Morphology of the canine pyloric sphincter in relation to function E. E. DANIEL, I. BEREZHN, M.D. ALEESCHEW, H. MANAKA, AND V. PBSEY-DANIEL Smooth Muscle Research Program, Faculty ofkPeQlth Sciences, McMaster University, 1280 Main Street Wrest, Hamilton, Ont. Canada U N 323 Received March 30, 1989 BANIEL, E. E., B E ~ Z I NI.,, AELESCHER, H,D., MANAKA, H., and POSEY-DANIEL, V. 1989.Morphology of the canine pyloric sphincter in relation to function. Can. J. Physiol. Phamacol. 67: 1560- 1576. The ultrastructure and imr~aunocytochemistryof the cmine distal pylofic muscle Iosp, the pyloric sphincter, were studied. Cells in this muscle were connected by gap junctions, fewer than in the antrum or corpus. The sphincter had a dense innewation and a sparse population of interstitial cells of CajJ.Most such cells were of the circular rnuscle type but a few were of the type in the myenteric plexus. Nerves were sometimes associated with interstitial cell profiles, but most nerves were neither close to nor assmiated with interstitial cells nor close to smooth muscle cells. Nerve profiles were characterized by an unusually high proportion of varicosities with a majority or a high proportion of large granular vesicles. Many of these were shown to contain material imunoreactive for vasoactive intestinal polypeptide (VIP) and some had substance P (SP) imunoreactive material. All were presumed to be peptidergic. VIP was present in a higher concentration in this muscle than in adjacent antral or duodenal circular muscle. Interstitid cells of Cajal made gap junctions to smooth muscle and to one another and might provide myogenic pacemaking activity for this muscle, but there was no evidence of a close or special relationship between nerves with VIP or SP and these cells. The absence of close relationships between nerves and either interstitial cells or smooth muscle cells Heaves unanswered questions about the structural basis for previous observations of discrete excitatory responses or pyloric sphincter to single stimuli or nerves up t s one per second. In conclusion, the structural observations suggest that this muscle has specid neural md myogenic control systems and that interstitial cells may function to control myogenic activity of this muscle but not to mediate neural signals. Key words: vassactive intestinal polypeptide, interstitial cells of Cajd, newopeptides, gap junctions, substance P. DANIEL, E. E., B E ~ Z I NI.,, ALEESCHEW, H. D.,MANAKA, H., et POSEY-DANIEL, V. 1989. Morphology of the cmine pyloric sphincter in relation to function. Can. J. Physiol. Phmacol. 67: 1568- B 573. On a CtudiC 19ultrstnactureet lqimunocytochiBimiedu muscle pylorique distal, le sphincter pylorique. Les cellules de ce muscle ktdent relikes par des jonctions lacunaires, dont le nombre Ctait plus restreint que dans I9antreou %ecorps. %R sphincter 6tait tr&sinnewk et contenait peu de cellules interstitielles de Cajal. Ces cellules ktaient pour la plupart du type muscle ckculaire; toutefois, p u de cellules de ce type 6taient csntenues dans le plexus myentkrique. Les neds ktdent parfbpis associes aux cellules interstitielles, wads la majorit6 d'entre eux ne se trouvaient pas a proximitt? de celles-ci ou w'y ttaient pas associkes, ou ne se trouvaient pas B proximitk des cellules du muscle lisse. Les patrons d9innervation se caractkksaient par une proportion inhabituellementClevCe de varicositks avec une majorit6 ou une forte proportion de grosses vtsicules grmuleuses. Plusieurs de celles-ci contenaient un $Ikment imunorCactif pour le peptide intestinal vasoactif (VIP) et certaines avaient un klkment imurmorCactif pour la substance P (SP). On a suppost5 qu9ellesCtaient toutes peptidergiques. Le VIP Ctait prksent en plus forte concentration dans ce muscle que dms Be muscle ckculaire duodCnal ou antral adjacent. %Rscellules interstitielles Ctaient relikes au muscle lisse et entre elles par des jonctions lacunaires, et pou~aientf o m i r une activitk pacemaker myoghne A ce muscle; toutefois, rien n'indiqudt I'existence d'une relation spkciale ou 6troite entre les nerfs avec VIP ou SF et ces celluBes. L'absence de relations Ctroites entre les nerfs et les cellules interstitielles ou Bes cellules du muscle lisse soaal&vetoujoasrs des interrogations sarr %eformdement structural d'observations mt6rieures de rCponses excitatrices discrktes daa sphincter pylskqwe B des stimuli neweux p u v m t Ctre appliquCs toute les secondes. En conclusion, les observations s6nncturalessuggbrent que ce muscle a des syst&mesBe contr6le myogkne et neuronal sp5ciaux et que Ies cellules interstitielles gourraient fonctioanmer pour contrdler I'activitk neurogene de ce muscle mrpis non pour rnkdier les signaux neuronaux. [Traduit par Ba revue] Introdnetion The generd morphology of the internal ring of circular smooth muscle at the gas~oduodend junction has recently been reviewed by SchuBze-Dekieu et a&.(1984), who designate this structure as the distal plylosic musele loop. Torgensen (1942) described this Hoop and mother large oblique loop of muscle over the distal mtmm which usually had a thickened Ping of s m m h muscle at its proximid component (called the proximal pylsric muscle Hoop by Schulze-Delrieu et ak., 1984). These two Imps join at the lesser curvature to form a pyloric toms. In dogs, the distal pylcsric rnuscle loop has its muscle bundles or ridge of muscle more obliquely arranged than in the human (Torgensen 1942), and is Hscated at the region from which basal 'Author to whom correspondence should be sent at the following address: HSC-4N5 1, Department of Biomedical Sciences, 1200 Main Street West, Hamilton, Orat. Canada L8N 325. active tonic with supekmpcssed phasic contractions caw be recorded (Allescher ef al. B 9 8 8 ~ )These . contractions are mysgewic in origin but can be both enhanced or inhibited by neural reflexes. An ascending chain of cholinergic