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The predominant immunoreactive cholecystokinin (CCK) forms in acid extracts of rat intestine eluted from reverse-phase high-performance liquid chromatography columns in the positions of CCK-8, CCK-33/39, and CCK-58. Control experiments indicated that smaller CCK forms did not arise from artifactual degradation of large CCK forms. Less than 10% of CCK-8 added to and extracted from intestine was recovered in acid; CCK-8 could not be recovered by subsequent alkaline extraction, but subsequent urea extraction yielded approximately 25% of the added peptide. This suggests that CCK binds to proteins during acid extraction and that the preponderance of large CCK forms in acid extracts is not due to inhibition of CCK degradation but results from poor extraction of small CCK forms. No evidence for a CCK-22-like peptide was found in acid or subsequent urea extracts of rat intestine, suggesting CCK posttranslational processing in adult rats is like that in humans and dogs.

Immunocytochemistry, radioimmunoassay, chromatography, and biological assay using a rabbit isolated duodenal muscle strip preparation were used in attempting to characterize motilin from the rat small intestine. Several different antisera and monoclonal antibodies directed against natural porcine motilin were used. A variety of fixation techniques using Bouin's, paraformaldehyde, and benzoquinone with different staining methods including, fluorescein-conjugated second antibody, peroxidase-antiperoxidase or peroxidase-conjugated second antibody techniques were used. All methods failed to detect immunoreactive motilin cells in the rat small intestine. The same antisera were used in radioimmunoassays for motilin to evaluate extracts of rat intestinal tissue. Two of these detected immunoreactive motilin in gut extracts, and these antisera showed a different distribution for the peptide. Samples containing immunoreactive motilin obtained from cation exchange chromatography on SP-Sephadex-G25 were concentrated and assayed for biological activity in a rabbit duodenal muscle strip preparation. Desensitization of duodenal tissue to porcine motilin could be demonstrated by pretreatment with this peptide. The biological activity of partially purified rat intestinal immunoreactive motilin was not prevented by pretreatment of the tissue with motilin. Further purification of this preparation on Bio-Gel P-10 yielded an immunoreactive motilin peak that co-eluted with natural porcine motilin. Rat intestinal immunoreactive motilin did not co-elute with natural porcine motilin following high pressure liquid chromatography on a Waters microBondapak C18 reversed-phase column using a linear gradient of water-acetonitrile (10-45%) over 30 min. Although of similar molecular size, rat motilin is probably structurally dissimilar to other mammalian motilins.

A new method for the preparation of smooth muscle thin filaments which include calponin was established. We found that calponin readily separated from thin filaments in the presence of 10 mM ATP. By preventing thin filament extract from exposing to ATP, we obtained thin filaments which contained actin, tropomyosin, caldesmon and calponin in molar ratios of 7:0.9:0.6:0.7. We studied myosin Mg-ATPase activity by using the thin filaments in comparison with classical thin filaments prepared by the method of Marston and Smith, which contained the same amounts of caldesmon and tropomyosin as our thin filaments but lost almost all calponin. The presence of calponin reduced the Vmax value for thin filament-activated myosin Mg-ATPase activity by 33% without a significant change in Km value. These findings suggest that calponin inhibits myosin Mg-ATPase activity by modulation of a kinetic step as an integral component of smooth muscle thin filaments.

We examined whether the gizzard MHC gene is expressed in other smooth muscle tissues and, if so, whether there exist any smooth muscle MHC isoforms at the mRNA level. Northern blot analysis showed that the gizzard MHC gene was also expressed in the aorta and jejunum, but not in the pectoralis muscle or in fibroblasts. This indicates that striated muscle and non-muscle MHC isoforms are encoded in genes distinct from the smooth muscle MHC gene. Further, nuclease S1 mapping showed that the aortic smooth muscle MHC mRNA was distinct from the gizzard mRNA in the 5'-terminal coding region. Both of these mRNA species are expressed in the jejunum. These observations suggest that there exist at least two chicken smooth muscle MHC isoforms, vascular-type and intestinal-type, and that these isoforms are generated from a single-copy gene, probably by an alternative mRNA processing mechanism.

A 5-HT3 binding site, with high affinity for (S-)[3H]zacopride, was solubilized from rabbit small bowel muscularis membranes utilizing 0.5% sodium cholate and 400 mM (NH4)2SO4. Approximately 72% of the (S-)[3H]zacopride binding activity was recovered in a form that retained the high affinity (Kd = 0.7 nM) and specificity for this radioligand that is characteristic of the membrane-bound receptor. ICS 205-930 and other 5-HT3 compounds were effective inhibitors and exhibited the same rank order of potency in the solubilized and membrane-bound preparations. The receptor-detergent complex did not sediment after centrifugation for 1 h at 150,000 x g and eluted between thyroglobulin (MW = 669,000) and apoferritin (MW = 443,000) when fractionated by high-performance liquid chromatography gel filtration. This is the first report of the solubilization of a 5-HT3 binding site.

