The isolation and primary culture of putative human root odontoblasts.

The use of procedures adapted from a routinely successful method of culturing bovine bone has led to the first system for the study of dentinogenesis in vitro. Two types of cells have been grown from pulp obtained from the growing root tips of impacted third molars extracted from 14- to 19-years olds: (1) epithelial-like cells that are probably derived from fragments of the epithelial root sheath and (2) odontoblast-like cells. The cultured epithelial-like cells grow out in distinctive rounded plaques while the odontoblast-like cells are tethered to and/or grow on top of the epithelial-like cells. The odontoblast-like cells produce mineralized matrix by 10 days when cultured on a defined mineralization formula containing conditioned medium obtained from fetal bovine bone cell cultures. Growth factors in this conditioned medium are important to cell proliferation and growth and to the synthesis of mineralized matrix. Sequential enzyme digestion in dispase and dispase/collagenase in serum-free Dulbecco's Modified Eagle's Medium is essential to obtaining adequate cell yields from the apical 3-5 mm of the developing root. Reduction of the number of fibroblasts by treating cultures with dispase in Tyrode's solution midway through the initial growth period enhances the purity of these cell cultures.


Dentin permeability and dentin sensitivity.

The hydrodynamic theory of dentin sensitivity is based on the premise that sensitive dentin is permeable throughout the length of the tubules. Such a condition may permit the diffusion of bacterial products across dentin to the pulp where they may cause irritation of pulpal soft tissues. However, the slow outward movement of dentinal fluid tends to flush the tubules free of exogenous substances. This balance between the inward diffusion of exogenous substances, whether bacterial or the active ingredients in desensitizing medicaments, and the cleansing action of dentinal fluid flow needs to be examined experimentally.


Odontoblast-mediated regulation of the progression of dentinal caries.

Osteoblasts and odontoblasts are both derived from the same mesenchymal cell line. Our aim was to investigate whether the processes of bone destruction and dentinal caries are biologically similar. The working hypothesis was that after the initiation of caries in the enamel, its rate of progression in the dentine is regulated by cell-mediated factors. Experimental caries was induced in the rat with a high sucrose diet combined with Streptococcus sobrinus infection. Both destruction of dentine and its apposition in the pulp under the carious lesions were measured after vital staining with tetracycline. Caries progression and dentine apposition were higher in developing teeth prior to apex "closure" than in adult, fully-formed teeth. Rats placed on a cariogenic diet during tooth development had an increased rate of caries progression. Fluoride administration via the drinking water was associated with decreased dentine apposition and little progression of dentine caries during the developmental stages. Dentine apposition was enhanced in adult rats placed on fluoride administration, while caries progression was reduced, whereas in animals subjected to metabolic acidosis dentine caries progression was enhanced, with reduced dentine apposition. In contrast, alkalotic animals had less dentinal lesions and smaller ones than the controls. Three theories are advanced to explain the observed changes: (i) They may be associated with changes in alkaline phosphatase activity in the pulpo-dentinal complex, (ii) they may be mediated by ionic changes in the dentinal fluid, or (iii) they may reflect the liberation of growth factors from dentinal matrix.


The hybrid layer: a resin-dentin composite.

During studies designed to improve the bonding of adhesive resins to tooth structure, it was found that methacrylates with both hydrophobic and hydrophilic groups promoted monomer penetration into suitably prepared dentin. The monomers impregnated and became entangled with the collagen fibrils of surface demineralized dentin, creating a hybrid layer after their polymerization. The identification, properties and function of the hybrid layer, a new biologic composite, are explained.


Extracellular fluid movement in the pulp; the pulp/dentin permeability barrier.

Fluid movement in the pulp depends largely upon the physiology of the blood vessels; normally there is a net efflux of fluid and proteins from the capillaries into the extracellular environment. Most pulp capillaries lie close to the odontoblast layer and in order to see whether fluid can pass between the odontoblasts into the predentin we have perfused the vessels of molar tooth germs in anesthetized piglets with the electron dense tracer lanthanum. The results show that the tracer permeates the capillaries but encounters a barrier to permeability at the apical (predentinal) ends of the odontoblasts. The completeness of the barrier to the tracer lanthanum is discussed together with structural evidence of tight junctions between odontoblasts in both pigs and humans and the presence of collagen fibers through the tight junctional zone. It is concluded that there is little or no evidence that pulp fluid is normally confluent with predentin. An advantage of this arrangement may be that by maintaining an enclosed microenvironment it permits regulation of the orderly process of matrix deposition and mineralization of predentin to dentin. In order to maintain constant vascular and extracellular fluid pressures the capillary efflux has to be balanced by fluid removal; recent work in cats has shown that lymphatic vessels are available to transport fluid out of the pulp. In this paper the differences in the intrapulpal distribution of these vessels have been extrapolated to human teeth in an attempt to explain certain variations in the symptoms and progress of pulpal inflammatory conditions.


Accessory cells in the immune defense of the dental pulp.

This communication focuses on the participation of accessory cells in the initial recognition and processing of antigenic substances in the dental pulp. Immunohistochemical analyses have demonstrated the presence of two types of accessory cells--one with a dendritic morphology located in the periphery of the pulp and one with a macrophage-like appearance located more centrally. Functional studies in vitro have provided evidence for the dendritic cells being the most significant of the two cells regarding their capacity to induce T-cell proliferation. Studies on ontogeny have revealed that the appearance of pulp accessory cells is delayed compared to other peripheral tissues. In experimentally induced pulp lesions a rapid increase of cells with morphologic and phenotypic features similar to normally occurring accessory cells was found. These data demonstrate that the dental pulp contains the necessary cellular constituents to mount an immunologic defense reaction. Future studies should focus on elucidating possible interactions between these immune cells and the neurovascular system of the pulp.


The role of growth factors in determination and differentiation of the odontoblastic cell lineage.

In developing teeth the differentiation of odontoblasts is triggered by the enamel epithelium and is tightly coupled with morphogenesis. There is substantial evidence that even in mature teeth the cells of the dental pulp retain the capability to differentiate into odontoblasts under suitable conditions. However, cells from other than the dental mesenchymal cell lineage apparently do not possess this potential. Thus, it is conceivable that the dental mesenchymal cells acquire cell type-specific potential to differentiate into odontoblasts during their developmental history. Therefore, the understanding of the mechanisms which regulate the terminal differentiation of odontoblasts requires that the molecular changes and mechanisms that are associated with their progressive determination be clarified. It can be speculated that there are key transition points in the developmental sequence during which the mesenchymal cells acquire new levels of differentiation. These include, (1) the condensation of the neural crest-derived mesenchymal cells around the epithelial bud, (2) their entrance into the dental papilla lineage during cap stage, and (3) the differentiation of the cells underlying the enamel epithelium into odontoblasts during bell stage. The transition points are conceivably characterized by amplification or onset of expression of new sets of genes encoding transcription factors, growth factors as well as structural proteins. We have applied in situ hybridization for localization of the expression of two growth factors during mouse molar morphogenesis: transforming growth factor beta 1 (TGF beta 1) and int-2 (a proto-oncogene coding for a fibroblast growth factor-related protein). During bud stage, expression of TGF beta 1 was first detected in the epithelium and shortly thereafter in the condensed dental mesenchyme. The expression was weak during early bell stage but a high number of transcripts appeared in secretory odontoblasts as well as in presecretory ameloblasts. int-2 mRNA appeared in the dental papilla mesenchyme at the onset of cap stage, persisted in the cuspal mesenchyme during bell stage and was lost upon completion of morphogenesis. Our findings suggest that cell type-specific expression of TGF beta 1 and int-2 is associated with phenotypic properties of the odontoblastic cell lineage. For instance, TGF beta 1 may regulate matrix deposition by increasing tenascin and syndecan expression in the condensed dental mesenchyme and by controlling dentin matrix deposition by odontoblasts. TGF beta 1 and int-2 may also be involved in signalling between the epithelial and mesenchymal tissues and in regulation of gene expression at the transition points of the developmental sequence that leads to the differentiation of odontoblasts.