nerves caused excitation of this muscle when activated by low frequency field stimulation sf the intrinsic duodenal nerves (Alleseher el ak. 1988~)or by intraduodenat acid (Alleseher et al. 1 9 8 9 ~ )Vagal . stimulation with low frequency and short duration pulses also caused excitation of the sphincter. Inhibitory reflexes sf the muscle were activated by field stimulation of the anmaan and were mediated in part by descending nonadrenergic, noncholinergic (NANC) intrinsic nerves and in part by adrenergic nerves acting via a-adrenoceptors (Allescher et ak. 1 9 8 8 ~ ) . Vasoactive intestinal polypeptide (VIP), a putative NANC mediator, was a direct inhibitor of contractile activity sf canine pyloms in vivo but was inconsistently effective in vitm (Alleschef et ak. 1989b). Nerves immunoreactive for VIP or galanin DANIEL ET AL. 1561 were dso present in greater density in the sphincter (Gonda et al. Methods 1989). Adrenergic nerves were densely distributed in the pyloric Tissuepreparation sphincter9 md activation of either aa-or a2-adrenoceptors lFof electron microscopical examination five dogs were eutk,anized appeared to mediate both neural inhibition of acetylcholine with sodium pentobarbital (100 mg/kg i.v.). The distal pyloric Bwp was identified by its structure and location and then cut out and placed release and acetylcholine actions on muscle. p-Adrenoceptor in oxyenioted normal Krebs solution at room temperature. After remsvoccupation acted to promote contraction by releasing acetylchoal, the distal pylsric Ioop was divided into four components: an inner line but also had direct inhibitory actions on muscle unmasked antral, inner duodenal, an outer a n d , and an outer duodenal compoby muscarinic blockade (Allescher et al. 1989~).Vagal fibers nent. Each pyloric muscle component was then slightly stretched, idso contained both inhibitory and excitatory fibers affecting the pinned out in petri dishes, and fixed by immersion at room temperature distal pyloric loop (Allescher et al. 1988~).In dogs, as in for 2 h with 2% glutaddehyde in 0.075 M cacodylate buffer (pH 7.4) humans (Wattchow et al. 1987) a variety of neuropeptide nerves containing4.5% sucrose. Additionally, the pylorus from one more dog are present in this muscle in dense supply to adjacent muscles was fixed by intra-arterial perfusion ia situ with the same fixture. After (E. E. Daniel, J. B. Fumess, and M. Costa, unpublished) in 5 min of the initid perfusion fixation, the distal pyloric loop was removed, divided into four similar muscle components, and immersed addition to adrenergic nerves (Allescher et al. 1989a, 1989b, in the same fixative for an additional 2 h at room temperature. The 1989~). structural preservation of tissue fixed by perfusion was similar to that There has been only one previous ultrastructural study of the fixed by immersion. Following fixation, tissues were washed overnight distal pyloric muscle loop. Cai and Gabella (1984) studied this in cacodylate buffer containing 6% sucrose md 1.25 d 4 CaCI2 (pH muscle and its innervation in the guinea pig. 'They found a high 7.4) at 4"C, post fixed with 2% 0 s 0 4 in 0.05 M cacodylate buffer (pH density of nerves innervating the circular muscle in this region 7.4) at room temperature for 98 mh, stained with saturated uranyl of the pyloms, but they did not distinguish the distal muscle loop acetate for 60 min at room temperature, dehydrated in graded ethanol from the overlying antrum. These authors also did not study the and propylene oxide, and embedded in Epon 812. Tissues were relationships between nerve varicosities and other structures oriented in molds to cut the circular muscle layer in a cross direction. such as interstitial cells of Cajal (ICC). Sections were strained for 2 min with lead citrate and examined in a Philips 301 electron microscope at 60 kV. Only tissues that were wdl In lower esophageal sphincter (LES), there are numerous fixed, appropriately oriented, and well stained were analyzed quantitanerves but most appear to supply ICC rather than muscle (Daniel tively. and Posey-Daniel 1984a, I984b). ICC are cells with a distincTissues to be used for immmc%histwhemistrywere initially fixed by tive ultrastmcture which are in gap junction contact with one intra-arterial perfusion in situ using 0.1% glutapaldehyde and 4% another and with smooth muscle (Thuneberg 1982). There are paraformaldehydecontaining 3% sucrose in 0. I M phosphate buffer as two physiologically relevant and structurally distinct sets of a fixative, and then removed, dissected, and further fixed for 1 h at ice ICC, located primarily in the myenteric plexus or in circular temperature. Tissues were embedded in LR m i t e resin (J. B. EM muscle. ICC have been proposed to play a role as pacemakers in Services Inc., h n d , Qu6.) using the "cold method" with accelerator myogenic activation of gut circular muscle (nuneberg 1982; as previously described (Berezin el al. 1987,1989). Thin sections were Hara et al. 1986) and as the sites of release and action of the cut on a ultramicrotome, mounted on collodium-coated 200 mesh NANC inhibitory mediator (Daniel and Posey-Daniel 1 9 8 4 ~ ~ nickel grids, and processed for the ianra~unocytschemicdlabelling using inmunogold techniques (Berezin et al. 1987, 198g2). 