Vascular Epithelium Research Articles

Mechanotransduction in Caenorhabditis elegans: the role of DEG/ENaC ion channels.

One of the looming mysteries in signal transduction today is the question of how mechanical signals, such as pressure or mechanical force delivered to a cell, are interpreted to direct biological responses. All living organisms, and probably all cells, have the ability to sense and respond to mechanical stimuli. At the single-cell level, mechanical signaling underlies cell-volume control and specialized responses such as the prevention of poly-spermy in fertilization. At the level of the whole organism, mechanotransduction underlies processes as diverse as stretch-activated reflexes in vascular epithelium and smooth muscle; gravitaxis and turgor control in plants; tissue development and morphogenesis; and the senses of touch, hearing, and balance. Intense genetic, molecular, and elecrophysiological studies in organisms ranging from nematodes to mammals have highlighted members of the recently discovered DEG/ENaC family of ion channels as strong candidates for the elusive metazoan mechanotransducer. Here, we discuss the evidence that links DEG/ENaC ion channels to mechanotransduction and review the function of Caenorhabditis elegans members of this family called degenerins and their role in mediating mechanosensitive behaviors in the worm.

Immunohistochemical localization of cytochrome P4501A induced by 3,3′,4,4′,5‐pentachlorobiphenyl (PCB 126) in multiple organs of northern leopard frogs, Rana pipiens

Monoclonal antibody 1-12-3 (MAb 1-12-3) recognizes an epitope exclusive to cytochrome P450s in subfamily 1A (CYP1A) from all vertebrates tested so far, including one amphibian species. In this study, we first tested the utility of MAb 1-12-3 for detection of presumed CYP1A proteins in hepatic microsomes of northern leopard frogs treated without or with 3,3',4,4',5-pentachlorobiphenyl (PCB 126). Statistical analysis showed that ethoxyresorufin-O-deethylase (EROD) activities and CYP1A equivalents in treated groups were significantly increased at doses > or = 2.3 mg/kg compared with the control groups (p < 0.05), and the increases were maintained for at least four weeks. This result confirmed that MAb 1-12-3 can be used for detection of CYP1A in northern leopard frogs and indicated that CYP1A is the primary catalyst for EROD in this species. In a subsequent experiment, sections of organs of PCB 126-treated frogs were immunohistochemically stained with MAb 1-12-3 to identify localization of the CYP1A in different cell types. The CYP1A staining was seen prominently in hepatocytes and epithelium of nephronic duct, while capillaries close to gastric epithelium and submucosal vascular epithelium in both stomach and intestine exhibited moderate to strong staining. The CYP1A was immunodetected in coronary endothelium and the vascular endothelium of lung and gonad. In skin, mild staining was seen in epithelial cells of mucous glands and serous glands and in vascular endothelium, demonstrating induction of CYP1A in the dermal layer.

TGF-beta1 and fibroblast growth factor-1 modify fibroblast growth factor-2 production in type II cells.

Fibroblast growth factor (FGF)-2, which stimulates DNA synthesis by type II cells in the lung, has been shown to be regulated by transforming growth factor (TGF)-beta1, an important inflammatory cytokine, in vascular epithelium. The goal of this study was to determine if FGF-2 production by alveolar type II cells is modulated by TGF-beta1 or FGF-1, which also stimulates DNA synthesis by type II cells. Isolated rat type II cells were exposed to 0-40 ng/ml of TGF-beta1 or 0-500 ng/ml of FGF-1 in serum-free medium for 1-5 days. With a specific immunoassay, significant increases of FGF-2 protein in type II cell lysates to levels above those in control cells were achieved after 1 day of exposure to 100 ng/ml of FGF-1 and after 3 days of treatment with 8 ng/ml of TGF-beta1. Similarly, transcripts for FGF-2 were dramatically increased above those in control cells with TGF-beta1 or FGF-1, as were those for FGF receptor-1. These results demonstrate important regulatory links between FGF-2 and both TGF-beta1 and FGF-1 in the alveolar epithelium that could contribute to the regulation of normal cell turnover, development, and the repair processes after injury in the lung.