A case of type I Gaucher's disease in a 39-year-old male is reported. Autopsy showed marked enlargement of the spleen (3,070 g) and infiltration of typical Gaucher's cells in the spleen, liver, bone, marrow, gastrointestinal tract, lymph nodes and adrenal glands. The diagnosis of Gaucher's disease was ascertained by the very low beta-glucosidase activity of cultured subcutaneous fibroblasts and the high content of glucocerebroside in the spleen tissue. A peculiar finding in this case was prominent deposition of brown pigment in endothelial cells of the spleen and smooth muscles fibers of the gastrointestinal tract, urinary bladder and prostate. Histochemical examination revealed that the granules in endothelial cells and smooth muscle fibers were ceroid. Such deposition of ceroid has never been reported previously in Gaucher's disease. Ceroid deposition in generalized smooth muscle fibers is known as brown bowel syndrome, and is highly suggestive of severe vitamin E deficiency. Although other symptoms of vitamin E deficiency were not noticed in this case, some malnutritional condition might play a role in prominent deposition of ceroid in lysosomes, possibly together with deficient activity of a lysosomal enzyme.

Receptors for cholecystokinin (CCK) on gallbladder muscularis smooth muscle have different apparent sizes in man (Mr = 85,000-95,000) and cow (Mr = 70,000-85,000). In this work, these receptors were demonstrated to represent N-linked complex glycoproteins with Mr = 43,000 protein cores, based on lectin-affinity chromatography and the deglycosylation of bands affinity labeled with 125I-D-Tyr-Gly-[(Nle28,31, pNO2-Phe33)CCK-26-33] using neuraminidase, O-glycanase and endoglycosidases H and F. Similarities in the core proteins were further demonstrated by Staphylococcus aureus V8 protease peptide mapping, in which both proteins yielded similar fragment patterns. Thus, gallbladder CCK receptors present in man and cow are both N-linked complex glycoproteins, with different carbohydrate domains and similar protein cores.

We have investigated, using indirect immunofluorescence techniques, the possibility that vinculin is a component of Sertoli cell ectoplasmic specializations. Affinity-purified polyclonal antibodies produced against human platelet vinculin were used to probe fixed frozen sections of rat testis. Specific fluorescence occurs in Sertoli cell regions adjacent to spermatids and to basally situated junctional complexes, sites at which ectoplasmic specializations are known to occur. Staining also occurs in Sertoli cell regions associated with tubulobulbar complexes. The antibody also labels focal contacts in cultured human dermal fibroblasts, apical junctional sites of rat epididymal epithelium, and dense plaques of smooth muscle. Our results are consistent with the prediction that vinculin is likely a component of ectoplasmic specializations and are also consistent with the hypothesis that these structures are a form of actin-associated adhesion complex.

Two major Ca2(+)-binding glycoproteins Mr 120,000 and 100,000 were isolated from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid -solubilized bovine heart sarcolemma membrane. Peroxidase-conjugated concanavalin A and wheat germ agglutinin lectins bind strongly to the isolated 120- and 100-kDa glycoproteins. Treatment with endoglycosidase F resulted in conversion of the 120-kDa glycoprotein to a form migrating at about 97 kDa. Treatment of the 100-kDa band with endoglycosidase F produced form of about 80 kDa. Endoglycosidase H digestion removes only 5% of the mass of both glycoproteins. the carbohydrate structure of both glycoproteins, is therefore, predicted to be at least 75% complex structure and 25% high mannose or hybrid structure. The 120- and 100-kDa glycoproteins are the major Ca2(+)-binding proteins in the sarcolemma membranes. Intact and endoglycosidase-treated glycoproteins bind 45Ca2+ as analyzed by a 45Ca2+ overlay technique. Using polyclonal antibodies, the 120- and 100-kDa glycoproteins were identified in muscle plasma membranes (ventricles, atria, and uterus smooth muscle). They were, however, not present in non-muscle tissues such as pancreas, liver, and kidney. The 120- and 100-kDa glycoproteins appear to be homologous molecules as judged by their similar V8 protease peptide maps, cross-reactivity with polyclonal antibody, and other physicochemical properties.

Postvagotomy (PV) gastroparesis is an infrequent but troublesome problem. To test the hypothesis that the rarity of the PV syndrome is due to compensatory up-regulation of muscarinic cholinergic receptors (mAChR), we measured changes in stomach mAChR and gastric acid secretion in dogs before and three weeks after truncal vagotomy. Maximum acid output dropped significantly one week PV and then partially recovered by three weeks PV. mAChR density changed in parallel and was significantly increased in body mucosa, body muscle, and antrum mucosa. In the body, changes in mAChR in mucosa correlated positively with changes in muscle, suggesting that mAChR binding in pinch biopsies of gastric mucosa might become useful in evaluating patients for postvagotomy syndrome. PV up-regulation of mAChR in the mucosa of the canine gastric body might explain PV recovery of gastric acid secretion.