Recent investigations on dentin specific proteins.

Several extracellular matrix (ECM) proteins have been isolated from dentin and shown to be synthesized by odontoblasts. Highly phosphorylated proteins (HP), the phosphophoryns, are specifically found in dentin and are synthesized only by odontoblasts. Phosphophoryns are probably involved in controlling the site and/or the rate of circumpulpal mineralization. Other moderately phosphorylated (MP) and low phosphorylated (LP) proteins have also been detected in dentin, but have not been studied extensively. A 53 kDal dentin sialoprotein (DSP) that resembles bone sialoproteins is expressed by odontoblasts and by pulp cells, but essentially by no other cell type. The function of this protein is unknown. Osteocalcin (bone Gla protein) is synthesized by osteoblasts and by odontoblasts and found in the ECM of bone and dentin. Other tissues or cells (including pulp) do not express osteocalcin. The highly specific nature of these dentin and bone ECM proteins suggests that antibody and molecular probes for the phosphoproteins, DSP and osteocalcin could be valuable in studies on the differentiation and identification of odontoblasts and their precursors. Other bone NCPs shown to be present in dentin may also prove to be important in dentinogenesis.


Regulatory factors in pulp biology: a reaction.

A number of developmental processes can be investigated in the mammalian pulp. Of particular interests are those developmental processes related to when, where and how specific phenotypes are expressed in the forming pulp tissues. This "reaction" attempts to highlight the basic processes related to immunological factors associated with accessory cells, growth factors and the determination and differentiation of odontoblasts, and dentin-specific non-collagenous proteins. The three papers associated with these developmental processes provide an exciting glimpse into the emerging advances being made in pulp biology.


Pulpal vascular changes in inflammation.

Changes in pulpal vessels in experimentally induced acute and chronic pulpitis in dog tooth were investigated using corrosive resin casts and scanning electron microscopic examination. Following a cavity preparation without water spray, increased permeability of blood vessels occurred in the primary stage of acute pulpitis. This was evidenced by the extravasation of resin from the vessel. This phenomenon was found initially in the venular network as well as in the capillary network located under the dentin. The morphological change was minimal in the vascular network underneath the cavity. This is in contrast to an expanded and tortuous vascular network representing an ulceration which was found around an abscess in chronic pulpitis. Furthermore, formation of vascular loops and AVAlc close to the inflamed region may represent a protective change in the pulp against inflammation.


Effects of selected inflammatory mediators on blood flow and vascular permeability in the dental pulp.

One of the major events involving inflammatory processes is the alteration of microcirculatory hemodynamics by inflammatory mediators released from tissue components. Using modern macrocirculatory techniques, 15 mu radioisotope labeled microspheres, 133Xe washout, laser Doppler flowmetry and double isotopes, 125 and 131I-albumin, and microcirculatory methods, intravital fluorescence microscopy with FITC labeled dextran, we have examined the effects of selected mediators, e.g. 5-hydroxytryptamine (5-HT), prostaglandin E2 (PG-E2), bradykinin (BK), substance P (SP), calcitonin gene related peptide (CGRP) and histamine on blood flow and vascular permeability in the pulp of experimental animals. Surprisingly, SP and CGRP caused weak albumin leakage in the pulp, while the opposite is true in high compliance tissues, such as muscles, suggesting that the vessels in a low compliance environment, such as the pulp, may not be as permeable in response to selected mediators. Intraarterial injection of 5-HT caused a strong vasoconstriction which was mediated by 5-HT1p receptor subtype. The pulpal 5-HT receptor subtype was identified by immunocytochemistry, receptor autoradiography and functional investigations. Intravital fluorescence microscopy observations of the rat incisor preparation showed that histamine, BK and PGE2 increased permeability, whereas isoproteranol caused partial inhibition of the BK-induced increase. In an induced pulpal inflammation model using plaque extract, blood flow increased over 40% in the moderately inflamed pulp, which demonstrated severe vasodilation and polymorpholeukocyte accumulation. In the partially necrotic pulp, blood flow decreased nearly 35%. Results of this study clearly show that there is a high structural/functional correlation in pulpal microcirculation in inflammation. As demonstrated in this presentation, the effects of inflammatory mediators on pulpal microcirculatory hemodynamics are complex.(ABSTRACT TRUNCATED AT 250 WORDS)


The role of selected cations in the desensitization of intradental nerves.

Dentinal sensitivity is a common complaint of patients. Removal of cementum or enamel leaves the dentin exposed allowing various stimuli to produce fluid movement through the dentinal tubules. These fluid movements are believed to activate pulpal sensory nerves leading to pain. Various therapeutic approaches have been investigated to treat this problem. Resins, fluoride compounds and oxalate solutions have been used to block the dentinal tubules. KNO3 and SrCl2 are active ingredients in desensitizing tooth pastes but neither compound reduces the permeability of dentine. The ability of various solutions to decrease hypertonic (3M NaCl) evoked nerve activity was tested in the canine teeth of anesthetized cats. Excitatory and desensitizing solutions were placed into a deep dentinal cavity prepared over the incisal pulp horn. An electrode used to record nerve activity was also placed into this deep cavity. KNO3 and other K+ ion containing solutions elicit a biphasic effect on nerve activity. Immediately following application of these solutions into the deep dentinal cavity there is a brief burst of high frequency spike activity. Following this brief period of excitation, the intradental nerves are silent and respond weakly to hypertonic NaCl. CaCl2 and other divalent cation solutions also inhibit hypertonic NaCl induced nerve activity, but have a weaker effect than that exerted by K+. These observations can be explained based on the effects of various ions on nerve excitability. Increasing the K+ ion concentration in the vicinity of the dentinal nerve terminal depolarize and activates the nerve fibers. Following a prolonged period of depolarization the action potential mechanism is inactivated. Divalent cation solutions stabilize the nerve membrane without changing the membrane potential. Desensitizing solutions do not decrease nerve activity by an effect on pulpal blood flow. This experimental model does not entirely explain the clinical action of these desensitizing agents. The role of inflammation in hypersensitivity and dental pain is currently being investigated.


Tissue pressure and blood flow in pulpal inflammation.