Blood Supply and Function of the Tegmentum Vasculosum (Stria Vascularis) of the Duckling Cochlea

Abstract On the basis of ultrastructural, biochemical, cytochemical, and electro-physiological evidence, the stria vascularis, a highly vascular epithelium of the mammalian cochlea, has been implicated as one site of endolymph synthesis. It would seem natural to suspect that its highly developed counterpart in the avian cochlea, the tegmentum vasculosum (TV), might have a similar function. This study utilizes routine light and electron microscopy, biochemistry, cytochemistry, and vascular corrosion casting to describe the structure and blood supply to the (TV) in the duckling and provide evidence for its involvement in endolymph synthesis. Four to 16 week-old ducklings (Anas platyrhyncos)were anesthetized and anticoagulated and perfused via the common carotid arteries with buffered, 2% glutaraldehyde for routine light microscopy or transmission or scanning electron microscopy, or with Mercox resin for vascular corrosion casting. Casts were macerated in 5% KOH and warm water to remove tissue, cleaned in formic acid and water, dried by lyophilization, mounted on stubs and viewed at 10-20 kv by SEM.

Morphogenesis of the first blood vessels.

The initial phase of vessel formation is the establishment of nascent endothelial tubes from mesodermal precursor cells. Development of the vascular epithelium is examined using the transcription factor TAL1 as a marker of endothelial precursor cells (angioblasts), and a functional assay based on intact, whole-mounted quail embryos. Experimental studies examining the role(s) of integrins and vascular endothelial growth factor (VEGF) establish that integrin-mediated cell adhesion is necessary for normal endothelial tube formation and that stimulation of embryonic endothelial cells with exogenous VEGF results in a massive "fusion" of vessels and the obliteration of normally avascular zones. The second phase of vessel morphogenesis is assembly of the vessel wall. To understand the process by which mesenchyme gives rise to vascular smooth muscle, a novel monoclonal antibody, 1E12, that recognizes smooth muscle precursor cells was used. Additionally, development of the vessel wall was examined using the expression fo extracellular matrix proteins as markers. Comparison of labeling patterns of 1E12 and the extracellular matrix molecules fibulin-1 and fibrillin-2 indicate vessel wall heterogeneity at the earliest stages of development; thus smooth muscle cell diversity is manifested during the differentiation and assembly of the vessel wall. From these studies it is postulated that the extracellular matrix composition of the vessel wall may prove to be the best marker of smooth muscle diversity. The data are discussed in the context of recent work by others, especially provocative new studies suggesting an endothelial origin for vascular smooth muscle cells. Also discussed is recent work that provides clues to the mechanism of vascular smooth muscle induction and recruitment. Based on these findings, vascular smooth muscle cells can be thought of as existing along a continuum of phenotypes. This spectrum varies from mainly matrix-producing cells to primarily contractile cells; thus no one cell type typifies vascular smooth muscle. This view of the smooth muscle cell is considered in terms of a contrasting opinion that views smooth muscle cell as existing in either a synthetic or proliferative state.

Patterns of bromodeoxyuridine incorporation and neuropeptide immunoreactivity during arm regeneration in the starfish Asterias rubens

Regeneration of the arm of the starfish,Asterias rubens(L.) (Echinodermata: Asteroidea) was examined using two preparations. The first involved regeneration of the entire arm tip and its associated sensory structures and the second examined regeneration of a small section of radial nerve cord in the mid–arm region. Cell cycle activity was investigated by incorporation of the thymidine analogue, bromodeoxyuridine (BrdU). Details of neuroanatomy were obtained by immunocytochemistry (ICC) using an antiserum to the recently isolated starfish neuropeptide, GFNSALMFamide (S1). BrdU labelling indicated that initial events occur by morphallaxis, with cell cycle activity first apparent after formation of a wound epidermis. As regeneration proceeded, BrdU immunoreactive (IR) nuclei revealed cell cycle activity in cells at the distal ends of the radial nerve cord epidermis, in the coelomic epithelium, the perihaemal and water vascular canal epithelia, and in the forming tube feet of both preparations. By varying the time between BrdU pulses and tissue fixation, the possible migration or differentiation of labelled cells was investigated. Neuropeptide ICC indicated the extension of S1–IR nerve fibres into the regenerating area, soon after initial wound healing processes were complete. These fibres were varicose and disorganized in appearance, when compared to the normal pattern of S1–IR in the radial nerve. S1–IR was also observed in cell bodies, which reappeared in the reforming optic cushion and radial nerve at later stages of regeneration. Double labelling studies with anti–BrdU and anti–S1 showed no co–localization in these cell bodies, in all the stages examined. It appeared that S1–IR cells were not undergoing, and had not recently undergone, cell cycle activity. It cannot be confirmed whether S1–IR neurons were derived from proliferating cells of epithelial origin, or from transdifferentiation of epithelial cells, although the former mechanism is suggested. Differentiation of the regenerating structures to replace cells such as S1–containing neurons, is thought to involve cell cycle activity and differentiation of epithelial cells in the epidermal tissue, possibly in association with certain types of coelomocytes which move into the regenerating area.