Using light and electron-microscopic immunolocalization techniques, and gel electrophoresis combined with immunoblotting, we have examined the expression of cytoskeletal proteins in normal human fetal, child and adult lenses, in human anterior capsular cataract and in bovine lens cells in vivo and in vitro. In this report, we focus our observations on the pattern of actin-isoform expression during normal and pathological situations in vivo and culture conditions. We have noted that cells of developing and mature human lenses as well as bovine lens cells in situ contain only beta- and gamma-actins. In contrast, alpha-smooth muscle (alpha-sm) actin, an isoform typical of smooth muscle differentiation, was demonstrated in bovine lens cells at different times of culture. Moreover, the multilayered cells observed in the subcapsular zone of human anterior capsular cataract were characterized by the presence of alpha-sm actin. Thus, extensive changes in actin-isoform expression take place in lens cells growing in culture and may also occur during cataractogenesis. The biological meaning of the appearance of a marker of myoid differentiation in the ectodermally derived lens-forming cells is discussed.

The distribution of estrogen and progesterone receptors (ER and PR, respectively) was studied immunohistochemically in the chick oviduct. Estrogen receptor immunoreactivity was found only in the nuclei of glandular epithelial cells. Progesterone receptor was found in the nuclei of glandular and luminal epithelia, stroma, smooth muscle cells and in the mesothelium. The dissimilar distribution of ER and PR suggests that either ER concentration in the luminal epithelium and smooth muscle is very low (below the sensitivity of ER immunostaining) or that estrogens control their PR synthesis indirectly via ER in glandular cells. A known estrogen-inducible protein, ovalbumin, was localized in the same glandular epithelial cells as ER. A progestin-inducible protein, avidin, was found in part of the luminal and glandular epithelium cells but not in other PR-positive cell types. This indicates the importance of cellular differentiation in the regulation of avidin synthesis. Estrogen and progesterone administration had effects also on ER and PR immunoreactivity. Estrogen and progesterone administrations for 24 h decreased markedly the immunoreactivity of their receptors. The decrease in receptor immunoreactivity is most likely due to a transient loss of immunoreactive receptor protein, since the antibodies (H222, PR6) react both with transformed (4 S) and non-transformed (8 S) receptor forms. At the subcellular level, PR was localized in the chromatin by immunoelectron microscopy. Progestin administration seemed to decrease PR immunoreactivity especially in the heterochromatin area, suggesting that conformational chromatin rearrangements occur during down-regulation of PR.

The distribution of mRNAs encoding muscarinic acetylcholine receptor (mAChR) subtypes (M1, M2, M3, and M4) was investigated in the bovine iris-ciliary body by Northern blot hybridization with subtype-specific oligonucleotide probes that were complementary to unique regions of the M1, M2, M3, and M4 mAChRs. Whole rat brain RNA, which contains all four subtypes, was employed as a positive control. Both the iris sphincter and the ciliary processes were found to contain predominantly the M3 mAChR subtype and minor amounts of the M2 subtype. Traces of the M4 subtype were detected also in the ciliary processes.

Two peptides with vasodilatory properties consisting of amino acids 1-30 and 31-67 of the 98 a.a. N-terminal end of the prohormone of atrial natriuretic factor (proANF) which circulates in man were investigated to determine if they have specific binding sites on membranes isolated from DDT1 MF-2 smooth muscle cells. Smooth muscle is a known biologic target of these peptides. Competitive binding experiments revealed that proANFs (1-30), (31-67), and (99-126) (i.e., C-terminus; ANF) each had specific and separate binding sites. The dissociation constants for proANFs (1-30), (31-67), and (99-126) binding were 0.11 nM, 4 nM, and 7.3 nM, respectively. The binding site concentrations for proANFs (1-30), (31-67), and ANF were 2.57, 59.91 and 40 fmols/10(6) cells, respectively. The number of binding sites per cell were 1548, 36,087, and 24,090, respectively, for proANFs (1-30), (31-67), and (99-126) (ANF). Each peptide bound to DDT1 MF-2 membranes between 10(-8) to 10(-11) M but could only bind to the other peptides' receptors at concentrations of 10(-6) and 10(-7)M. These results suggest that proANF(1-30) and proANF(31-67) do not work through the ANF receptor but rather have their own separate and distinct receptors that mediate their biologic effects.

Receptors for galanin in membranes from the rat gastric and jejunal smooth muscle were studied using [125I] radioiodinated synthetic porcine galanin. Specific binding was time and temperature dependent. At 32 degrees C radioligand was degraded in the presence of smooth muscle membranes in a time-dependent manner. At optimal experimental conditions, the equilibrium binding analyses showed the presence of a single population of high affinity binding sites in both the rat stomach and jejunum (Kd value of 2.77 +/- 0.78 nM and 4.93 +/- 1.74 nM for stomach and jejunal smooth muscle membranes, respectively). The concentration of the high affinity binding sites was 58.19 +/- 11.04 and 32.36 +/- 5.68 fmol/mg protein, for gastric and jejunal preparations, respectively. Specific binding was completely inhibited by 10(-6) M of nonradioactive galanin; was 75% blocked by 1 microM of galanin(9-29); it was 10% blocked by 1 microM of galanin(15-29). Galanin(1-15) at a concentration of 1 microM was ineffective for inhibiting [125I]galanin binding. Deletion of four C-terminal amino acid residues from galanin(9-29) to give galanin(9-25) also resulted in almost complete loss of affinity. Radioiodinated galanin and N-terminally deleted fragments had receptor binding potency in the following order: galanin(1-29) greater than galanin(9-29) greater than galanin(15-29). We conclude that the C-terminal part of the galanin chain is important for the rat gastric and jejunal smooth muscle membrane receptor recognition and binding and that N-terminal amino acid sequences are probably not so important, since galanin(1-15) was not active but galanin(9-29) retained most of the receptor binding activity.