The initial vascular reactions during inflammation are vasodilation and increased vessel permeability. Both these basic reactions cause increased pulpal fluid volume. In the dental pulp the inflammatory vascular reactions take place in a rigid enclosed dentin chamber, which to some extent makes the pulp vulnerable. Due to this lack of distensibility any gain in pulpal volume will necessarily increase the pulpal tissue pressure. If the tissue pressure rises to the same level as the blood pressure it will compress the pulpal vessels, thus counteracting a beneficial blood flow increase during inflammation. Using the micropuncture technique and laser Doppler flowmetry we have performed simultaneous measurements of tissue pressure and blood flow in the cat dental pulp during neurogenic inflammation. Sensory nerve stimulation caused a rise both in blood flow and tissue pressure. Our findings thus strongly suggest that the increased pulpal tissue pressure promote fluid absorption back into the blood. If, in theory, plasma proteins and other macromolecules had leaked out during sensory nerve stimulation they must have been successfully removed by lymphatics, unless the tissue pressure would have risen to the same level as the capillary blood pressure causing a fall in PBF. This was not found. On the contrary, increased blood flow was measured, even in experiments lasting for more than 8 hours. It is therefore concluded that the pulp may have a beneficial blood flow increase during inflammation in spite of simultaneously increased tissue pressure.


Involvement of sensory nerves in hemodynamic reactions.

Small diameter afferent nerves in the pulp contain vasoactive peptides (tachykinins and CGRP) which are released upon nerve stimulation causing long lasting vasodilation. The aim of the present experiments was to study the contribution of afferent nerves in vasodilator reactions in the cat dental pulp when clinical procedures were applied. For this purpose the inferior alveolar nerve (IAN) was cut unilaterally 10 days prior to study. Lower canines were then subjected to various stimuli and blood flow responses in the pulp were monitored by laser Doppler flowmetry. Bipolar electrical tooth stimulation (5 impulses, at 2 Hz, 50 microA) and application of capsaicin (10(-4) M) in a deep cavity instantaneously induced long lasting increases in pulpal blood flow in control teeth but not in denervated teeth. Bradykinin (10(-3) M) applied locally produced a more rapid and larger pulpal vasodilation in innervated than in denervated teeth. Intermittent grinding (3 times, 1 sec each) of surface dentin with a round diamond bur instantly increased flow in control teeth by 53%, whereas in denervated teeth the response was delayed and significantly smaller (16% increase). Deeper preparations produced long lasting responses of similar magnitude on both sides but the onset was much slower in denervated teeth. Percussion and low intensity ultrasonic stimulation (1 sec) instantly caused increased flow in control teeth but not in denervated teeth. The effect was abolished by local anesthetic (mepivacaine) injection apically. Sympathectomy and acute IAN denervation did not alter the responses. The results show that afferent (sensory) nerves contribute importantly to hemodynamic reactions in the pulp in response to clinical procedures.


Colocalization of TGF-beta 1 and extracellular matrix proteins during rat tooth development.

Recent studies suggest that TGF-beta 1 controls events in embryogenesis via its modulation of the extracellular matrix (ECM). During tooth development, histomorphogenetic and cytodifferentiation events are regulated by ECM-mediated epithelial-mesenchymal interactions. In this study, we have investigated the in vivo relationship of the localization of TGF-beta 1 to various ECM proteins, including glycosaminoglycans (GAGs), fibronectin, collagens I and III and to the enzyme alkaline phosphatase, at the early and late bell stages of rat molar tooth development. We have used polyclonal antibodies specific to peptides present in the precursor and processed forms of TGF-beta 1, fibronectin and Collagens I and III and immunohistochemical methods of detection. GAGs were stained with Alcian Blue GX and alkaline phosphatase was detected with the enzyme Naphthol-AS B1. Our results indicate that both the intracellular and extracellular forms of TGF-beta 1 are expressed in a site-specific manner, and that the growth factor colocalizes with alkaline phosphatase, fibronectin, collagens I and III and GAGs during the bell stages of tooth development. These data support the hypothesis that in tooth development, the actions of TGF-beta 1 may contribute to important qualitative changes in the composition and structure of extracellular matrices.


Characterization of bone resorptive mediators in active periapical lesions.

The mechanism of bone destruction in periapical lesions was studied using a rat model system. Periapical lesions were induced by pulp exposure and infection from the oral environment. Lesions expanded most rapidly between induction on day 0 and day 15 ("active phase"), with enlargement occurring at a slower rate thereafter (days 20 and 30, "chronic phase"), as assessed by radiography and automated image analysis. Pooled extracts of day 15 periapical tissues contained significant levels of bone resorbing activity (BRA), as determined by 45Ca release from fetal rat long bones. Normal rat dental pulp and periodontal ligament contained no activity. In kinetic experiments, highest levels of BRA were detected in active phase tissues on days 10 and 15, with BRA declining thereafter in chronic phase tissues to near baseline levels by day 30. In characterization studies, BRA in pooled day 15 tissue extracts was unaffected by treatment with polymyxin B, but was completely abolished by proteinase K treatment or heating to 70 degrees C, indicating an active moiety distinct from bacterial LPS, probably a protein(s). FPLC gel filtration chromatography revealed that BRA could be resolved into four major peaks, of MW 30-60 kD (Peak I); 15-20 kD (II); 1-2 kD (III); less than 1kD (IV), consistent with the presence of the following bone resorptive mediators: (I) cytokines TNF alpha, TNF beta and/or unprocessed IL-1 alpha; (II) processed IL-1 alpha and/or IL-1 beta; (IV) PGE2. These findings demonstrate that bone resorbing activity is temporally related to bone destruction, and that the activity present during the rapid phase of periapical lesion expansion is primarily attributable to bone resorptive cytokines.


Pathophysiological roles of arachidonic acid metabolites in rat dental pulp.

Arachidonic acid metabolism in normal rat incisor pulp was examined by measuring the conversion activity of exogenously added arachidonic acid in pulpal homogenates. It was demonstrated that the major metabolites were 12-hydroxyeicosatetraenoic acid and prostaglandin (PG) I2. Immunohistochemical studies revealed that PGI2 synthase was distributed in the pulpal blood-vessel cells, fibroblasts and odontoblasts, suggesting that PGI2 may contribute to regulating the function of these cells. When the incisor pulp was experimentally inflamed by applying lipopolysaccharide, arachidonic acid metabolism in the pulp showed overall increase. Change in the pulpal vascular permeability, which was assessed by quantifying the amount of extravasated dye, was almost parallel to the changes in PGE2 and PGI2 production. When production of the PGs was inhibited by indomethacin, the increase of vascular permeability in the inflamed pulp was also suppressed. Topically-applied PGE2 and PGI2 methyl ester abolished the suppression of increase in vascular permeability by indomethacin. These results suggest that PGE2 and PGI2 may be involved in the increase of vascular permeability in the experimental pulp inflammation. We further measured the production of leukotriene (LT) B4 in the inflamed pulp by incubating isolated pulp samples with Ca ionophore A23187, followed by radioimmunoassay. Change in LTB4 production was revealed to be almost parallel to that of neutrophil infiltration. BW755C, an inhibitor of both cyclooxygenase and lipoxygenase, reduced both LTB4 production and neutrophil infiltration. Accordingly, it was suggested that LTB4 may be involved in neutrophil infiltration in the experimental pulp inflammation.