CARBONIC ANHYDRASE ACTIVITY AND ZINC DEFICIENCY

A 60-year-old female was hospitalized because of anemia, edema, and diarrhea. She was diagnosed as having short bowel syndrome. The patient developed acrodermatitis enteropathica and taste impairment. Laboratory data showed that the serum zinc level was 21 μ g/dl and erythrocyte CAI specific activity was 0 units/mg isozyme (normal range 0.37 ± 0.08 units/mg isozyme) and CAII specific activity was 2.99 units/mg isozyme (normal range 3.02 ± 0.05 units/mg isozyme). The patient was diagnosed as being in a zinc deficient status. Zinc supplementation resulted in the disappearance of these complications. The serum zinc level reached 50 μ g/dl and erythrocyte CAI specific activity was recovered to 0.26 units/mg isozyme and CAII specific activity was 1.60 units/mg isozyme. CAI is found in gastrointestinal epithelial cells, in vascular epithelium, corneal, lens, ciliary body epithelium, and in sweat glands. CAII is found in virtually all tissues and is especially abundant in secretory and absorbing epithelia. The tissue distribution of CAI corresponds to clinical and physiologic indicators of zinc deficiency. Thus, the erythrocyte CAI specific activity may reflect the actual tissue zinc deficiency status.

Apport de l'angiographie au Vert d'Indocyanine (ICG) dans la localisation des néovascularisations chororoidiennes occultes

To determine the role of Indocyanin Green (ICG) angiography in localizing occult new vessels associated with age-related macular degeneration (ARMD) and assess the possibilities of ICG guided laser photocoagulations. Fluorescein and ICG angiographies (IMAGEnet system) of 62 patients with occult new vessels (ONV), serous (SPED) or vascular (VPED) pigment epithelium detachment have been studied. Based on fondoscopic examination and fluorescein angiography, 43 eyes (69%) disclosed ONV, 8 (13%) SPED and 11 (18%) VPED. Choroidal neovascularisation was confirmed by ICG angiography in 37 ONV cases (86%), in 8 (72%) VPED cases, but in no SPED. Conversion of ONV in classical neovascular membranes was possible in 19 ONV cases (44%) and in 6 (54%) VPED cases, making a laser photocoagulation possible in 9 eyes (36%). ICG angiography plays an important role in the evaluation, classification and laser treatment of patients with ONV secondary to ARMD.

The immunohistochemical composition of the human corneal basement membrane.

Frozen sections of human cornea and conjunctiva were stained by immunofluorescence and immunoperoxidase methods with a panel of antibodies against various basement membrane (BM) and extracellular matrix proteins to study the composition of the human corneal BM. Laminin (Ln) immunoreactivity was detected in the BMs of the blood vessels and conjunctival, limbal, and corneal epithelia by using a polyclonal antiserum and monoclonal antibodies (MAbs) against Ln chains alpha 1, beta 1, and gamma 1, but in the cornea the reaction with MAbs against alpha 1, beta 1, and gamma 1 chains was clearly weaker than in the other BMs and in some sections totally absent. Immunoreactivity to Ln alpha 2 chain was detected only in the BM of the limbus, whereas Ln beta 2 chain was detected in the BMs of blood vessels and that of conjunctival and limbal epithelia. Ln-5 and Ln alpha 3 and beta 3 chains were seen in the BMs of the corneal, limbal, and conjunctival epithelia coaligning the integrin beta 4. Immunoreactivity for collagen type IV was lacking in the BM of the corneal epithelium, but present in the BMs of the limbal and conjunctival epithelia as well as in the vascular BM. Collagen type VII immunoreactivity was seen in the BM areas of the corneal, limbal, and conjunctival epithelia. BMs of corneal, limbal, conjunctival, and vascular epithelia expressed fibronectin (Fn), including extradomain A cellular Fn and oncofetal cellular Fn isoforms, all lacking, however, extradomain B cellular Fn. The present results give new information on the Ln chain distribution and show that the BM of the corneal epithelium differs from that of surrounding epithelia by the lack of collagen type IV.

