.3*4In the present study we have examined whether intestinal peristalsis is associated with a release of imm~oreactive SP into the vascular bed of the intestine. We also tried to obtain information as Substance

to whether the released SP originates from intrinsic or extrinsic neurons. The possibility of SP release into the vascular system seemed to be plausible because in the cat, in uiuo, the intestine is likely to be a major source of circulating SP.”

EXPERIMENTAL PROCEDURES Tissue preparation

Adult guinea-pigs, of either sex (300-SOOg) and fasted overnight, were anaesthetized with sodium pentobarbitone (Wmg kg-’ i.p.) and kept on artificial respiration. The abdomen was opened by a midline incision, the mesenteric artery quickIy cannulated and perfused with Tyrode solution at 37°C (for details see below). After the portal vein had also been cannulated the whole small intestine was removed from the animal and placed in an organ bath containing oxygenated Tyrode solution at 37°C. The oral and aboral ends of the intestine were connected to inflow and outflow cannulae respectively. The lumen of the intestine communicated, via the inflow cam&a, with a Mariotte bottle containing Tyrode solution to allow adjustment of the intraluminal pressure. The set-up for eliciting peristalsis has been described in detail bv Holzer and Lembeck.20 In one set of experiment; guinea-pigs were pretreated with a total dose of 62 mg kg-’ capsaicin. They received successive subcutaneous injections of 2, 10 and 50mg kg-’ capsaicin on the 6th, 5th and 4th day prior to the experiment, respectively. Vehicle for capsaicin was 10% ethanol, 10% Tween 80 in saline (IOmgml-’ capsaicin). Ten minutes before each capsaicin or vehicle injection the animals were anaesthetized with a combination of sodium pentobarbitone (25 mg kg-’ i.p.), haloperidol(5 mg kg-’ i.p.) and lofentanil (I pg kg-’ i.p.).

Abbreviations:

DMPP, dimethylphenylpiperazinium; i.a., intraarterial; I-SP, immunoreactive substance P, SP, substance P. 913

014

J. Donnrrer (‘I II/.

Experimentalprorocol Vascular perfusion of the small intestine was performed with oxygenated Tyrode solution (for composition see Holzer and Lembeck”). The Tyrode solution also contained O.lo/, bovine serum albumin to reduce adsorption of SP to surfaces, 20/1M bacitracin to protect SP from enzymatic cleavage,” and 1.5 p M naloxone to overcome the inhibitory influence of endogenous opioids on the release of SP.‘.” The perfusion rate was maintained at I.5 ml min ’ by means of a peristaltic pump. The preparation was allowed at least IOmin for equilibration. Then 1.5 ml fractions of the venous et&ate were collected into tubes standing on ice and containing glacial acetic acid to give a final concentration of 2 M acetic acid. Immediately after collection the samples were frozen and freeze-dried. After five fractions were collected to observe the basal SP release, peristalsis was evoked by raising the intraluminal pressure to 5 mbar for 5 min. The venous effluate during these and the following 5 min was also collected. Then the intestine was allowed 20 min to recover before the next stimulation was performed. This and one further stimulation consisted of intraarterial (i.a.) infusions of capsaicin, dimethylphenylpiperazinium. or 108 mM K+ (isotonically replacing Nat ). At the end of the experiment the small intestine was freed of the mesentery. blotted and weighed. Radioimmunoassay

The SP content of the samples was determined by radioimmunoassay according to the protocol of Mroz and Leeman.36 The antiserum used, Rd,, is directed towards the C-terminal part of SP” and shows full cross-reactivity with SP sulfoxide.*’ The detection limit of the radioimmunoassay

was I4 pgjsample corresponding to 12””displacement of the tracer. None of the drugs used in the experiments interfered with the assay. Since the nature of the immunoreactivity uas not further characterized, it is referred to as immunoreactive SP (I-SP). The amount of I-SP released was calculated as pg;min and g wet wt. For quantitative evaluation of drug effects. the total evoked release was calculated as the “sum of I-SP released by the stimulus minus the mean release of the two fractions preceding the stimulus.” Materials

Capsaicin (Merck), dimethylphenylpiperazinium (Aldrich), hexamethonium (Fluka), haloperidol. lofentanil (Janssen), substance P (Peninsula).