A new low molecular weight calcium-binding protein, designated as SMCaBP-11, has been isolated from chicken gizzard using a phenyl-Sepharose affinity column followed by ion-exchange and gel filtration chromatographies. The isolated protein was homogeneous by the criteria of gel electrophoresis in the absence and presence of sodium dodecyl sulfate (NaDodSO4). Molecular weight studies by both sedimentation equilibrium in 6 M guanidine hydrochloride and 15% polyacrylamide-SDS gels indicated the subunit molecular weight to be 11,000, and since a molecular weight of 21,000 was obtained in native solvents, the protein exists as a dimer in benign medium. The amino acid composition of this protein is similar but distinct from other known low molecular weight Ca2(+)-binding proteins. Ca2(+)-binding assays using Arsenazo III (Sigma) indicated the protein to bind 2 mol of Ca2+/subunit. In non-SDS gels, the protein moved faster in the presence of EDTA, suggesting that Ca2+ binding affects its mobility in a manner similar to other smooth muscle calcium-binding proteins such as calmodulin and 67-kDa calcimedin. Upon binding calcium, the protein underwent a conformational change as revealed by UV difference spectroscopy and circular dichroism studies in the aromatic and far-ultraviolet range. When the protein was excited at 280 nm, the tyrosine fluorescence emission maximum was centered at 306 nm. Ca2+ addition resulted in a nearly 15% decrease in intrinsic fluorescence intensity. Fluorescence titration with Ca2+ exhibited two classes of calcium-binding sites with Kd values of 0.2 and 80 microM, in agreement with UV difference spectral data.(ABSTRACT TRUNCATED AT 250 WORDS)

Type VI collagen is a component of 100 nm long periodic filaments with a widespread distribution around collagen fibers and on the surface of cells. It is an unusual collagen constituted by three distinct chains, one of which (alpha 3) is much larger than the others and is encoded by a 9-kb mRNA. The amino acid sequence of the alpha 3(VI) deduced from the present cDNA clones specifies for a multidomain protein of at least 2648 residues made of a short collagenous sequence (336 residues), flanked at the N-terminus by nine 200 residue long repeating motifs and at the C-terminus by two similar motifs that share extensive identities with the collagen-binding type A repeats of von Willebrand factor. Type VI collagen and alpha 3(VI) fusion proteins bound to insolubilized type I collagen in a specific, time-dependent, and saturable manner. The alpha 3(VI) chain has three Arg-Gly-Asp sequences in the collagenous domain, and cell attachment was stimulated by the triple helix of type VI collagen and by alpha 3(VI) fusion proteins containing Arg-Gly-Asp sequences. This function was specifically inhibited by the Arg-Gly-Asp-Ser synthetic peptide. The type I collagen-binding and the cell-attachment properties of the alpha 3(VI) chain provide direct information for the role of type VI collagen in connective tissues.

Acid extracts of human intestines obtained from surgical samples or from organ donors contain cholecystokinin (CCK) immunoreactivity. From surgical samples, extracted and eluted quickly, greater than 75% of the CCK immunoreactivity eluted in the same region as purified canine CCK-58 during analytical reverse-phase high-pressure liquid chromatography (HPLC). A major portion of the CCK immunoreactivity from donor intestinal extracts also eluted in this region. This immunoreactivity has been purified from human intestinal extracts by a series of several reverse-phase and cation-exchange chromatographies. Amino acid and microsequence analysis showed that this immunoreactivity is human CCK-58. Tryptic digestion of purified human CCK-58 produced another immunoreactive form that eluted in the position of CCK-8 during analytical reverse-phase HPLC. The immunoreactivity of the trypsin-digested material was 2.6-fold higher than that of an identical sample of CCK-58 incubated without trypsin. Thus the carboxyl-terminal antibody used for radioimmunoassay cross-reacts greater than twofold less with human CCK-58. This diminished cross-reactivity would lead to an underestimation of the relative proportions of CCK-58 in tissue and plasma extracts. If CCK-58 is the major circulating form this diminished cross-reactivity would also lead to underestimations of the circulating levels of total CCK. Determination of human CCK-58 structure confirms that one of the major components of human CCK that expresses biological activity is CCK-58.

Cells containing smooth muscle myosin were localized in the human aqueous outflow pathway by immunohistochemical techniques. In the majority of eyes, immunoreactive cells were observed adjacent to the collector channels and slightly distal to the outer wall of Schlemm's canal. In a few eyes, smooth muscle myosin was localized to cells in the juxtacanalicular tissue and the trabecular meshwork. The immunoreactive cells from these regions may be true smooth muscle cells or pericytes, which can contain smooth muscle myosin. No obvious differences were observed in the pattern of distribution of smooth muscle myosin-containing cells in a comparison of age groups. In the majority of eyes, we observed an apparent direct insertion of the longitudinal portion of the ciliary muscle in the corneoscleral meshwork far internal to the scleral spur.