Expression of nitric oxide synthase in the human nasal mucosa.

The nasal mucosa plays an important role in defense of the lung against harmful agents. It has been suggested that this is partly mediated by the production of nitric oxide (NO). We have investigated the localization of the messenger ribonucleic acids (MRNAs) for human endothelial NO synthase (Type III NOS) and inducible NO synthase (Type II NOS) and the immunoreactivities of these enzymes in human nasal mucosa by immunohistochemistry, in situ hybridization, and reduced nicotinamide adenine diphosphate (NADPH) diaphorase histochemistry. Inferior nasal turbinates were obtained from 27 patients at the time of surgery for local disease. Strong immunostaining for Type III NOS was localized to vascular endothelium, surface epithelium, and submucosal glands in all subjects. Moderate immunostaining for Type II NOS was seen in surface epithelium; glandular, inflammatory, and vascular endothelial cells; and smooth-muscle cells in the specimens from patients with chronic rhinitis only. In situ hybridization showed expression of the mRNA for Type III NOS in similar sites to those shown by immunohistochemistry, whereas the mRNA for Type II NOS was predominantly localized to inflammatory cells. The sites of NOS expression were further confirmed by NADPH histochemical staining. These findings demonstrate the cellular expression of NOS in the human nasal mucosa and suggest a possible role for Types II and III NO synthase in the regulation of blood flow, nasal secretion, and ciliary movement in health and disease.

Differential expression of complement proteins and regulatory decay accelerating factor in relation to differentiation of cultured human colon adenocarcinoma cell lines.

Self protection of host cells against inadvertent injury resulting from attack by autologous complement proteins is well reported for vascular epithelium. In intestinal epithelium, the expression of C complement proteins and regulatory proteins remains currently poorly reported. This study looked at the distribution of C complement proteins and regulatory decay accelerating factor (DAF) in four cultured human intestinal cell lines of embryogenic or colon cancer origins. C3 and C4 proteins and DAF were widely present in human colon adenocarcinoma T84, HT-29 glc-/+ cells compared with human embryonic INT407 cells. In contrast, no expression of C5, C5b-9, and CR1 was seen for any of the cell lines. Taking advantage of the Caco-2 cells, which spontaneously differentiate in culture, it was seen that the C3, C4, and DAF were present in undifferentiated cells and that their expression increased as a function of the cell differentiation. These results, taken together with other reports on the presence of C complement proteins and DAF in the intestinal cells infer that the expression of regulatory C complement proteins develops in parallel with the expression of C proteins to protect these cells against the potential injury resulting from the activation of these local C proteins. Moreover, the finding that the pathogenic C1845 Escherichia coli binds to the membrane bound DAF in the cultured human intestinal cells synthetising locally C proteins and regulatory C proteins supports the hypothesis that E coli could promote inflammatory disorders by blocking local regulatory protein function.

Alpha-V/beta-3 and alpha-V/beta-5 integrin distribution in neoplastic kidney.

Integrins are transmembrane glycoproteins that mediate cell-cell and cell-matrix interactions. Altered integrin expression may contribute to tumor progression, invasiveness and metastases. The alpha-V/beta-3 (alpha v beta 3; osteopontin/ vitronectin receptor) has recently been implicated in neovascularization and tumor-induced angiogenesis. alpha v-Subunit also associates with beta 5 to form an alpha v beta 5-complex, another vitronectin receptor. We studied tissue distribution of alpha v beta 3-and alpha v beta 5-integrins, as well as alpha 1- and beta 1-subunits in nephrectomy samples from 7 subjects with localized renal cell carcinoma. Grossly and histologically uninvolved regions ('normal') from the same nephrectomy specimens were used for comparison. Integrin expression was studied with specific monoclonal antibodies and the immunoperoxidase technique. alpha v beta 3 was expressed in the glomerular epithelial cells, Bowman's capsule, vascular endothelium, and weakly in tubular epithelial cells. alpha v beta 5 had a similar distribution except for minimal expression on vascular endothelium. alpha 1-Expression was observed in mesangium and but weakly in Bowman's capsule. beta 1-Expression was seen in glomerular epithelial cells, Bowman's capsule, vascular epithelium and tubular epithelial cells. Unlike in 'normals', neoplastic expression was more heterogeneous alpha v beta 3 was expressed in tumor cells in 4/7 cases, vascular endothelium in 6/6, and in stroma in 4/7. alpha v beta 5 was weakly expressed in tumor cells in 4/5, vascular endothelium in 5/5, and stroma in 4/5 cases. alpha 1-Expression was seen in tumor cells in 3/7, vascular endothelium in 4/7 and in stroma in 7/7 cases. beta 1-Expression was seen in tumor cells in 7/7 cases, vascular endothelium in 7/7, and in stroma in 4/7 cases. This study delineates the pattern of expression of the alpha v beta 3-and alpha v beta 5-integrins in 'normal' and neoplastic human kidney. Variations in alpha v beta 3-and alpha v beta 5-integrin expression may play a role in normal and neoplastic processes of the kidney.