1984b; Daniel et al. 1984). The ICC have been found to be closely innervated by nerves containing VIP immunoreactivity Antisera in large granular vesicles in the canine LES (Berezin et al. Antibodies to VIP and substance P (SP) were raised in rabbits against 1988), in the canine colon at the circular muscle submucosaI the synthetic peptides, and details of their specificity are given in border (Berezin et al. 1989')~ and in the canine small intestine publications by Dimaline et a1. (1980) for R501 and in Manaka et al. deep muscular plexus (Daniel 1977; B e ~ z i net sl. 1985). (1989) for R24M. Each was a gift sf Professor N. Yanaihara, Shizuoka The purpose of this study was to define the ultrastructu~of College of Pharmacy, Shizuoka, Japan. The use of R501 for imthe muscle and nerves of the canine distal pyloric muscle loop or munocytochemistry is also described by Dimaline et a!. (1980). In sphincter, the relationships of ICC to these structures, and to brief, R5Ol is directed against a large proportion of the mid- and determine if VIP or substance P immunoreactivity was present C-terminal region of VIP an8 in ianmunocytochemistry fails to recognize either secretin or glucagsn. R M 2 (N. Yanihara, personal comin large granular vesicles in nerve endings and had a special munication) is directed against the entire sequence of SP and recogrelationship to ICC or to muscle. nizes a variety of SP fragments except SP 6- 1 1. It cross-reacts with Thomas (1957) in his review of gastric emptying stated that neurokinin A (NKA) less than 2%. No bsts of its actions in i m u n w y the evidence to that time did not support that the pyloric sphinctochemistry have been made, so it might interact with N U . We have ter was a muscle with a nerve supply and functional capacities found SP and N U but not NKB in synaptosomes from canine gut different from that of the adjacent gastric muscle. After 30 enteric nerves. The dilution of each antiserum used has been found to years, it appears on functional (Allescher et wl. 1988a, 1988c, yield the following optimal results: high intensity of the labelling over 1989s-1989e) and on morphological (this study) grounds that some nerve vesicles with little labelling over the other parts. In all this muscle may in fact have these properties and may meet the experiments, ovalbumin (final concentration, 0.5%) was added to definition of a sphincter (Code and Schlegel 1968) possessing a phosphate-bufferedsaline for find dilution of antisera. For immunwytocheIIpicd labelling, either the protein A - gold (10 special nerve supply, a special basis for control of rhythmic nun) or the goat anti-rabbit imunoglobulin G - gold (10 mnm) (GARactivity, and special functional properties. Another question IgG geld) markers (Janssen Pharmaceutical, Beerse, Belgium) were also enunciated by Thomas (1957) still remains unresolved, used. The sections were treated as previously described (Berezin et al. i.e., what role does this muscle play in regulating normal gastric 1987, 1989). Staining with many1 acetate and lead citrate was done emptying? before examination in an electron microscope. '~erezh, I., Wuizinga, J. D., and Daniel, E. E., submitted for publication. al To demonstrate the spificity of ~ u n o c ~ h e m i c labelling, several controls were performed for each antiserum used: (i) incubation of sections with gold complex done, omitting the step with the anti- 1562 CAN. J . PHYSIBL. P%IAIWBaCOL.VQL. 67, 1989 serum; (ii) replacement of the antiserum by the same antiserum, which was previously absorbed with 0.6- 1.5 X 1 r 5M synthetic VIP (20-50 pg/mL) (Sigma Chemical Co., St. Louis, MO) (SP antiserum was M (10 y g / d ) synthetic SP (Sigma preabsorbed with 0.74 x Chemical Co.); and (ddi) replacement of the primary antiserum by diluted normal rabbit s e m (Sigma Chemical Co.). None of these procedures resulted in positive staining. Morphologs'calprocedures Sections were initially examined under the low magnification mode of the Philips 301. A complete thin section was chosen for examhation. Grid squares covered with smooth muscle as completely as feasible were scanned systematic~yfor nerves and gap junctions at 3300 magnification ( l o x higher on the screen). The fist ten gap junctions were photog~aphedat 7200X to measure their length and the remainder were counted. Nerve profiles within 2.5 pm of muscle were also photographed, classified, and counted. Classification was based on the system used previously by (Daniel and Posey-Daniel 8984a, 1984b); i.e., nrom (less than w e e synaptic vesicles and usually containing neurofilments and (or) neurotubules); and varicosities (thee or more synaptic vesicles) which were subclassified as containing (i) small granular vesicles (SGV) when one or more was clearly of this type, (ii) small agranular vesicles (SAV) when the majority were of this type (unless SGV were present), and (iii) large granular vesicles (LGV) when the majority were of this type. Only very rare profiles contained m<sst.Iy mitochondria and these were counted for convenience with axon profiles. No myelinated nerves were found near smooth muscle cells. Interstitial cells of Cajal ICC were identified by their characteristic ultrastructure (Thuneberg 1982; Daniel and Posey-Daniel 198$a, 1984b; Berezin et al. 1988). When fixed by immersion, ICC in circular muscle are usually election dense; they contab many mitochondria and fdments especially in their processes, dark granules, and rough endoplasmic reticulum. ICC have ansaray cavmlae, and frequently make gap junction contact with smooth muscle and with om another. They are often close ( S 30 m)to nerve profiles. Another class of ICC primarily located in the myenteric plexus may also be present (Thuneberg 1982). These are more fibroblast-like, less electron dense, have fewer caveolae, fewer granules, and are associated with but are less comrnonIy close (<38 m)to nerves. They do occasionally make gap junctions to one another and. to muscle. All ICC were classified as to their location: close to nerves, close to smooth muscle, presence of a nucleus, or presence of gap junctions. ICC of the fibroblast-like type were difficult to identify and were counted when they were in gap junction contact with smooth muscle or one another; these were very rare. Each grid square studied was photographed in its entirety by overlapping photographs at 550X magnification. These were printed at 4 0 0 x and prepared as a montage; the length of smooth muscle plasma membrane