Mineral-associated adhesion proteins are linked to root formation.

There is a general agreement that the extracellular environment plays a critical role in controlling cell behavior. Thus, significant research efforts have focused on understanding the effects of extracellular matrix proteins on cell function. In particular we have focused on determining the role of adhesion proteins in the regulation of root formation. Using an OPN antibody, 2arN (generously provided by Drs Craig and Denhardt), the expression of OPN during root formation was determined. OPN (osteopontin) is a bone-associated adhesion protein. OPN was expressed in the dental follicle region of molars obtained from 3 day old CD-1 mice, but was not expressed in the odontoblast layer. In contrast by day 8, positive staining was noted in the odontoblast layer, as well as in the area of Hertwig's epithelial root sheath. However, at this same time point no positive labeling for 2arN was observed in the enamel organ or in the dental papillae cells. By day 15 positive staining for OPN was seen in the area of the periodontal ligament, as well as the region of primary deposition of extracellular matrix onto dentin. Also determined was the ability of fibronectin, OPN and dentin sialoprotein (DSP) to promote the attachment of dental ectomesenchymal cells, in vitro. Interestingly, these cells attached remarkable well on bacteriological dishes (control) in the absence of an adhesion protein. DSP did not increase cell attachment beyond that observed for control cells. In contrast, both fibronectin and OPN enhanced cell attachment. These studies, while preliminary indicate that OPN is expressed in a unique fashion during root development, thus suggesting a regulatory role for such adhesion proteins during root formation.


The effects of capsaicin on pulpal blood flow.

One of the initial events in pulpal inflammation has been characterized as neurogenic inflammation: the release of neuropeptides following excitation of sensory C-fibers by noxious stimuli which alters microcirculatory parameters, that is, vasodilation and plasma extravasation. Thus, the purpose of the study was to investigate the effect of capsaicin on pulpal blood flow (PBF) with the aim of understanding neurogenic inflammation in the dental pulp by characterizing the response of the pulpal vasculature to repeated applications of various concentrations of capsaicin. Experiments were performed on canine teeth of cats anesthetized with sodium pentobarbital. PBF was measured by the laser Doppler flowmeter following the application of capsaicin into the dentinal cavities of the canine teeth. The increases of PBF to capsaicin were 31.8 +/- 6.3% (n = 6) and 54.2 +/- 6.2% (n = 9), for 1 microM and 100 microM capsaicin, respectively. The increase of PBF peaked at about 50 sec after the capsaicin placement and the PBF returned to control level within 20 min following capsaicin removal. Four repeated applications of 1 microM capsaicin caused a small change in the peak PBF amplitude. In contrast, a second application of 100 microM capsaicin caused a significantly smaller increase of PBF than the first application (26.8 +/- 6.4% vs. 54.2 +/- 6.2%). Results of the present study show that local application of capsaicin caused the increase of PBF and that repeated capsaicin stimulations inhibited PBF responses. Smaller PBF increases in response to high concentrations of capsaicin also suggested the depletion of vasoactive substances, e.g. substance P, from the sensory nerve terminals, which appears to be dose-dependent. The PBF response to capsaicin suggests that neurogenic inflammation in the dental pulp involves capsaicin-sensitive nerve endings.


Temperature related changes in pulpal microcirculation.

During dental treatment temperatures can be reached which might possibly damage the tooth pulp. To determine the effect of both thermal stimulation on pulpal microcirculation and local anesthesia on thermoregulation we measured the pulpal blood flow by using laser Doppler flowmetry. Experiments were carried out on lower incisors of Wistar rats anaesthetized with thiopental. The rats were divided into three groups, with one remaining untreated, and the others being either desensitized with capsaicin or sympathectomized with guanethidine. In a range between 33 degrees C and 42 degrees C there was no substantial change in blood flow, which, however, was the case below 33 degrees C. Up to 49 degrees C an increase could be recorded in both untreated and guanethidine pretreated animals, whereas the capsaicin groups showed almost no reaction. This increase in blood flow can be blocked reversibly by local anaesthesia. For this purpose we tested articain 5% and mepivacain 3%, both without constrictor. Intravital microscopic studies show that the temperature related increase in blood flow is also associated with plasma extravasation. From these results we draw the conclusion that pulpal thermoregulation is linked to nociceptive sensory neurons and can be described as "neurogenic inflammation".


Changes in pulpal blood flow and in fluid flow through dentine produced by autonomic and sensory nerve stimulation in the cat.

We have recorded the flow of fluid through exposed dentine in cat canines. The effects on this flow of electrical stimulation of the cervical sympathetic trunk (CST) and of the inferior alveolar nerve (IAN) were investigated. In some preparations, pulpal blood flow was also monitored with a laser Doppler flow meter and the compound action potentials evoked by IAN stimulation were recorded from the tooth. CST stimulation (0.5-5/s) resulted in a significant decrease in the rate of outward flow of fluid through the dentine or, with the higher frequencies tested, a reversal of the flow. CST stimulation also produced vasoconstriction. IAN stimulation caused a significant increase in the rate of outward flow of fluid from the dentine. This effect required the recruitment of A delta fibres: A beta fibres alone had no effect. Pulpal vasodilatation occurred with the same threshold. Mechanical stimulation of dentine in an acutely denervated tooth also caused vasodilatation, indicating that pulpal afferents were involved. The results show that the rate of fluid flow through dentine, and hence the rate of inward diffusion of chemicals from the oral cavity, can be influenced by changes in the state of the pulpal microvasculature.


Effects of inflammation on dental sensory nerves and vice versa.

A brief review of the normal sensory innervation of teeth is presented, especially concerning the fibers that are immunoreactive (IR) for calcitonin gene-related peptide (CGRP). Numerous CGRP-IR fibers innervate coronal dentin at sites populated by primary odontoblasts and associated pulp cells that synthesize nerve growth factor (NGF). If the primary odontoblasts and adjacent pulp cells are lost as a consequence of injury, CGRP-IR dentinal innervation is greatly reduced. The responses of CGRP-IR nerve fibers to pulpal injury are reviewed. Those reactions show that pulpal sensory nerve fibers can alter the size, shape and immunoreactivity of their terminal branches in response to different stages of inflammation and healing.


Role of intradental A- and C-type nerve fibres in dental pain mechanisms.