The distribution of cells expressing the integrins VLA-1 to 6 in human intestine was examined by alkaline phosphatase immunohistochemistry using monoclonal antibodies specific for the individual alpha-chains of the VLA heterodimer. VLA-2,3, and 6 were expressed on all epithelial cells in the small and large bowel. VLA-1 was expressed on crypt cells in the small and large bowel, but was only weakly expressed or was absent on villus epithelial cells in the small bowel and colonic surface epithelial cells. All epithelia were negative for VLA-4 and VLA-5. Intraepithelial lymphocytes were VLA-1+ and VLA-4+. VLA-1,3, and 5 were expressed uniformly by muscularis propria, muscularis mucosae, pericrypt cells, and smooth muscle fibres within the villi. By contrast, VLA-2 and 4 were present only in pericrypt cells and fibres within the villi; they were absent from the muscularis mucosae. VLA-1,3,5, and 6 were expressed by endothelium. Staining of muscle fibres and endothelium in the lamina propria made it difficult to determine the extent of VLA expression on lamina propria lymphocytes. However, VLA-1+ cells with lymphoid morphology were only rarely seen. All mononuclear cells in the lamina propria were VLA-4+.

P19 embryonal carcinoma (EC) cells can be induced in vitro to differentiate into cells resembling those normally formed in the embryo. Among these cell types is one whose morphology is fibroblast-like. Using indirect immunofluorescence and Western blot analysis with antibodies directed against various isoforms of actin, many of these fibroblast-like cells were found to express smooth muscle actin isoforms. Northern blot analysis of RNA indicated the presence of a smooth muscle-specific isoform of myosin heavy-chain mRNA in immortal lines of these fibroblast-like cells. These results suggest that these fibroblast-like cells resemble fetal myofibroblastic or myoepithelial cells, which have a wide distribution during embryonic development.

Using the surgically separated epithelium and muscle preparation of the guinea pig seminal vesicle, quantitative determinations were made of the hormonal sensitivities of three components of male accessory sex organ fibromuscular stroma. The parameters measured were epithelial collagen, muscle tissue cell number, and muscle tissue collagen. Epithelial collagen increased approximately tenfold between one week of age and adulthood. This growth was completely prevented by castration and was sustained at normal levels of maintenance of prepubertal castrates with dihydrotestosterone (DHT). Castration of adult animals reduced epithelial collagen 40%; such reductions were prevented by DHT treatment. Estrogen treatment of adult castrates, either alone or in combination with DHT, had no anabolic effect on the epithelium. In all of these studies, changes in epithelial collagen correlated with changes in epithelial wet weight, DNA content, and RNA/DNA. From 1 week of age to adulthood, growth of seminal vesicle muscle involved marked increases in wet weight, DNA content, RNA/DNA, and collagen content. Increments in all parameters were prevented by prepubertal castration and were sustained at normal levels in castrates by DHT treatment. However, in adult animals, muscle DNA content and collagen levels were insensitive to castration and androgen replacement; only tissue weight and cell size (RNA/DNA) were androgen sensitive. Estrogen treatment of adult castrates caused supranormal increases in muscle DNA and collagen; changes in collagen were irreversible. The effects of simultaneous treatment of DHT and estrogen did not differ from those of DHT alone. Therefore, normal postnatal development of the stromal components, epithelial collagen, muscle cells, and muscle collagen was androgen dependent, but in adult animals, only epithelial collagen retained androgenic sensitivity. Estrogen induced supranormal and irreversible growths in the muscle.

Pig small intestine was used as starting material for a batchwise isolation of a peptide fraction enriched in antibacterial activities against Escherichia coli (anti-Ec factor) and against Bacillus megaterium (anti-Bm factor). Separation and further purification were by different types of chromatography. Sequence analysis showed the anti-Bm factor to be apparently similar to vasoactive intestinal peptide. The anti-Ec factor was found to have a 31-residue sequence that was cecropin-like. It was named cecropin P1 and its structure was confirmed by solid-phase synthesis. Synthetic cecropin P1 with and without C-terminal amide was assayed on eight different bacteria. Mobility comparison between synthetic and natural cecropin P1 indicates that the natural peptide has a free C-terminal carboxyl group.

Using pure leukotriene D4 (LTD4) as the agonist, we determined the dissociation constants, Kb and pA2 values, for the selective leukotriene antagonist FPL 55712 on guinea pig ileum, trachea, and parenchyma. Responses of the 3 tissues to LTD4 were competitively antagonized by FPL 55712. Kb and pA2 values were similar for trachea and parenchyma. However, these values differed from those obtained in ileum. We propose the existence of multiple LTD4 receptors, with those in lung differing from LTD4 receptors in ileum.