was measured using a k i t z Videoplan System. Gap junction lengths and d l other quantities were normalized to plasma membrane length as previously described (Allexher et al. 1988b; Agrawal and Daniel 1986). Cross-sectioned smooth muscle cells were also counted PO allow comparison to earlier nomalization procedures (e.g., Daniel et al. 1984). Determination CPf VIP in pyloric sphincter Stomachs with the proximal duodenum intact were obtained from four dogs killed with an overdose of pentobarbital; stomachs were placed promptly in ice-cold sucrose - 3-[N-morpholino]propanesulfonic acid (MOPS) solution (8% sucrose, 50 d'd MOPS). The lumen was opened dong the lesser curvature. The opened specimen was pinned flat on an ice-cold dissection plate with the serosa down so that the mucosa could be dissected off. Muscle strips were prepared from the antral region 3-5 cm oral to the pylorus, the duodenum 2-5 cm aboral to the pyloms, the outer pyloric muscle layers, and the distinct inner pyloric muscle ring. The strips from these regions were then treated separately as follows. They were boiled imediately for 18 minat 100°Cin distilled water, then weighed (average weight, 0.4-0.6 g), put into 5 d of 0.5 M acetic acid, minced with scissors, and homogenized with a PI0 Polytron homogenizer. The homogenate was then extracted for 10 mirn at 100°Cin a boiling water bath, and after this it was centrifuged at 10 800 x g for 10 min. The supernatant was collected and assayed for VIP imunoreactivity. Tissue concentration of VIP-like imunoreactivity was measured using the VIP-specific antiserum W501 at a final dilution of 1 :14 000, '2s~-labelledVIP was prepared by the chloranrrine T method (see Manslka et al., 1989). The radioimunoassay buffer consisted of 10 rrnM phosphate, 140 mFM NaCI, 25 mFM EDTA, 0.5% bovine serum albumin, and 250 K d l h e i n inhibitor u n i t s / d aprotirain at a final pH of3.4. Buffer, antiserum, sample, and tracer were incubated at 4'C for 48 h in a final volume of 0.7 I12L. Antibody-bound and unbound peptide were separated using anti-rabbit gamma-globulin goat serum. The radioactivity was counted in a Beckman 5500 gamma counter. Under these conditions, the antiserum cross-reactswith PHI peptide less than 2% when PHI was present at 250 ng/mL. FIG.1. (a) A small bundle of nerves near profiles of smooth muscles (SM) of the inner antrd segment of the pyloric sphincter. Note one varicose sfHhlCfure(mow) with large granular vesicles (EGV) enclosed by glid cell cytoplasm and several axons, two of which are not wholly enclosed by the glial cell. (b) A similar nerve bundle near SM of the inner duodenal segment of the pyloric sphincter. Note the LGV-containing varicose structure (mow) which is incompletely covered by glial cytoplasm and a profile probably of interstitial cells of Cajal (ICC; double arrow). (c) A large bundle of nerves near smooth muscle cell profiles (SM) of the toms. Profiles of nerves with a predominance of small agranular vesicles (sav) are marked by curved white mows. Note that they alw have several EGV as well in all cases. One nerve profile with a predominance of LGV is marked with a straight white arrow. (4A large nerve bundle with a Schwann cell (SC) from the inner duodenal partion of the sphincter. Most nerve profiles are of axons, but two contain predominantly sav (one or no LGV). At bottom left a profile (IC) which may be part of a fibroblast or a fibroblast-IikeICC. At top right, a profile of a smooth muscle cell. The calibration b a s all correspond to approximately 292 nm. FIG.2. (a)Interstitial cells of Cajd (IC) profile in the inner duodenal segment of the pyloric sphincter showing a typical circular muscle ICC. Note the electron dense cytoplasm, the lobulated nucleus with condensed chromatin, dense particles resembling ribosomes scattered between packed mitochondria (m), golgi apparatus (g) and many membrane caveolae (c). In the same grid square, cytoplasm extensions of this cell made two gap junction contacts to smooth muscle (SM). No nerves were near this ICC. (b) An ICC profile (IC) in the inner antral segment making gap junction contact (mow) with another IC near SM. No nerves were present. (c) An ICC profile (IC) making gap junction contact (arrow)with SM of the inner anman. Two nerve profiles (N) in a nerve bundle are labelled. This is as close as nerves and ICC are usually found. The bar at top.correspnds to a b u t 2 16 nm and applies to all panels. FIG.3. (a) A long gap junction (mow) between two interstitial cells of Cajal (IC) near smooth muscle (SM) of the inner mtral segment of the pyloric sphincter. Note caveolae (c). Another cell profile (labelled IC?) may be a profile of a fibroblast-likeICC; it does have some cavmlae (c), but they are fewer than usual a d the cytoplasm is less electron dense than usual. The calibration bar corresponds to about 216 nm. (b) A long gap junction (mow) between two ICC profiles. One of the ICC (IC) is a typical ICC of circular muscle with ribosome-like particles, cavwleae, and a typical lobulakd nucleus with a nucleolus. A nerve bundle with one axon labelled (a) was associated with but not close to the ICC. %he calibration bar comesponds to a b u t 288 m. (c) A fibroblast-like ICC (IC) in the inner antral segment makes gap junction contact (mow) with a smooth muscle cell (SM). Note the absence of caveolae, the absence of mitochondria, and the presence of much endoplasmic reticulum and goHgi apparatus. There is also an associated Schwann cell (SC) with nerve profiles including one labelled axon (a). The calibration bar corresponds to about 508.5 m. DANIEL ET AE. 1563 1564 CAN. J. WYSHOL. PHARMACBL. VOL. 67, 1989 DANIEL ET AL. 1565 1566 CAN. J. PWSHBL. PMARMACOL. VOE. 67, 1989 FIG.4. Two mast cells (A%) surrounded by smooth muscle cells (SM) and without nearby nerve profiles. (a) The cell has large membrane-bounded electron-dense (e.g. at curved mow) which is interpreted as only slightly activated. (b)The cell contains few such electron dense granules, but does contain many membrane-bounded regions with residual Bight granular contents (e.g., curved mow). This structure is interpreted as nearly fully activated. Note, however, the lack of evidence of exocytssis. Both are in the inner antraI segment*The calibration b a s csrrespnd to about 389 nm. Results Qualitative observations All segments of the pyloric sphincter contained smooth muscle cells with gap junctions as well as other junctions such as close appositions and intermediate contacts. In no region was there a deep muscular plexus, since duodenal muscle was not included. In all regions there were also numerous nerve profiles near but not close (130 nm) to muscle (Fig. I), and occasional profiles of ICC (Figs. 2 and 3). These were mostly of the type found in ckcular muscle (Figs. 2a, 2b and 3a, 3b) but occasionally fibroblast-like HCC were found (Fig. 3e). An unexpected feature was the frequent presence of mast cells in the midst of muscle bundles sometimes but often not (Fig. 4) near nerves. Some appeared to be minimally activated (Fig. 4a); some were markedly activated (Fig. 4b). There were also other immune cells such as neutrophils a d macrophages present in several instances and these were always near nerves. Smooth muscle cells had no unusual features (Figs. l -4). The nerve profiles were, however, characterizedby the presence of a large number of varicose profiles containing a large number of LGV (Fig. l a and lb); even when not in the majority in a given varicose profile there were frequently many of them (Fig. Ic). There were almost no instances of a very cose (<30 nm) relationship between bare nerve profiles and smooth muscle cells. Nerve profiles were usually clustered in bundles and partially surrounded by glial cell cytoplasm (Fig. I). In contrast to observations in the lower esophageal sphincters (Berezin et ale 1988; Daniel and Posey-Daniel 1984; Daniel et al. 1984; Allescher et ak. 1988b), h e deep muscular plexus of the small intestine (Daniel et al. 1977), and the submucosd brder of the colon circular muscle (Berezin et a&.1988), ICC were not usually closely associated with nerves. Instead they were often distant from any nerve profile (Figs. 2a92b, a d 3a) and never as close as 30 nm to nerves (Figs. 2c, 3b9and 3c) even ma. 5. (a) Thin sections through the smooth muscle layer stained w i h VIP antisenam followed by the protein A - gold complex showing W-imanunopositive nerve profiles (N)containing LGV (arrow). Note a significant distance (at least 8.8- 1.O pm) between VIP-inmunopositive nerve and surrounding smooth muscle cells (SM). (b)Enlarged view of a portion of VIP-immunopsitive nerve (N) (shown in Fig. 5s) showing gold labelling over large granular vesicfes (mows). The calibration bars are 8.5 pm. FIG,6. (Q) Electron micrograph showing VIP-imunopsitive nerve varicosity (N)containing LGV (mow) near the interstitial cell (IG). Note m absence of a close contact between the interstitial cell and VIP-irppmunopsitive nerve. No VIP imunoreactivity was found in the interstitial cell. (b) Same section as in Fig. h.Higher magnification micrograph of a gortion of meme vahicosiay (N)showing concentration sf gold particles mostly over LGV (mows). The calibration b a s are 0.5 pn. DANIEL ET AL. 1567 1568 CAN. J. PHYSIQL. PHAMACOL. VOL. 67, 1989 when nerves were nearby. It was not possible to determine if they formed a network or a layer, as in the colon. They seemed to be scattered sparsely, usually at the periphery of the muscle bundles. They occasionally made gap junctions with one another (Figs. 2b and 3b) or with smooth muscle cells (Figs. la, lb, 2c and 3a). Quantitativejindings Smooth muscle cells in d l regions had a moderate density of gap junctions (Table 1). In dl cases but one, the average profile length of gap junctions were near 200 nm; in the outer duodenal portion of the sphincter it was smaller but not significantly so. In all cases except the inner antrd segments, there was a similar high density (>30 per 1000 pm of smooth muscle plasma membrane) of nerve profiles; in that segment, there were significantly (p < 0.05) fewer nerve profiles based on one-way analysis of variance and subsequent tests (Table 1). A notable aspect of d l tissues was a high proportion of nerve profiles with mostly LGV compared with those with SAV. Usually in gut (Daniel and Posey-Daniel 1984a, 1984b; Dmiel et al. 1977; Roboehm et al. 1985;Allescher et al. 1988)there is anegligible or much smaller number of these compared with profiles with SAV. In addition, as noted above, many classified as SAV had a mixture of vesicles including many LGV (Fig. lc). In most gut tissues, nerve profiles have a few LGV mixed with SAV (Daniel et al. 1977; Cai and Cabella 1984; Gibbins 1982; Cook and Bmstock 1976; see Fumess and Costa (1987) for review). There were very few varicose nerve profiles with SGV, probably reflecting a failure of preservation of the central dense core with the method of fixation used. Loss of this core may have inflated the number classified as SAV, but this would not have influenced the finding of a high proportion of LGV found. The LGV found in combination with SAV in vesicles classified as SAV were on average significantly (p < 0.05) larger in size (13 1.8, n = 113) compared with those in varicosities classified as EGV (12 1.3, n = 321) based on an unpaired t-test. Table 2 sumarizes some of these data in which there were sufficient well-preserved LGV of each type in the same grid square for comparison. ICC were relatively sparse, fewer than 1.5 per 1000 ylm of smooth rnuscle plasma membrane (Table 1). There were a significantly smaller number in the inner duodenal segment. Overall, less than hdf of the ICC were associated with nerves and none made close contact (<30 nm) with nerves, but most were ass~ciatedwith smooth muscle cells. There were few ICC profiles with nuclei or with gap junctions, and gap junctions between ICC were larger (p < 0.05 in unpaired t-test) in profile length (402.3 & 48.0 (SE) nm; n = 41) than those between ICC and smooth muscle cells (249.3 k 35.2 (SE) nm, n = 28). In addition t~ typical ICC found in circular smooth muscle (Thuneberg 1982; Daniel and Posey-Daniel 1 9 8 4 ~1984b; ~ Be~ z i et n al. 1988), there were ICC that resembled fibroblasts but that made gap junction contact with smooth muscle and were close to nerves (Fig. 3c). Imunoreactive VIP content ofantrak circular muscle, pylorus, and duodeml circular muscle The imunoreactive VIP contents of antral muscle, duodenal muscle, and muscle tissue from the muscle layer overlying the pyloric region were similar. Tissue contents of VIP ranged from 180to 290 ng/g wet weight and were as follows (mean values 2 standard deviations) antrum, 2 12 2 35 ;pylonas outer layer, 220 2 56; and duodenum, 238 2 81. However, the inner pyloric muscle ring had a significantly (p < 0.01 based on one-way analysis of variance and t-tests) higher VIP content (766 2 I25 DANIEL FiT AL. TABLE 2. Size of large granular vesicles (nm 9 SE) - - Region NOTE:n , ~ IBUvesicles. In SAV n n In LGV h of sections r studied; EGV, Large granular vesicles;SAV, small agranu- TABLE3. Percentage of varicose nerve profiles with mostly large granular vesicles (relative to those with small agrmular vesicles) Reference Daniel et al. 1977 Daniel et al. 1989 Daniel et al. 1984 h o t o et al. 1983 Robotham et al. 1985 Mlescher et al. 1988b Site % Rabbit small intestine Longitudinal muscle Circular muscle Myenteric plexus Australian opssom esophagus circular muscle Canine corpus North American opossum Esophagus LES Body circular muscle Es~phagusmuscularis rnucosae Canine LES NOTE: LES, Bower esophageal sphincter. ng/g wet weight) than duodenum, which had the next highest VIP content. All other tissues did not differ significantly from the duodenum in their VIP content. lmmunocytochemica& observations There were numerous nerve profiles with LGV imunoreactive for VIP (Figs. 5-7) and some were ifnrnunoreactive for SP (Fig. 8). LB1 all immunopositive nerve profiles, VIP and SP inmunoreactivities were primarily associated with LGV (Figs. 5b, Bb, 7, and 8). The density of gold labelling differed from one LGV to another within the same varicosity, and in each immunopositive nerve profile a few LGV always remained unlabelled (Figs. 5b, Qb,7, and 8). In canine pyloric sphincter both peptides were observed in nerve profiles with a predominance of LGV (Figs. 7 and 8). However, on each section only a fraction of the nerve varicosities containing LGV demonstrated either VIP or SP immunoreactivity; other varicosities containing morphologically similar LGV remained unlabelled. The sections examined and stained either by the protein A - gold complex or by GAR-IgG gold revealed that regardless of the staining technique used there was a significant difference in the proportion of labelled and dabelled nerve varicosities between sections incubated with VIP amtiserum compared to those incubated with SP antiserum. after staining with VIP antiserum the majority of nerve varicosities containing LGV were found to be immunolabelled, whereas the sections stained with SP antiserum displayed only a small proportion of immunoreactive nerve profiles. There was no consistent distinction either in the morphology (electron 1569 density of LGV or their diameters) or in the relationship to ICC or to smooth muscle cells between SP- and VIPimmunoreactive nerve profiles (Figs. 7 and 8). In canine pyloric sphincter VIP- and SP-immunoreactive nerve varicosities were observed outside (Figs. 6a, 7, and 8) as well as inside muscle bundles (Fig. 5a). In both locations imunopositive nerve profiles were never seen cbsely associated (less than 50 nm) with smooth muscle cells (Fig. 5a). Also, no close contacts were revealed between ICC and VIP- or SP-imunoreactive nerves. There was always a significant distance (at least 0.5 pm) between ICC and imunopositive nerve varicosities (Fig. 6s). No VIP or SP immunoreactivity was observed in ICC (Fig. 6Q). Peptidergic innervation olfpyloric sphincter The observations of this study indicate that the structure of nerve, muscle, and interstitialcells of Cajd in the canine pyloric sphincter differs in several respects from these structures in canine lower esophageal sphincter or canine stomach circular muscle (Daniel and Posey-Daniel1984a; Allescher er al. 1988b; Daniel et al. 1984, 1989; Oki md Daniel 1974). First, compared with both tissues there was a higher proportion of nerves with a predominance or a substantial number of large granular vesicles (Table 3). This is also in contrast to a wide variety of other gut tissues studied by similar techniques and with similar vesicle profile classification. Gibbins (1982) summarized the proportion of profiles with greater than 20% or no LGV in a number of smooth muscle tissues from rat, rabbit, and guinea pig. Rabbit and rat mococcygeus had 22 and 6596, respectively, of such profiles, and rabbit portal vein had 22% (all other tissues had less than 10% and most tissues had 20-4596 of varicose profiles with no LGV at all). Canine pylof-ic sphincter had the highest of varicose structures with mostly LGV, and in all -proportion cases at least 40% of the number of those with mostly SAV. Considering that many of the varicosities classified as SAV had a substantial number of LGV, the implication is strong that this muscle has a major neural input from peptidergic nerves, since these mediators are believed to be located in peripheral nerves normally in LGV (see Baumgarten et ak., 1970; Pelletier et a&. 