The arousal of the two components of pain (the first rapid or sharp pain and the second dull pain) are considered to be related to activation of A delta- and C-type nociceptive primary afferents, respectively. The same dichotomy of pain sensations may also exist in teeth, although due to the short distance between the site of stimulation and the brain the two sensations might not be as clearly separated as in stimulation of, for example, the extremities. The sensations evoked by stimulation of human teeth vary according to the type of the stimuli applied. Low-intensity electrical stimulation is able to induce non-painful (prepain) sensations. At high current intensities pain is evoked. Drilling, probing and air-drying of exposed dentin induce only pain. Most studies also indicate that thermal stimulation only induces painful sensations. The quality of dental pain can vary. Typically, dentinal stimulation of teeth with healthy pulps induces sharp pain. On the other hand intense heat stimulation can result in dull pain which radiates to a wider area of the face and jaws. This component of the stimulus-induced pain seems to share some characteristics of toothache associated with painful pulpitis. Single fibre recordings of intradental nerve activity in experimental animals have shown that in addition to A-fibres a considerable number of C-type primary afferents innervate the dental pulp. This is in accordance with the results of neuroanatomical studies, which indicate that 70-80% of pulpal axons in human, monkey, dog, and cat teeth are unmyelinated. Intradental A- and C-fibre groups seem to be functionally different and can be activated separately by certain external stimuli. Comparison of the response characteristics of the pulp nerve fibres and the sensations induced from human teeth indicate that: 1) A-fibres are responsible for the sensitivity of dentine and thus for the mediation of the sharp pain induced by dentinal stimulation, 2) Prepain sensations induced by electrical stimulation result from activation of the lowest threshold A-fibres some of which can be classified as A beta-fibres according to their conduction velocities. Comparison of the responses of the A beta- and A delta-fibres indicate that they belong to the same functional group, 3) Intradental C-fibres are activated only if the external stimuli reach the pulp proper. Their activation may contribute to the dull pain induced by intense thermal stimulation of the tooth and to that associated with pulpal inflammation.


Studies on the presence and functional properties of afferent C-fibers in the cat's dental pulp.

The present investigation describes how intradental afferent C-fibers can be identified and characterized in the anaesthetized cat. Functional single fibers innervating the lower canine tooth were recorded from filaments split from the inferior alveolar nerve. Fibers responding to monopolar electrical stimulation of the tooth were classified as slowly and fast conducting according to their conduction velocity (c.v.). The axonal c.v. was determined for 31 slowly conducting fibers by electrical stimulation of the nerve. Sixty-eight percent of these proved to be C-fibers. The slowly conducting fibers had higher activation thresholds, smaller amplitudes and longer durations of the action potentials than the fast conducting fibers. Many of the fast but none of the slowly conducting fibers were repetitively activated by a single stimulus pulse. The highest frequency at which the fibers responded to every stimulus pulse was much lower for the slowly than for the fast conducting fibers. Most of the slowly conducting intradental fibers tested were activated by both hot and cold stimulation of the tooth, by pressure applied to the pulp chamber and by the application of KCl or bradykinin to the pulp. Eight C-fibers responded to mechanical stimulation of the pulp. For some fibers the response magnitude correlated with the stimulus intensity. Many slowly conducting intradental fibers developed an ongoing discharge after heat and pressure stimulation. The results indicate that there exists in the dental pulp of the cat afferent C-fibers that resemble polymodal C-nociceptors. They might be involved in the generation of the dull aching pain present in inflammatory conditions of the pulp. The activation of intradental C-fibers in pulpitis might result either from the elevated pressure and/or by sensitization of the C-fiber endings by inflammatory substances released.


Immunoassay evidence for a role of nerve growth factor in development of dental innervation.

Nerve growth factor (NGF) is known to have an essential role in prenatal development of spinal and trigeminal primary sensory neurons serving nociceptive functions. Investigation of a possible function of NGF in development of intradental innervation has recently begun with the demonstration by others of NGF mRNA and NGF receptor in the pulp. The pulp is of special interest in this respect because of its late development and unusual properties of its innervation. In the present study, use of a sensitive ELISA for NGF has made it possible to detect and measure NGF antigen in pulps of developing rat molars. Pulps contained relatively high concentrations of NGF before and during the time of innervation development.


Immediate physiological response of odontoblasts.

The normal structure and function of the odontoblast cell layer is often perturbed by a variety of pathological processes as well as by the restorative procedures and materials required to treat them. It is well accepted and documented that a number of acute histological changes may take place under these circumstances resulting in the loss of integrity of the odontoblast cell layer. To date, our understanding of the immediate physiological response of the odontoblast to pathological processes or iatrogenic trauma remains limited. This paper reviews: first, the evidence for and against the existence of physiological barrier between the distal segments of odontoblast cell bodies; second, the alteration of this physiological barrier following routine restorative procedures; third, the role junctional complexes may play in this barrier function, and finally, the role that dental innervation, both afferent and efferent, may play in influencing and regulating the response of the dental pulp to external stimuli.


Increase in c-fos-like immunoreactivity in the trigeminal nucleus complex after dental treatment.

An immunohistochemical study was made of the temporal changes in the expression of the c-fos oncogene protein in the spinal trigeminal nucleus complex (STNC) after three different types of dental injury. One hour after tooth extraction or pulp exposure, c-fos neurons appeared in the ipsilateral subnucleus caudalis, but there were very few c-fos neurons in the ipsilateral subnuclei oralis and interpolaris, and in the trigeminal main sensory nucleus and contralateral STNC. The great number of c-fos neurons was found 2-4 hours after injury, levels decreased by 8 hours after injury. Twenty-four hours after injury, the number of these neurons in subnucleus caudalis became very small. Shallow cavity preparation, however, did not induce an increase in the number of c-fos neurons at any selected postoperative time in STNC. These results indicate that c-fos neurons appear mainly in subnucleus caudalis very soon after severe dental injury.


Patterns and plasticity of dental afferent inputs to trigeminal (V) brainstem neurons in kittens.

In the young cat, the natural replacement of deciduous teeth with a permanent dentition may be accompanied by extensive peripheral nerve degeneration and reinnervation. Since we have previously reported significant physiological effects of tooth pulp deafferentation on V brainstem neurons, we wished to determine if deafferentation associated with deciduous tooth exfoliation was accompanied by comparable neuroplastic changes in the brainstem of kittens. We have examined the pulpal afferent terminating pattern within the brainstem as well as the electrophysiological properties of V brainstem neurons during this naturally occurring deafferentation phenomenon. The pattern of the HRP-labelled terminals in different parts of V brainstem subnuclei in kittens was similar to that in adult cats. The highest density of labelled terminals was found in the subnucleus oralis. The properties of subnucleus oralis neurons also showed many similarities between kittens at age of 3-4 months old and adult cats, e.g., (1) in the proportions of neurons activated by tactile stimulation of 1-4 teeth (mandibular and maxillary canines and premolars), (2) the ratio of neurons showing fast adapting or slow adapting responses to dental tactile stimulation, (3) the proportions of neurons activated by electrical pulp stimulation of 1-4 teeth, and (4) the minimal latencies of pulp-evoked responses. However, the incidences of pulpal inputs as well as the convergent pattern to oralis neurons of afferent inputs evoked by periodontal stimulation and electrical pulpal stimulation were less common in kittens than adult cats.


Functional alterations in pulpal microcirculation in response to various dental procedures and materials.

Using the quantitative 15 microns microsphere injection method, the effects of several restorative procedures on pulpal microcirculation in dog canine teeth were investigated. Pulpal blood flow (PBF) decreased steadily as the remaining dentin thickness (RD) became smaller with crown preparation without water spray. One hour after the preparation wit 1/3 RD (approximately 1 mm) PBF was reduced to 10% of the control, indicating that dry deep preparation has a deleterious effect on PBF. A careful preparation to an RD of 1 mm under copious water spray had a negligible effect on PBF. Dry preparation to an RD of more than 50%, on the other hand, caused a significant increase in blood flow through shunt vessels, AVAs and "U"-turn loops as determined with the 9 microns and 15 microns microspheres. These shunt vessel activities were especially prominent in the apical portion of the pulp, suggesting that the activation of the shunt vessels may be one of the compensatory mechanisms of the pulp in response to inflammation. In rat incisor teeth observed by intravital microscope, shunt vessels opened up as the incisor tips were drilled. Impression procedures after tooth preparation with water spray using the copper band with wax caused severe flow fluctuation as compared to rubber base material impression. In anesthetics, the use of epinephrine was found to be the single most important factor affecting pulp circulation. Whether it is by infiltration or mandibular block, the use of epinephrine containing anesthetic caused a severe reduction in PBF.(ABSTRACT TRUNCATED AT 250 WORDS)


Etiology of dentin hypersensitivity.