RESULTS Pressure-induced perbtalsis The basal release of I-SP was sometimes below, sometimes just above the detection limit of the radioimmunoassay (corresponding to about 4 pg g-’ min- ‘). Raising the intraluminal pressure to 5 mbar for 5 min initiated peristalsis (see Holzer and Lembeckm) and caused the appearance of I-SP in the venous effluate. The release of I-SP outlasted the pressure stimulus for some minutes (Fig. I). When the ganglion blocking drug hexamethonium (24OpM) was infused i.a., the infusion starting

32 p’M DMPP

Fig. I. Time course of the release of I-SP into the venous cffluate colIectcd from the vascularly perfu%%l, isolated small intestine of the guinea-pig. The dotted horizontal line indicates the detection limit of the

radioimmuaoiuaay; a meamuaW bout &+e of I-SP was seen in only a few experiments. DMPP, dilnethylj&aly~um; ~OllaMK+,46tisaesthcnormalK+~;CAP,~n.The infusion of apt&In was pnxz~W by infusion of its solvent (1%ethanol, 1%T\veen 80 in satin@ kr 3 min. Means f S.E.M., n as indicated.

Substance P release from guinea-pig intestine Table

I. Total

release of I-SF from the vascularly perfused, isolated small intestine of

the guinea-pig .-~~I..5 mbar intraluminal pressure Untreated control Hexamethonium infusion (240 p M) Systemic vehicle pretreatment Systemic capsaicin pretreatment Capsaicin infusion (22 $M) Untreated control Hexamethoaium infusion (240 $vff Systemic vehicle pretreatment Systemic eapsaicin pretreatment Dimethyfphenylpiperazinium infusion (32 PM) Untreated control Hexamethouium infusion (240 fr M) Systemic capsaicin pretreatment

pgg-’

I-SP

n

l12&4 60&g*

15 5

lOOi:19 93 i: 18

:

75214 74* 11

12 6

80*11 22 1*

5 7

117~12 4+2’ 126 & 20

23 8 6

Means f S.E.M., *P < 0.05, two sample r-test. The total evoked release was calculated as the sum of I-SP released by the stimulus minus the mean rehase of the 2 fractions preceding the stimulus. Systemic capsaicin treatment: se. injections of 2, 10, and 50 mg kg-’ capsaicin on the 61,5th and 4th day prior to the experiment. Systemic vehicle pretreatment: se. injections of 10% ethanol too/, Tween 80 in saline in volumes equivalent to those used for systemic capsaicin pretreatment.

fOmi~ before pressure stimulation, the release of I-SP was s~~i~~~tly (P c 0.05, r-test) reduced by 46% (Table I) and almost no propulsive peristalsis was observed. The release of I-SP was not si~fi~ntly reduced when the ~inea-pigs had been pretreated with capsaicin or its vehicle (Table 1). Caps&in I.a. infusion of capsaicin (22 @5) far 3 min produced intestinal contractions and stimulated the release of I-SP (Fig. 1). Hexamethonium (240 PM) had no effect on the I-SP release caused by capsaicin infusion. However, when the guinea-pigs from which the intestine was obtained had been systemically pretreated with capsaicin, La. infusion of capsaicin (22 ,uM) was no longer capable of evoking peristalsis or stimulating the release of I-SP (Table 1).

La. infusion of the ganglion stimulant drug dimethylphenylpi~~inium (DMPP, 32 FM) for 3 min produced a large increase in the I-SP content of the venous outflow (Fig. 1). The motor response to DMPP was muscle spasm rather than coordinated peristalsis. La. infusion of 24OgM he~amethonium (starting 10 mm before the infusion of DMPP) completely prevented the stimulation of I-SP release by DMPP whereas systemic pretreatment of the guineapigs with capsaicin was without effect (Table 1). 108 mM K+ Raising the K* concentration of the perfusion solution by a factor of 40 (by isosmotie replacement of Na’) for 5 min stimulated the venous outflow of I-SP most effectively (Fig. 1). The accompanying motor response was an uncoordinated spasm of the intestinal muscle.

The present findings show that stimuli which increase the motor activity of the small intestine result in a release of I-SP into the vascular bed. In the resting intestine I-SP was usually not detectable in the venous effluate, most likely because the basal release of I-SP from intramural neurons was too small to result in measurable concentrations of I-SP in the perfusion medium. Possible sources of the released I-SP are intestinal SP-containing neurons aad mucosal endocrine cell~.“~~~It is, however, likely that the bulk of the released I-SP originated from neurons, since the I-SP content of the mucosa is very small as compared with that of the whole wall of the guineapig small intestine.24 The most important finding was that a rise in intraluminal pressure, which is the adequate stimulus for triggering peristalsis,3o increases the release of I-SP. This corroborates the concept that substance P is one of the excitatory neurotransmitters that mediate the peristaltic retlex, at least the atropine-r~istant pathway of peristalsis. 3,4Interestingly, the release of dynorphinr’ and methionine-enkephalin,9 which are possibly involved in the peristaltic refIex as inhibitory neurotrans~tt~~ has been found to be suppressed during peristaltic activity. The notion based on pharmacological evidence” that the ganglionic stimulant DMPP releases SP in the gut has been directly confirmed in this study. The ganglio~c blocking drug hexamethonium totally prevented the release of I-SP induced by DMPP whereas it reduced the release associated with pressure stimulation by only about 50%. This might indicate that part of the enteric I-SP release during peristaltic activity is triggered by endogenous acetylcholine acting at nicotinic receptors on SP-releasing neurons. I3 The pressure-induced release of I-SP remaining in the presence of heK~ethonium