Neurogenic bladder dysfunction was created in male rats by removal of the pelvic ganglia. The bladders were then emptied manually once daily, and kept free from infection. After 10 days or six weeks the bladders were taken out and the mucosa-submucosa was removed. The detrusors were then weighed and used for collagen assay. Detrusor weight increased 4.5 and six times after 10 days and six weeks, respectively. Total detrusor collagen increased 2.3 and 3.7 times, but due to the increase in detrusor weight the concentration decreased to 60 per cent of normal. The electron microscopic investigation showed that the cross-sectional area of the smooth muscle cells had increased fourfold after six weeks. The collagen fibrils were found mainly in the interstitial tissue between the muscle bundles. As these increased in size following the neurogenic lesion, the collagen-rich tissue component decreased relatively. Our conclusion is that frequent emptying and the avoidance of bladder infection protects the denervated rat urinary bladder wall from injuries that would otherwise lead to an increased collagen concentration.

The ATP-induced disassembled molecules of chicken gizzard myosin from "thick filaments" have been examined in the electron microscope using the rotary-shadowing technique and have been compared with the myosin molecules disassembled by high concentrations of ammonium acetate without ATP. Both myosin molecules consisted of two globular heads and a long tail. However, two remarkable differences between these myosin molecules were observed: 1. Most of the myosin molecules disassembled by ATP had intramolecular hairpin "loops" in the tails. The length of the hairpin loop was about 510 A and that of the remaining part of the tail was about 600 A. On the other hand, no myosin molecules disassembled by ammonium acetate had the intramolecular loop in the tail. 2. The two globular heads of myosin molecules disassembled by ATP tended to bend back towards the tail, but those of the myosin molecules disassembled by ammonium acetate tended to bend forwards.

The relationship between isometric tension and the increase in cyclic GMP level associated with the initial phase of drug-induced contraction of taenia coli was studied. Maximal contraction induced by 100 micrometers carbachol or 124 mM KCl occurred in 30 sec and was associated with an increase in cyclic GMP levels of at least ten fold. However, 100 micrometers serotonin or low concentrations of carbachol (30 microM) produced nearly maximal contraction but only 10--20% of the maximal elevation in cyclic GMP induced by high concentrations of KCl. Furthermore, in the presence of a low concentration of Ca2+, or verapamil, both 124 mM KCl and 100 micrometers carbachol increased cyclic GMP with little elevation in isometric tension. Thus, large contractions were obtained with only small increases in cyclic GMP, and under other conditions, large increases in cyclic GMP were obtained with little increase in isometric tension. The results suggest that the initial increase in cyclic GMP level caused by these agents is not directly dependent on Ca2+ in the cellular pool containing the contractile proteins.

Three muscarinic acetylcholine receptor subtypes may be defined on the basis of functional and binding studies using selective antagonists. The subtypes may be solubilized in a stable form in digitonin. In solution, the subclasses still exhibit different structure-binding relationships but these have been perturbed by solubilization. The binding of the selective antagonist, pirenzepine, to the purified cortical receptor is complex and similar to that found in membranes. The muscarinic receptor subclasses thus appear to be different molecular entities. Possible explanations for the molecular heterogeneity are discussed. It has also been possible to solubilize receptor-GTP binding protein complexes which have higher sedimentation coefficients (13.4 S) than the apparently monomeric receptor (11.6 S).

The localization of Ca-accumulating structures in the longitudinal body wall muscle (LBWM) of the opisthobranch mollusc Dolabella auricularia and their role in the contraction-relaxation cycle were studied by fixing the LBWM fibers at rest and during mechanical response to 400 mM K or to 10(-4)--10(-3) M acetylcholine in a 1% OsO4 solution containing 2% K pyroantimonate. In the resting fibers, electron-opaque pyroantimonate precipitate was mostly localized at the peripheral structures, i.e., along the inner surface of the plasma membrane, at the membrane of the surface tubules, and at the sarcoplasmic reticulum. In the fibers fixed during mechanical activity, the precipitate was diffusely distributed in the myoplasm in the form of numerous particles with corresponding decrease in the amount of the precipitate at the peripheral structures. Electron-probe X-ray microanalysis showed the presence of Ca in the precipitate, indicating that the precipitate may serve as a measure of Ca localization. These results are in accord with the view that, in the LBWM, the Ca stored in the peripheral structures is released into the myoplasm to activate the contractile mechanism.

The penile artery of the bull contained significant amounts of acetylcholine, noradrenaline and dopamine, and its axon profiles contained either numerous small granular or agranular vesicles, as well as some large granular vesicles. In the dorsal metatarsal artery, only noradrenaline and dopamine were detectable, and the axon profiles observed contained numerous small granular vesicles. In the penile artery, the axons with small agranular vesicles, probably cholinergic axons, were in close contact with axons containing small granular vesicles. It is suggested that, in the penile artery of the bull, one of the functions of the cholinergic nerves is suppression of excitatory adrenergic neurotransmission.