Hypersensitive dentin appears to be mainly the result of an activation of the dentinal pain fibres, the A-fibres, at the pulpal wall. The stimuli which activate these nerves are primarily those which remove fluid from the dentinal tubules and mobilize capillary forces, causing a rapid outward flow. Application of a cold stimulus causes the fluid to contract, resulting in a similar rapid outward flow in the pulpal region of the tubules. Hypersensitivity to cold is also marked when there is a fluid-filled gap containing bacteria in the tooth. Experiments have shown that a freshly exposed dentin surface with patent tubules is more sensitive than a surface contaminated by a smear layer. Inflammation in the adjacent pulpal region can also increase sensitivity. The development of hypersensitive cervical and occlusal surfaces is due to mechanical and acidic effects from the oral environment, toothbrush abrasion, erosive components in the diet, plaque and bacterial invasion of dentin. Sometimes dentin is exposed by restorative therapy and occasionally eccentric occlusal loads can contribute to hypersensitivity. The sensitivity may persist unless the open tubular apertures are sealed.


Reactions of sensory nerves to dental restorative procedures.

The reactions of sensory nerves to restorative procedures can be classified as immediate, early and late. For each of these, the neural response depends upon the severity of pulpal injury and the stages of inflammation and healing. Immediate responses in the first few minutes include destruction of nerve fibers in the injured dentin and pulp, hypersensitivity of surviving fibers, release of neuropeptides into the pulp and neurogenic inflammation. Early responses occur during the first few days after cavity preparation, with nerve fibers sprouting in the surviving pulp and gaining increased axonal transport and neuropeptide contents. Sensory fibers containing calcitonin gene related peptide (CGRP) greatly outnumber those with substance P (SP); but both types grow toward the surviving odontoblasts and associated pulp tissue surrounding the lesion. Later during subsequent weeks the nerve fibers accompany granulation tissue as it replaces acute inflammation; and nerve sprouting subsides when inflammation is reduced and when reparative dentin covers the injury site. An important response to tooth injury that may regulate nerve sprouting reactions is the increased production of nerve growth factor (NGF) by pulpal fibroblasts near the lesion. The timing of the nerve sprouting reactions suggests that they may contribute to tooth hypersensitivity after restorative procedures.


Microleakage related to restorative procedures.

A current Med-line search from 1966 to present cited over 344 juried publications employing the term microleakage. Dentistry recognizes microleakage as a multifaceted biological phenomenon. Brännström et al. (1967), Trowbridge (1982), Närhi (1983), and others have reported the sensory component of microleakage as a consequence of hydrodynamic fluid movement within the dentinal tubule complex. This movement of dentinal fluid has been demonstrated to displace nociceptive receptors which stimulate the nerves of the Group A fibers which run and terminate within the odontoblastic layer (see Matthews 1992). Penetration of oral fluids, bacteria and their toxic products within the preparation-material interface following material insertion accounts for the pathological component of microleakage (Browne and Tobias 1986). Continued microleakage of bacterial infiltrates eventually present as an inflammatory process which may initially signal the dentin complex to respond by deposition of a hypermineralized or sclerotic dentin. The pulp-dentin interface will repair with a specialized zone of reparative dentin. An overwhelming carious lesion often results in pulp infection and eventual necrosis. Vital dentin is an extension of the pulp, presenting the first line of defense to the consequences of microleakage. Recent publications have demonstrated that microleakage of dental materials in non-exposed and exposed pulps is a function of controlling bacterial infection. In an exposed mature dental pulp, the mesenchymal tissue permits the reorganization of pulp tissue and regeneration of a new dentin bridge in the presence of a biological seal. New odontoblastoid cells appear to regenerate from deeper pulpoblasts in the presence of various dental materials, apparently without an epithelial stimulating factor (Yamamura 1985). This inherent healing of the dental pulp and regeneration of a new dentin bridge is expressed in the presence of various dental materials, but only in the absence of bacterial infection. Data which evaluates the biological deposition of reparative and dentin bridges as either repair or regeneration are presented as a basis for considering the clinical selections of dental materials. Recent data demonstrate that dentin and pulp healing are ensured when a proper biological seal is provided to control and prevent microleakage.


Integration of human and animal concepts of energy metabolism.

With relatively few limitations, findings derived from the study of the energetics of farm animals provide extremely valuable information in the application of human energetics to the solution of dietary questions. The nature of the body's response to variation in energy intake and the nature of variation among individuals are good examples. It is important to appreciate the differences in the usual experimental constraints of studies with animals and humans. Human requirements are based on the summation of the energy costs of several different activities performed throughout the day whereas animal requirements are based on steady-state conditions at various production levels. Although the factors which influence specific biochemical or physiological events may be very similar, the manner in which those effects are incorporated into energy systems and the terminology used to describe them may differ substantially.


Role of gastrointestinal factors in hunger and satiety in man.

There is, we feel, a strong case to be made for a major influence of gastrointestinal factors in the control of eating behaviour in humans. Moreover, the findings suggest that satiety and hunger are controlled by different gastrointestinal mechanisms. Gastric distension is perhaps the most important factor that stops people eating, but a true feeling of satiety also requires the presence of nutrients in the small intestine. Hunger appears to be little influenced by the amount of food in the stomach and appears to be more dependent on the nutrient load in the small intestine. I do not mean by this to suggest that the gastrointestinal mechanisms are the only mechanisms controlling eating behaviour in humans or even the most important. Nevertheless they constitute an important and fundamental control system that links with the post-absorptive mechanisms and a plethora of social and conditioning factors to influence what we eat.


Nutrition and socio-economic development in Southeast Asia.

While most Third World countries, particularly in Africa and Latin America, have experienced a deterioration in child welfare as a result of the severe economic downturn in the 1980s, Southeast Asia in general managed to sustain improvements in the situation of its children because it has maintained satisfactory rates of economic growth. However, there were exceptions within Southeast Asia. The Philippines, Vietnam, Dem. Kampuchea and Laos had unsatisfactory growth rates and, consequently, unsustained nutritional gains from the 1970s through the 1980s. Economic factors exerted a big impact on the Philippine nutrition situation, particularly on the dietary status of the households and the nutritional status of children. As a result of the economic dislocation occurring in the country, the nutritional gains of 1978-82 were not maintained in succeeding years. Unlike the case of Thailand, it has been estimated that the solution to nutritional problems in the Philippines is far from being achieved in the immediate future (Villavieja et al. 1989). On the other hand, the nutrition improvements in Thailand have been as remarkable as the economic growth over the last decade. Long-term investments in health, nutrition and other social services in Thailand (as well as in Indonesia) have paid off according to the assessment by the United Nations (1990). It appears, therefore, that the nutrition situation in developing countries is highly dependent on the economic situation, globally and nationally (Cornia et al. 1987), as well as on investment in social services. Adjustment policies should, therefore, consider their implications on distribution and poverty in order that they could positively contribute to the improvement of the nutrition of the people.