Sudden infant death syndrome and superoxide/ nitric oxide

In a sudden infant death syndrome review Valdès-Dapena describes Naeye's report of increased medial muscle mass in walls of small pulmonary arteries and increased weight of cardiac right ventricles. These findings point to cardiorespiratory insufficiency, a problem in fast growing chicks raised at high altitudes. The vascular epithelium lining all blood vessels synthesises nitric oxide which induces relaxation of smooth muscle in vessel walls, and is possibly an important neurotransmitter. Others demonstrate that nitric oxide is involved in regulating vessel calibre, blood pressure and blood flow, as well as falls in ventricular outputs. Superoxide interacts with nitric oxide and removes it from the circulation. Superoxide is thus a vasoconstrictor. Superoxide is produced by activated phagocytes and possibly lymphocytes and other cell types in the immune response. Elevated immunoglobulins in mucus secretions are a hallmark in sudden infant death syndrome and hypoxic chicks. Our approach therefore is that cardiorespiratory insufficiency may be induced by superoxide in small pulmonary arteries preventing nitric oxide from acting as a muscle relaxant in vascular walls.

The involvement of endogenous nitric oxide in vagal-cholinergic stimulation of exocrine and endocrine pancreas in dogs.

Previous studies showed that nitric oxide (NO), synthesized from L-arginine (L-arg) by NO synthase (NOS) in vascular epithelium and nerve terminals, affects exocrine pancreatic secretion, but its role in control of endocrine pancreas has not been studied. In this study, the role of NO in the control of pancreatic secretion in response to vagal-cholinergic stimulation and duodenal infusion of nutrients was determined in conscious dogs with chronic pancreatic fistulas. Sham feeding (SF), urecholine iv infusion, and duodenal perfusion with nutrients were used to stimulate the pancreatic protein secretion, and insulin and glucagon release in tests without and with iv infusion of NG-nitro-L-arginine (L-NNA), an inhibitor of NO synthase, L-arg, a substrate of NOS, or their combination was used. SF, urecholine, and duodenal nutrient resulted in the stimulation of pancreatic protein secretion reaching, respectively, 50, 20, and 42% of cerulein maximum. Infusion of L-arg almost doubled the basal protein secretion and tended to increase the secretory response to SF and duodenal nutrient. After infusion of L-NNA, the pancreatic secretory responses to SF, urecholine, and duodenal nutrient were inhibited by about 70, 30, and 75%, respectively. When L-arg was combined with L-NNA, the reduction in pancreatic secretion by L-NNA was significantly attenuated. SF resulted in a significant rise in plasma insulin and glucagon, and this response was completely abolished by L-NNA infusion. Urecholine and duodenal nutrient also resulted in a marked increment in plasma insulin and glucagon, the insulin (but not glucagon) increment being abolished by the pretreatment with L-NNA and reversed by the addition of L-arg.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocompetent cells of the upper airway: Functions in normal and diseased mucosa