Though the therapeutic efficacy of glucocorticoids on bronchial asthma has been attributed mainly to their anti-allergic and anti-inflammatory effects, the possibility of intervention of glucocorticoids in bronchial asthma might be an important parallel process. However, little is known about the mechanism of glucocorticoid action on tracheal muscle and there is insufficient basic information about the glucocorticoid receptor of tracheal muscle. Accordingly we set out to analyze the glucocorticoid receptor of bovine tracheal muscle. Bovine tracheal muscle was used because it is plentiful enough for biochemical analysis. Our results showed that a small amount (0.08 pmol/mg.protein) of glucocorticoid binder of high affinity (kd = 4.6 nM) was present in the cytosol of bovine bronchial muscle. The binder did not show any affinity for estradiol, dihydrotestosterone or andronsterone, but showed a low affinity for progesterone. Furthermore, the binder underwent salt-dependent variation of its molecular form (9.2S under low salt condition and 4.6S under high salt condition) typical of steroid receptors. These results indicated the presence of a glucocorticoid receptor in the tracheal muscle, suggesting that glucocorticoid acts on bronchial muscle in therapeutic practice on bronchial asthma.

Adenosine triphosphate (ATP) caused contraction of the resting isolated rat anococcygeus muscle. Non-phosphorylated purines did not cause contraction of the resting muscle but did so in muscles in which the tone was raised by carbachol or guanethidine. Adenosine, (-)N6-phenylisopropyladenosine (PIA) and 5'-N-ethyl-carboxamide adenosine (NECA) were approximately equipotent, and these responses were not prevented by theophylline, quinidine, 2,2'-pyridylisatogen tosylate, phentolamine, methysergide, dipyridamole, hexobendine or indomethacin. The contractions became smaller as muscle tone progressively declined, and it is suggested that this effect may explain the apparent blockade of ATP responses by indomethacin reported previously. Adenosine, 2-chloroadenosine, ATP, PIA and NECA inhibited contractile responses of the anococcygeus to field stimulation of the excitatory adrenergic innervation. This inhibitory action was blocked by theophylline, and as PIA was easily the most potent purine tested, it may involve activation of an A1/Ri receptor. It is also argued, however, that the A/R scheme of classification may be inappropriate for the description of responses of intact tissues. As response to noradrenaline were not changed by the purines, the inhibitory effect on stimulation-evoked contractions is probably mediated at a presynaptic site. None of the purines tested had any effect on the neurally mediated inhibition of the anococcygeus which is seen when intrinsic tone is raised and the excitatory adrenergic nerves are blocked.

The ultrastructural localization and relations of substance P- and met-enkephalin-labeled neuronal structures were examined in the wall of the human gastric antrum during early fetal life. By 14-16 weeks of gestation, clearly discernable neural plexuses and a well developed external muscle coat were present. In the submucous coat, neural plexuses varied from immature forms consisting of 1-4 neurites partially enveloped by Schwann cell processes to more mature plexuses where neurons were completely enclosed by Schwann cell processes. Neuronal profiles with substance P- and met-enkephalin-like immunoreactivities were observed in the submucous plexus. In the myenteric plexus met-enkephalin-like immunoreactivity was seen within cell bodies and neurites. By contrast, although substance P-like immunoreactivity was observed in neurites in the myenteric plexus, no substance P-labeled somata could be identified. Unlabeled terminals were seen in contact with both unlabeled dendrites and met-enkephalinergic neurons. An increase in electron density was observed at the sites of contact. These structures probably represent early stages in the development of synaptic specializations. In addition, met-enkephalin-labeled varicosities were seen in apposition to smooth muscle cells of the circular muscle coat. This suggests that antral smooth muscle cells are directly innervated by met-enkephalin neurons.

The microfibrils associated with elastic tissue have been shown to be predominantly proteinaceous. On the basis of their affinity for cationic stains, including ruthenium red, they have been assumed to be glycoprotein, but more evidence to support this claim has not been adduced. Despite repeated investigation of glycoprotein materials obtained by extraction of elastic tissues with reagents that appear to remove microfibrils, the chemical composition of elastin-associated microfibrils remains obscure. An electron microscopic study of the microfibrils in two elastin-rich tissues (bovine nuchal ligament and aorta) during their development was pursued using more specific histochemical methods. The periodic acid-alkaline bismuth stain (analogous to the periodic acid-Schiff stain for glycoproteins in light microscopy) has been adapted for this study. Specific aldehyde groups (confirmed by blocking with m-aminophenol or sodium borohydride) were identified after periodate oxidation as fine granules of bismuth stain. These were shown to localize specifically along the elastin-associated microfibrils in a finely punctate form. Staining of the amorphous elastic component did not occur except for a fine rim adjacent to the microfibrils. Lectin binding with concanavalin A (with ferritin markers) confirmed that there are glucose- or mannose-containing proteins associated with the microfibrillar component of elastic tissue. This was true of these microfibrils in all layers of the aortic wall and throughout the ligament. It was also true of mature adult tissues in which there was a lesser proportion of microfibrils. It is concluded that elastin-associated microfibrils really are associated with glycoprotein(s).

Antibodies reacting with chicken gizzard caldesmon were used to determine the distribution of caldesmon on smooth muscle thin filaments. Antibodies developed against both the intact caldesmon molecule and a 40 kilodalton proteolytic fragment cause thin filaments to aggregate laterally. Aggregates produced with the latter antibody display regular periodic labelling with a repeat of approximately 38 nm, a distribution characteristic of proteins associated with tropomyosin on thin filaments. The stoichiometry of caldesmon on thin filaments has been critically reevaluated and alternative models of caldesmon distribution on thin filaments are proposed.