Eicosapentaenoic acid inhibits endothelium-dependent relaxation to acetylcholine in guinea pig coronary resistance vessels.

Dietary supplementation with eicosapentaenoic acid (EPA) alters arachidonate metabolism. This study characterizes the effect of dietary EPA on endothelium-dependent vasodilation to acetylcholine (ACH) and ATP in guinea pig coronary resistance vessels. Guinea pigs were fed standard chow (n = 6), standard chow+sesame seed oil (n = 6), or standard chow+menhaden fish oil (17% EPA; n = 6). Coronary vasodilations were examined in the isolated, potassium-arrested heart utilizing a modified Langendorff preparation. Coronary vessels were constricted with prostaglandin F2 alpha and relaxed with ACH (5.5 x 10(-9)-10(-6) moles) or ATP (10(-10)-10(-7) moles). Endothelium-dependent dilations to ACH, but not ATP, were attenuated by dietary supplementation with EPA. To assess the role of the endothelium in modulating vascular responses to agonists following dietary manipulation, the perfusate was stimulated by electrolysis (9 V, 4 Hz, 2 msec) in order to generate free radicals, which we have shown to preferentially damage the endothelium. After endothelial damage, responses to ACH, ATP, and nitroprusside were similar between the dietary groups. In an additional group of standard diet animals (n = 6) experiments were performed to assess the role of prostanoid metabolism in affecting coronary vascular reactivity. Perfusion of hearts with indomethacin (14 microM) reduced endothelium-dependent vasodilations to ACH (5.5 x 10(-9)-10(-6) moles), but not to ATP (10(-10)-10(-7) moles). After endothelial damage, infusion of ACH resulted in vasoconstriction, whereas vasodilation responses to ATP were absent. We conclude that dietary supplementation with EPA inhibits endothelium-dependent dilations to ACH in guinea pig coronary microvessels. These diet-related differences in vascular reactivity may be related to the fish-oil-induced alteration of vasodilator prostaglandin metabolism. In the coronary bed, different endothelial factors appear to mediate relaxation to ACH and ATP.


Effects of stearic acid and beef tallow on iron utilization by the rat.

Two experiments were done in which anemic rats were fed diets containing safflower oil or stearic acid and low (10 ppm) or adequate (39-42 ppm) iron. Diets were 24% fat by weight. In the stearic acid diets, 2% (Experiment 1) or 4% (Experiment 2) of the fat was supplied by safflower oil to satisfy essential fatty acid requirements. Repletion of hemoglobin, hematocrit, and liver iron was assessed. Compared with safflower oil in both experiments, stearic acid had a significant positive effect (P less than 0.0001) on repletion of hemoglobin (Hb), hematocrit (Hct), and liver iron concentration; the effect on Hb and Hct was most pronounced when dietary iron was low. When expressed as g Hb/mg Fe intake, Hb repletion was affected by a significant interaction between fat and Fe (P less than 0.002) and was greatest in rats fed low iron stearic acid diets. In a third experiment, rats were fed low dietary iron and 24% safflower oil, 20% stearic acid + 4% safflower oil, 3.2% stearic acid + 20.8% safflower oil, or 20% beef tallow + 4% safflower oil. The 20% beef tallow provided 3.2% stearic acid in the total diet. The response of Hb and Hct were similar to those in the first two experiments for rats fed safflower oil or stearic acid. Rats fed beef tallow had significantly greater (P less than 0.05) Hb and Hct repletion than did rats fed safflower oil, although the degree of repletion was less than that observed in rats fed 20% stearic acid. There was no difference in iron repletion of rats fed 3.2% stearic acid and rats fed beef tallow. We conclude that stearic acid enhances iron utilization by rats.


Effects of H-2 on neural tube defects in congenic mice.

Pregnant mice congenic with C57BL/10 (B10.A, B10.BR, B10.D2, B10.A[2R], B10.A[5R], B10.A[15Rd, B10.A[1R], B10.A[18R], and B10.0L) were fed Purina Mouse Chow or the same diet plus 200 IU of vitamin A daily. The pregnant dams were sacrificed on the eighteenth day of gestation, and the fetuses were sexed and examined for defects in neural tube development. The frequency of neural tube defects was low (mean frequency of all strains, 0.36%) and was not affected by the addition of vitamin A (200 IU/day) to the diet. Twenty-seven of the 29 defects observed occurred in the anterior tube (exencephaly); fourteen were identified in female fetuses, but the sex could not be determined in the other 15 cases because of fetal death and early autolysis. Variations in frequency among the strains suggest that a locus between E beta and H-2D has a moderate influence on the occurrence of neural tube defects. Strains that had H-2d alleles in this segment of the H-2 complex had relatively high frequencies, and those with H-2b or H-2k alleles had significantly lower frequencies.


Role of extracellular magnesium in insulin secretion from rat insulinoma cells.

Magnesium (Mg2+) is an abundant intracellular cation that participates in the regulation of the intracellular concentration of ATP. In this study, we examined the relationship between insulin secretion and intracellular free Mg2+ ([Mg2+]i) in a rat-insulinoma cell line (RIN m5F), using a fluorescent dye (Mag-fura-2). KCI, forskolin, and D-glyceraldehyde increased [Mg2+]i and insulin secretion from RIN m5F cells in a dose-dependent fashion. Verapamil, a voltage-dependent Ca2+ channel blocker, inhibited the increase of [Mg2+]i that was evoked by KCI, forskolin, and D-glyceraldehyde. In a Mg(2+)-free buffer, these agents failed to cause an elevation in [Mg2+]i; however, the insulin response to KCI and forskolin was enhanced, compared with that in the presence of Mg2+ (1.25 mM). Our findings suggest that [Mg2+]i is dependent upon extracellular Mg2+, and the influx through the voltage-dependent Ca2+ channel. Mg2+ may competitively inhibit the voltage-dependent Ca2+ channel, which is known to play a role in insulin secretion. An absence of Mg2+ in the extracellular space may result in enhanced insulin secretion. [Mg2+]i may play a role in insulin secretion from RIN m5F cells.


Effects of cold acclimation and fasting on thyroxine 5'-deiodinase in brown adipose tissue of ob/ob mice.

Gradual acclimation to mild cold for 6 weeks increases the total activity of thyroxine 5'-deiodinase in brown adipose tissue (BAT) of genetically obese (ob/ob) mice to a level greater than that in similarly acclimated lean mice. This increase is largely due to the growth of the BAT in the ob/ob mouse, because specific activity of the enzyme is only slightly increased. In similarly cold-acclimated lean mice, the specific activity of thyroxine 5'-deiodinase was not altered. BAT mitochondrial GDP binding increased to the same high level in the gradually cold-acclimated ob/ob mouse as in cold-acclimated lean mice. We conclude that the growth and maintenance of BAT in the cold-acclimated ob/ob mouse, as in the cold-acclimated lean mouse, does not require greatly increased activity of thyroxine 5'-deiodinase. Fasting for 48 hr did not alter thyroxine 5'-deiodinase activity of BAT in either lean or ob/ob mice. The fasting-induced increase in activity seen by others in lean mice is probably due to thermoregulatory stimulation of BAT occasioned by the low environmental temperature at which the fasting occurred.