1974; Pickel et al. 1977; Larssen 1975; Berezin et ab. 1985, 1987 for other references). These findings are in agreement with those of Cai and Gabella (1984) who studied the guinea pig pylorus and reported that most nerve varicosities in the pylorus contained a mixture of LGV and SAV. They did not distinguish those that had a predominance of either type of synaptic vesicle. The implication that an unusually high propoation of vesicles containing neuropeptides is present in this muscle is reinforced by the immunocytwhemicd studies. Previous stwdies with light microscopy (E. E. Daniel, M.Costa, a d 3. B. Fmess, unpublished) showed the presence of higher density nerves immunoreactive for VIP, substance P, enkephdins, and neuropeptide M in the canine pyloric sphincter than in s ~ o u n d i n g muscles. Similar observations were made in the human pyloric sphincter (Wattchow et al. 1987) and in the cat pylorus (Min 1980; Lidkrg et ai. 1982, 1983). Recently a higher density of catecholamine-containing nerves was also found in canine pyloric sphincter (Allescher et al. 1989a) and these presumably overlapped in part the nerves with neuropeptide Y. Also in this study we found the content of VIP by radioimmunoassay to be higher than in nearby muscles (3-4 times higher). Our ifnmunocytochemical studies were consistent with this obse:rvation, since a large number of nerve profiles containing LGV were imrnunoreactive for VIP. Substance P and VIP were 1570 CAN. B. PHYSIBL. PHAMACOL. VOL. 67, 1989 RG.'9. Thin section though a large nerve bundle stained with VIP antiserum followed by the protein A - gold complex. Note the predominant LGV-containing varicose structure (mows) of VJP-imunoposigive nerve. m,mitochondrion. The calibration bar is 0.2 pm. Rc. 8. Thin section stained widh substance P antiserum fo1Iowed by GAR - IgG gold showing SP-hmunoreactive nerve bundle. Gold particles are concenbated over LGV (mows) of nerve varicosities. The calibration bar is 0.2 Fm. DANIEL ET AL. 1571 located in these vesicles, and VIP-imunoreactive material was present in a high proprtion of LGV. Substance P imrnunoreactivity was present in a smaller but significant fraction of LGV. The observation that LX3V present in nerve profiles together with a majority of SAV were larger than those present in profiles with mostly LGV suggests that there may be different neuropeptides in these two nerve types. However, Furness and Costa (1987) have pointed out that this is probably an unreliable basis for differentiating LGV with regard to their neuropeptide content. Our invnunocytochemical technique does not lead to staining of membranes of SAV. Therefore we could not determine if VIP or substance P was present in profiles with or without SAV or in both. Although cholinergic nerves provide the final common pathway for most excitatory input of the canine pyloric sphincter (Allescher et al. 1988a, 1988~;1989a- 1989e), our study could not distinguish if there was an increased density of these nerves. For reasons s u m m h z d by Gibbons (1982), by Furness and Costa (1987), and by Daniel et al. (1977), those vakcose nerve profiles with a predominance of SAV cannot be equated exclusively with cholinergic nerves. Future studies will have ts address this question. Although demonstrating a dense innervation of the pyloric sphincter with peptidergic nerves, previous light microscopic studies do not reveal the relationship between nerve v ~ c o s i t i e s and the muscle or ZCC cells. Conceivably nerves viewed by light microscopy have few varicose endings related to control of muscle or ICC function. Cai and Gabella (1984) did not mention ICC or their relationship to nerves. Also it has k e n unclear whether these peptidergic nerves mainly innervate muscle or innervate ICC or both. ICC and gastric pacesetter activity The relative lack of close innervation of ECC, their dispersion throughout the muscle, and their gap junction connections to smooth muscle cells lead to the hypthesis that they function primarily as the "myogenic'pacemakers of this mu3cle and are modulated by the neuropeptides released in their environments. The arrangement of ECC as a complete network would help explain the observed close coupling of pacesetter activity throughout the canine stomach (Daniel and S m a 1978). From ultrastructural studies it is impossible to judge if such an interconnected network of ICC exists or if they function as mutually independent inputs coupled though the syncytium of smooth muscle cells. A reliable and selective stain for ICC would help resolve these issues. Earlier stwdies (Torgensen 194%)showed that the longitudinal muscle and myenteric plexus were continuous across the gastroduodenaljunction in the dog; we (E. E. Daniel, M. Costa, and J. B. Furness, unpublished) have observed ganglia connected to the myenteric plexus located deep within the circular muscle overlying and in the sphincter. Whether ICC from the rnyenteric plexus accompany these ganglia, explaining the occasional presence in the sphincter of fibroblast-like ICC (Thunekrg 1982), is unclear. If ICC of the myenteric plexus form a network of coupled cells across the pyloms and coupled to ICC of circular muscle, this would provide a mechanism for the transmission of both duodenal and gastric myogenic pacemaking activity to the sphincter. This would explain the very high frequency of contraction (up to 68 per minute) sometimes found during reflex stimulation of the sphincter (Allescher et al. 1988a), even though the phasic contractile activity of the sphincter is often at the gastric rate (4 to %/min) when there is no neural input (Allescher et al. 1988~). ICC and nerves Our studies show that in contrast to the remainder of the circular muscle of canine gut studied to date, only a small proportion of the nerves of the pyloric sphincter innervate the ICC. En canine small intestine deep muscular plexus (Daniel et al. 1977; Duchon et al. 1974), canine colon circular muscle (Berezin et al. 1987), and canine LES (Allescher et a1. 