The presence or absence of autoantibodies in acute hepatitis B was investigated longitudinally in a prospective study of 38 patients, 37 of whom recovered completely. Antibodies to nuclei, bile canaliculi or mitochondria were not found in any of the 354 investigated sera. Smooth muscle antibody (SMA) was present in 23 patients for median 4 weeks and from (median) -1 to +3 weeks from peak SGPT. Titers reached from 1:10 to 1:400, with a median of 1:50. In the patient with persistent HBs-antigenemia, SMA - present in low titer (1:10) - persisted as well. Besides smooth muscle cells, other localisations were: glomerular (15 patients), around hepatocytes ('polygonal' 11 pts), around renal tubuli (10) and in the gastric mucosal layer (8). These fluorescence patterns, the presence of which was not correlated to the SMA titer height, disappeared either earlier than or simultaneously with smooth muscle cellular fluorescence. A maximal titer greater than 1:50 was associated with a thymol turbidity ten times the upper limit of normal or more (i.e. greater than 25 S-H U) and with the peak serumgammaglobulin about simultaneously with peak SGPT. The presence of SMA was not correlated with extrahepatic manifestations nor with the peak values attained for ESR, SGPT, alkaline phosphatase or bilirubin.

The neuromuscular junctions and nonjunctional sarcolemmas of mammalian skeletal muscle fibers were studied by conventional thin-section electron microscopy and freeze-fracture techniques. A modified acetylcholinesterase staining procedure that is compatible with light microscopy, conventional thin-section electron microscopy, and freeze-fracture techniques is described. Freeze-fracture replicas were utilized to visualize the internal macromolecular architecture of the nerve terminal membrane, the chemically excitable neuromuscular junction postsynaptic folds, and the electrically excitable nonjunctional sarcolemma. The nerve terminal membrane is characterized by two parallel rows of 100-110-A particles which may be associated with synpatic vesicle fusion and release. On the postsynpatic folds, irregular rows of densely packed 110-140-A particles were observed and evidence is assembled which indicates that these large transmembrane macromolecules may represent the morphological correlate for functional acetylcholine receptor activity in mammalian motor endplates. Differences in the size and distribution of particles in mammalian as compared with amphibian and fish postsynaptic junctional membranes are correlated with current biochemical and electron micrograph autoradiographic data. Orthogonal arrays of 60-A particles were observed in the split postsynaptic sarcolemmas of many diaphragm myofibers. On the basis of differences in the number and distribution of these "square" arrays within the sarcolemmas, two classes of fibers were identified in the diaphragm. Subsequent confirmation of the fiber types as fast- and slow-twitch fibers (Ellisman et al. 1974. J. Cell Biol.63[2, Pt. 2]:93 a. [Abstr.]) may indicate a possible role for the square arrays in the electrogenic mechanism. Experiments in progress involving specific labeling techniques are expected to permit positive identification of many of these intriguing transmembrane macromolecules.

Exposure to the beta-adrenergic agonist, metaproterenol, elicits extensive receptor loss and desensitization of adenylate cyclase activity in the hamster DDT1, MF-2 cell line. The reappearance of beta-adrenergic receptors and restoration of adenylate cyclase activity were investigated. Receptor reappearance was investigated under conditions in which lost receptors were not detectable either on the cell surface or within the cell. Exposure to metaproterenol resulted in a 3-5-fold decrease in beta-adrenergic receptor affinity for agonist, an 85% reduction in beta-adrenergic receptor number per cell, and a 65% reduction in isoproterenol-stimulated adenylate cyclase activity without any change in NaF-stimulated enzyme activity. The rate of reappearance of the lost receptors was proportional to the concentration of metaproterenol to which the cells were initially exposed. Metaproterenol, at a concentration of 250 microM, induced long-term receptor loss which required 16 days in fresh media devoid of metaproterenol before the full complement of receptors reappeared. This prolonged receptor loss may be due to residual metaproterenol; however, the resensitization of isoproterenol-stimulated adenylate cyclase activity was restored 2 days after removal of metaproterenol. The lag period for the reappearance of receptors was shortened by incubation with either the beta-adrenergic antagonist, nadalol, or the glucocorticoid, methylprednisolone. Both pharmaceuticals reversed receptor down-regulation and up-regulated receptor number in control cells, although the extent and time course of restoration were different. These data suggest that the process of resensitization in DDT1 cells involves rapid restoration of adenylate cyclase activity and a slower reappearance of receptors over a time period of six population doublings.

It was found that thin filaments from chicken gizzard muscle activate skeletal muscle myosin Mg2+-ATPase to a greater extent than does the complex of chicken gizzard actin and tropomyosin. The protein factor responsible for this additional activation has been now identified as the high Mr actin binding protein, filamin.

In this study of the human dental pulp, substances were found in smooth muscle cells, myoendothelial cells, fibroblasts, and odontoblastic processes that demonstrate staining and extraction characteristics identical to myosin in muscle. It is concluded that the myosinlike material similar to that found in cells elsewhere in the human also occurs in cells of the dental pulp.