Secretory immunity is central in primary defense of the airway mucosa. B cells involved in this local immune system are initially stimulated in mucosa-associated lymphoid tissue, including tonsils and adenoids, and then migrate to secretory effector sites where they become immunoglobulin (Ig)-producing plasma cells. Locally produced Ig consists mainly of J-chain-containing dimers and larger polymers of IgA (pIgA) that are selectively transported through glandular cells by an epithelial receptor called secretory component or pIgR. Secretory antibodies perform surface protection by immune exclusion of soluble antigens as well as infectious agents. IgG can also participate in this primary defense because it reaches secretions by passive diffusion similar to IgE. However, the inflammatory properties of antibodies belonging to the latter two classes explain their involvement in mucosal immunopathology when elimination of penetrating antigens is unsuccessful. T helper (Th) cells activated in this process may by a Th2 profile of cytokines promote persistent inflammation with extravasation and priming of eosinophils. This mechanism appears to occur in the late-phase allergic reaction, perhaps driven mainly by interleukin-4 (IL-4) released from mast cells subjected to IgE-mediated degranulation. Eosinophils are potentially tissue-destructive cells, particularly after priming with IL-5. Cytokines also up-regulate adhesion molecules on vascular endothelium and epithelium, thereby enhancing migration of eosinophils and other leukocytes into the mucosa.(ABSTRACT TRUNCATED AT 250 WORDS)

PCR-dependent identification of Ig genes encoding autoantibodies

Autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, polymyositis, myasthenia gravis, and Hashimoto's thyroiditis result from abnormalities in immune regulation culminating in the stimulation of autoreactive lymphocytes. The pathogenicity of the autoantibodies in these diseases is a function of Ig specificity, isotype, p l , and affinity. Some autoantibodies directly damage host tissue by binding to determinants expressed on host cells. Others recognize intracellular or intranuclear antigens (such as Sm, Ro/SSA, and Jo-1). In the latter case, target molecules are not readily accessible to the autoantibodies. However, when released into immunologically accessible locations, the autoantigens complex with circulating Ig to form pathogenic immune complexes capable of inducing inflammatory reactions in the kidneys, vascular epithelium, and other organs. The function and character of autoantibodies have been studied extensively in animal models of autoimmune disease. In humans, such studies have primarily involved the analysis of long-lived monoclonal autoantibody-producing cells, generated by EBV transformation. Yet the production of EBV-transformed clones may be subject to a selection bias, such that lines cloned in vitro may be unrepresentative of the repertoire expressed in vivo . Studies of autoantibody-secreting human cells have also been limited due to the relatively small number of clones that can be derived and maintained from individual donors. The development of a rapid and unbiased technique for identifying autoantibody-secreting human B cells would facilitate analysis of the origin and character of such lymphocytes. Identifying the genes responsible for the production of specific autoantibodies would also improve our understanding of the etiology and pathogenesis of human disease. In the present work, we describe a technique for (i) identifying individual autoantibody-secreting cells and then (ii) PCR-amplifying their variable heavy (and light) chain genes. Individual clones of autoantibody-secreting lymphocytes were identified using an antigen-specific ELIspot assay. These clones were generated during short-term in vitro culture, thereby reducing the potential for repertoire bias compared to EBV-transformation and hybridoma formation. This method permitted relatively large numbers of Ag-specific B cells from individual patients to be identified. Messenger RNA from those clones was reverse-transcribed and amplified to identify and analyze the Ig genes responsible for encoding specific autoantibodies.

Tachykinin receptors in rabbit airways--characterization by functional, autoradiographic and binding studies.

1. In many species, both NK1 and NK2 tachykinin receptors appear to be important in mediating the contraction of airway smooth muscle. We have examined the distribution and characterization of receptors for tachykinins in rabbit airways using functional length tension studies, autoradiography and radioligand binding studies. 2. Contractile responses to tachykinins were elicited in four different areas of the respiratory tree--trachea, and three progressively more distal areas of the right bronchus. The NK2 receptor-preferring agonists, neurokinin A (NKA), neuropeptide gamma (NP gamma) and the NK2-selective [Lys5 MeLeu9, Nle10]-NKA(4-10) [NKA (4-10) analogue] produced similar contraction in all four areas. Substance P (SP) and the NK1-selective [Sar9,Met(O2)11]-SP (Sar-SP) exhibited a marked location-dependence in the magnitude of contraction, producing minimal contraction in the trachea and more proximal bronchi with contractions becoming progressively larger in the more distal airways. Senktide (which is selective for the NK3 receptor) produced negligible contraction in all areas. 3. The NK2-selective antagonist, MDL29,913, was a weak antagonist of NKA and NKA(4-10) analogue. At a concentration of 2 microM, it produced a small but significant shift in the response curve to NKA and a greater shift (8 fold) in the curve to NKA(4-10) analogue, but it had no effect on responses to Sar-SP. The non peptide NK1 receptor antagonist, CP-96,345, was also unexpectedly weak in this preparation. The pD2 value for Sar-SP was decreased 27 fold by CP-96,345 at a concentration of 1 microM, without alteration in the maximum response.4. Autoradiographic binding sites to ['251I]-NKA were sparse over smooth muscle in proximal airway preparations and markedly increased in density in the more distal airways. There was negligible binding over vascular smooth muscle and epithelium.5. Radioligand binding studies revealed binding to ['251I]-NKA which was 82% specific. The order of potency for inhibition of ['251I]-NKA binding was SP> = Sar-SP> NKA = NPy>CP-96,345> NKA(4-10) analogue >NKB>>>MEN 10207 (the NK2 subtype selective antagonist) >MDL 29,913> senktide. This profile indicates binding predominantly to NK, receptors.6. These results suggest that there are at least two types of tachykinin receptors in rabbit airways, a population of NK, receptors, the density of which is greatest in the periphery and, in addition, NK2 receptors which are uniformly distributed throughout the airways. These receptors have unusual characteristics in that the NK, antagonist, CP-96,345 and the NK2 antagonist, MDL 29,913 respectively exhibited only weak potency.