1 9 8 8 ~ ; Berezin et al. 1988), a substantialproportion of nerves innervate these cells. In canine pyloms,they do not (Table 2). Few nerves closely innervate ICC and simple cdculations from Table 2 indicates that most nerves were not found near ICC. However, nerve varicosities were also not epecially close to smooth muscle cells nor did there seem to be a specific relationship between my type of nerve a d any structure. The same conclusions were reached based on immunwytochemical studies of the bases of profiles with VIP-immunoreactive and substance P-immunoreactive materid In light of these structural relationships, we postulate that geptidergic neural control of canine pyloms are executed by the release of mediator in the environment of ICC, smooth muscle cells, and other nerves and that no special wiring arrangement relates nerves to particular structures. For ICC elsewhere it has been suggested that they are the sites of direct action of the NANC inhibitory nerve mediator (Daniel and Posey-Daniel 1984~ 19846). ~ The lack of a direct close innervation of muscle or ICC by peptidergic nerves may account for some of the failure to date to implicate VIP, tachykinins, galanin, or enkephdins in physiological control of the pyloms (see Allescher et al. 1988a, 1989b91889c, 1989d, 1889e). These neuropeptides may have longer term modulating actions on myogenic and neural control systems rather than act as primary neurotransmitters for reflexes. Relationship o$ICC, nerves, and reflexes to pylorkes Allescher et al. (1988~)found that with certain low parameters of vagal stimulation, the pyloric sphincter contracted with each stimulus applied up to 1 pulses per second (pps). Furthermore, each excitatory stimulus through an orally directed chain of cholinergic myenteric neurons from the duodenum could Brive individual contractions of the pyloric sphincter up to a rate of 0.7 pps. There was also an inhibitory response of the pyloric sphincter to low frequency stimuli of antral nerves. Reflexes in which one stimulus or a train of very few leads to a response imply a direct neural control rather than the neural modulation of myogenic activity postulated above on structural grounds. Ascending excitation from duodenum and excitation from vagal stimulation clearly involved more than one synapse (responses were blocked by hexamethonium or by atropine), but there must have been transmission without a requirement for temporal summation. How this direct neural control is achieved at the final synapse without close contact between one set of nerve varicosities and responding muscle is unclear from our structural observations. P1. Comparison to other GI muscles It is of interest to compare the structures of the canine pyloric sphincter to those found in a corpus and antral circular muscle in earlier studies (Oki and Daniel 1974; Daniel et al. 1984). In those studies normalization was to the number of crosssectioned smooth cells (expressed per 100 such cells). Conversion factors for the present data (see Table 1) were determined. Pyloric sphincter clearly contained more nerves close to muscle (3- to 6-fold more), more nerves with LGV (Table 3), more ICC profiles with nuclei (0.2 to 0.8 per 100 cells compared to 0.1 per 108 muscle cells), except for the outer anbum which had a 1572 CAN. J. PHYSIOL. PHARhlACOL. VOL. 67, 1989 similar smdl number, and smaller numbers (4.3-7.4 compared with 32.4 per 180 muscle cells) of similar size ( a b u t 200 nm) gap junction profiles in smooth muscle. In an earlier study of the opossum esophagus we also observed a lower density of gap junctions but a similar density of nerve profiles in the sphincter compared with the proximal muscle (Daniel and Posey-Daniel 1984a). h the earlier study of corpus muscle (Daniel e6 d. 19841, the number of %CCwith gap junctions was too small to allow size comparison to those in smooth muscle. In the present study, the gap junctions between ICC amd smooth muscle cells were similar in size to those between smooth muscle cells, but those between ICC were about twice as long (<4gbdB nm compared with about 280 nm). This raises the question of what properties of the coupled cells determine maximum gap junction size. In conclusion, this study shows that the canine pyloric sphincter has a distinctive structure compared with other canine gut circular muscles. St is richly innervated with nerves containing an unusually high proportion of profiles with a majority or substantial numbers of large granular vesicles. These probably contain peptides as demonstrated by the exclusive and frequent occurrence in them of substance P-imunoreactive or VIPi m u n o r a c t i v e material and by the high content of VIP in this muscle. These nerves do not make close contact with either muscle or other sphincteric structures. Muscle contains fewer gap junctions than the corpus but more interstitial cells of Cajal. In contast to the lower esophageal sphincter or the deep muscular plexus of smdl intestine, these are not major targets for nerves, but do make gap junctions to smooth muscle and to one mother. Most are typical of the ICC of circular muscle but a few are more fibroblast in type resembling ICC of the rnyenteric plexus. Overall the findings are consistent with the possibility that ICC control the "myogenic" activity ~f this muscle. The establishment of a structural basis for previously observed neur d reflexes will require further studies. AGRAWAL, R., and DANIEL,E. E. 1986. Control of gap junction formation in canine trachea by xachidonic acid metabolites. Am. J. PhysioB. 250: C495-C505. BLLEEx~R, W. D., AHMAD,S., DANIEL,E. E., DENT, 9., KOSTOLANSKA, F., and FOX,J. E. T. 1988a. Inhibitory opioid receptors in canine pyloms. Am. J. Physiol. 255: G352-G3g0. W. D., BEREZIN, I., JURY,J., and DANIEL, E. 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