916

J. Donnerer cv ul.

may be explained in more than one way. (i) Since the same dose of hexamethonium virtually abolished peristaltic activity it seems possible that I-SP is released from at least two different synapses of the peristaltic reflex, i.e. from a pre- and postganglionic site. (ii) Some intramural SP neurons might be activated by a non-cholinergic excitatory input. (iii) Possibly some of the intestinal SP neurons respond directly to intestinal distension. Systemic capsaicin treatment was used to solve the question as to whether I-SP was released from neurons intrinsic or extrinsic to the intestine. Capsaicin has become established as a specific neurotoxin for primary sensory neurons, particularly for those containing SP (for reviews see Lembeck and Gamse,” Nagy,37 SzolcsLnyi4). In the intestine, SP-containing neurons of extrinsic (presumably sensory) origin are functionally and morphologically impaired by capsaicin pretreatment.‘.” The SP-containing neurons intrinsic to the intestine are, however, not affected by capsaicin treatment.‘.“.“.‘“.” The finding that the I-SP release induced by pressure stimulation was not reduced after systemic pretreatment with capsaicin indicates that the I-SP released originated mainly from neurons intrinsic to the intestine. The same was true for the I-SP release induced by the infusion of DMPP. These data are in line with the well-accepted concept that the peristaltic reflex of the intestine is coordinated by the intrinsic nerve plexuses (see Kosterlitz and Lees’“). Acute administration of capsaicin excites primary sensory neurons and causes the release of I-SP from these neurons.‘6.‘*.29.43.47.48 In the isolated small intestine, acute administration of capsaicin causes contraction’,4s and facilitates the peristaltic reflex.46 These motor effects seem also to be due to release of

SP from primary sensory nerve endings.’ ‘: The present result that capsaicin induced the release of I-SP into the venous effluate presents direct evidence for this notion. In preparations obtained from capsaicin-pretreated guinea-pigs i.a. infusion of capsaicin was no longer capable of releasing I-SP, a finding that strongly suggests that the I-SP released by capsaicin originated from sensory nerve endings in the gut. The I-SP releasing effect of capsaicin was not affected by hexamethonium, which is in line with the observation of Barth6 and Szolcsanyi’ that the contractile effect of capsaicin in the gut is not inhibited by ganglion blocking drugs either. Overall depolarization by increasing the extracellular K’ concentration was most effective in stimulating the release of I-SP. It is likely that the I-SP released by 108 mM K+ originated from both intrinsic and extrinsic SP-containing neurons. Conclusions

This paper has shown that intestinal peristalsis elicited by raising the intraluminal pressure is associated with the release of I-SP into the vascular bed of the gut. It appears likely that the released I-SP originates mainly from enteric neurons intrinsic to the gut. These findings support the concept that SP is a neurotransmitter involved in the coordination of the peristaltic reflex. Acknowlerlgements-This study was supported by the Austrian Scientific Research Funds (grant 4641), the Pam Research Commission of the Austrian Academy of Sciences and the Jubilee Funds of the Austrian National Bank. The SP antibody used in this study was kindly donated by Dr. S. E. Leeman, University of Massachusetts, Worc&.er. The authors would also like to thank Mrs. S. Schm for able technical assistance.

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46. Szoicslnyi J. and Barth6 L. (1985) The capsaicin-~nsitive non-parasym~thetic excitatory innervation of the small intestine. In ~o~~~u~~o~ of ~~roc~~jc~ ~r~s~j~s~o~ (ed. Vizi E. S.), pp. 311-325. Akademiai Kiado, Budapest. 47. Theriault E., Otsuka M. and Jesse11 T. M. (1979) Cap&&-evoked release of substance P from primary sensory neurons. 3rain I&s. 170, 209-213. 48. Yaksh T. L., Jesse11T. M., Gamse R., Mudge A. W. and Leeman S. E. (1980) Intrathecal morphine inhibits substance P release from mammalian spinal cord in uivo. Nu?ure 286, 155-1.57. 49. Yau W. M. and Youther M. L. (1982) Direct evidence for a release of acetylcholine from the myenteric plexus of guinea-pig small intestine by substance P. Eur. J. Phurmac. 81, 66.5-668. (Accepted

10 October 1983)