Soft-tissue localization of 99mTc-hydroxymethylene diphosphonate due to interaction with calcium.

Soft tissue localization of 99mTc-labelled bone imaging agents is often associated with high calcium levels either locally or systemically. This could be due to the formation of a large molecular complex after administration of the radiopharmaceutical. We have investigated the formation of such a complex between 99mTc-hydroxymethylene diphosphonate (HMDP) and calcium by a number of techniques. The results indicate that 99mTc-HMDP is a colloidal preparation which may contain particles up to 280 nm in size, and that the labelled colloid may aggregate in the presence of calcium salts forming particles up to 3 microns. Such particles are too large to permit free diffusion across vascular epithelium but large enough to localize in the liver or lungs. It is suggested that this interaction may be responsible for soft tissue localization of the technetium-labelled bone imaging agents. In vivo the radiolabelled complex in a mixture of 99mTc-HMDP and calcium chloride localizes to a significantly larger extent in liver, spleen and muscle and less in bone compared with 99mTc-HMDP diluted with saline. The increased liver and spleen uptake can be prevented by prior administration of non-radioactive colloid but this does not significantly improve the bone uptake of the 99mTc-HMDP/calcium chloride mixture. The clinical relevance of these findings lies in their possible explanation for soft-tissue uptake and poor bone accumulation of the technetium-labelled diphosphonates in some patients. This may be particularly important in evaluating quantitative bone scans, especially if serial scanning is done to monitor disease progress or therapy since local or systemic calcium levels may change between scans.

Ras p21 in breast tissue: associations with pathology and cellular localisation

Immunocytochemistry with monoclonal antibody Y13-259 demonstrated p21 ras in paraffin sections of breast tissue from 171 women: 85 with invasive breast carcinoma, 14 with non-invasive carcinoma and 72 with benign changes only. Many different tissue elements contributed to ras expression. Semiquantitative assessment showed that intensity of immunostaining in the normal epithelium of large ducts, small extralobular ducts and terminal duct lobular units (TDLU) was usually exceeded by that of myoepithelial cells. Vascular smooth muscle and apocrine epithelium also stained strongly, but the flat epithelial cells lining cysts did not express detectable p21 ras. There was a progressive increase from normal epithelium through epithelial hyperplasia of usual type and atypical hyperplasia to carcinoma in situ, without further increase in invasive carcinoma. Expression in carcinomas was inversely related to oestrogen receptor content but independent of the prognosis-associated variables of size, histological type, vascular invasion or lymph node metastasis.

Entry of the synthetic ACTH(4–10) analogue into the rat brain following intravenous injection

In view of known central effects of N-terminal ACTH fragments, a possibility of their entry into the brain was studied. Rat blood and brain extracts after intravenous injection of the tritiated synthetic ACTH(4–10) analogue, Met-Glu-His-Phe-Pro-Gly-Pro, were subjected to a high-performance liquid chromatographic analysis. At two time points the labelled peptide was detected in brain extracts. The brain to blood ratios of peptide content in brain and blood were found to be significantly higher than those calculated for a distribution of labelled bovine serum albumin in rat brain capillaries and blood. This strongly suggests that this peptide penetrates into the brain tissues, its quantity not exceeding 0.01% of dose injected. Peptide diffusion through the vascular epithelium of brain capillaries could account for the data obtained.