Temporal effects of estradiol and diethylstilbestrol on pituitary and plasma prolactin levels in ovariectomized Fischer 344 and Holtzman rats: a comparison of radioimmunoassay and Nb2 lymphoma cell bioassay.

An Nb2 lymphoma cell bioassay (Nb2BA) and a radioimmunoassay (RIA) were used to compare plasma and pituitary levels of prolactin in ovariectomized Fischer 344 (F344) and Holtzman rats treated with either diethylstilbestrol (DES) or estradiol for up to 8 weeks. The objectives were to determine whether there were temporal differences in prolactin responses in strains with different genetic predispositions to estrogen-induced pituitary tumor formation and to determine whether the results of the two assay methods were equivalent. All rats were ovariectomized for 7 days and all except controls received subcutaneous Silastic implants of DES or 17 beta-estradiol and were sacrificed at intervals from 2 days to 8 weeks later. Pituitary content and plasma levels of prolactin were determined by Nb2BA and RIA and the ratio of these measurements was calculated. DES induced a significant increase in pituitary prolactin in F344 rats by 2 days of treatment, as measured by RIA. Pituitary content increased to a peak by Day 4, after which a gradual decline occurred until the end of the experiment. Nb2BA measurements were similar to those obtained by RIA, except at 8 weeks, when the content determined by Nb2BA was significantly higher than the content determined by RIA. When estradiol was given to F344 rats a pattern of increase and subsequent decrease in pituitary content similar to that seen with DES was observed and levels measured by Nb2BA and RIA were essentially equivalent. Plasma levels of prolactin in DES-treated F344 rats increased exponentially through the 8 weeks, and the Nb2BA measurements were significantly greater than levels determined by RIA throughout the treatment period. Estradiol treatment produced a pattern of change in plasma levels of prolactin similar to that observed with DES, except that RIA and Nb2BA measurements were not different. Comparable results were obtained in Holtzman rats, except plasma levels were not increased to the same degree as seen in F344 rats. From these results, we conclude that DES, but not estradiol, can selectively increase the secretion of prolactin that is more bioactive than immunoreactive and that this effect of DES is observed in F344 and Holtzman rats, although F344 rats released more prolactin in response to estrogens than did Holtzman females.


Interleukin 1 pretreatment reduces the insulin response to glucose in chronic sucrose-fed rats.

The present studies were undertaken to determine whether interleukin 1 beta ([IL-1] 1.0 micrograms/kg, ip) pretreatment for 3 days impairs the adaptive response to sucrose feeding in rats. One week after the last IL-1 injection, when no differences in plasma glucose and serum immunoreactive insulin (IRI) levels were observed, sucrose feeding was started. Sucrose feeding for 4 weeks did not affect basal glucose levels, whereas basal IRI levels were increased in sucrose-fed rats without IL-1 pretreatment. Eight weeks later, plasma glucose levels were increased before and at 15 min after intravenous bolus of 0.5 g/kg of glucose in sucrose-fed rats with IL-1 pretreatment. Only in IL-1-treated sucrose-fed rats were basal and glucose-stimulated IRI levels significantly reduced, compared with those levels in sucrose-fed vehicle-treated rats. IL-1 decreased pancreatic IRI contents at 1 and 9 weeks after the injection. These data suggest that pancreatic damage by IL-1 attenuated insulin response to glucose stimulation after long-term sucrose feeding.


Naloxone reversal of ischemic arrhythmia is stereospecific and suggests role of endogenous opioid peptides in ischemic heart disease.

The effects of the stereoisomers of naloxone during myocardial ischemia were studied. (-)-Naloxone (but not the (+)-isomer naloxone) attenuated the ischemia-induced cardiac arrhythmias, hypotension, and bradycardia that result from coronary artery occlusion in anesthetized rats. From these findings, it may be inferred that endogenous opioid peptides may play a role in the pathophysiology of myocardial ischemia. It is also suggested that naloxone may have therapeutic value in the prevention and treatment of ischemic heart disease.


Iron metabolism is modified by the copper status of a human erythroleukemic (K562) cell line.

Copper deficiency is known to result in a microcytic, hypochromic anemia. Red cells of copper-deficient animals have less hemoglobin than their copper-adequate counterparts. The objective of this work was to determine what role copper plays in maintaining hemoglobin levels. It was hypothesized that the primary defect lies in intracellular iron metabolism. The influence of copper supplementation on iron uptake and storage was examined in a cell line capable of hemoglobin synthesis. The results demonstrated that copper supplementation of human K562 cells was associated with higher cytosolic iron levels and ferritin levels. Copper supplementation of the cell culture altered the initial rate of iron uptake from transferrin and enhanced iron uptake in noninduced cells; however, in hemin-induced K562 cells, which express fewer transferrin receptors on the cell surface, copper appeared to reduce iron uptake. Subsequent studies showed that the cells were able to take up the same amount of iron from transferrin when incubated over a longer period of time (24 hr). In the noninduced (non-hemoglobin synthesizing) cells, proportionally more iron was associated with the ferritin. We concluded from these studies that copper affects both uptake and storage of iron and that copper supplementation reduces cellular iron turnover.


Regulation of duodenal insulin-like growth factor I and active calcium transport by ovariectomy in female rats.

There is a significant body of data that supports the concept that reproductive hormones in females have effects on duodenal calcium transport that are not mediated via altered circulating concentrations of 1,25-dihydroxyvitamin D (1,25(OH)2D). Previously, we have shown parallel alterations in duodenal Ca transport and longitudinal bone growth rate in sexually maturing female rats in response to ovariectomy and estradiol (E) treatment of ovariectomized (OVX) rats (OVX+E) without any change in circulating levels of 1,25(OH)2D or parathyroid hormone. Results are presented here from experiments designed to: (i) further explore the relationship between 1,25(OH)2D and ovarian status in the regulation of duodenal calcium transport, and (ii) determine whether OVX and E replacement alter circulating and duodenal levels of insulin-like growth factor I (IGF-I) that might be related to effects on Ca transport. Growth hormone, which has been shown to affect intestinal Ca absorption and vitamin D metabolism, is thought to act indirectly by stimulating IGF-I. Six-week-old female rats were OVX, given estradiol implants (OVX+E), and fed a diet containing either 0.5% or 0.1% Ca for 3 weeks. In both diet groups, the OVX animals exhibited a higher level of Ca transport, as measured by the everted gut sac method, than either the intact controls or the OVX+E group; there was no difference in calcium transport between the different diet groups. Although there was no difference in circulating levels of 1,25(OH)2D among the intact, OVX, and OVX+E groups fed either diet, animals fed the 0.1% Ca diet had higher circulating levels of 1,25(OH)2D than those fed the 0.5% Ca diet. There was no difference in duodenal levels of calbindin9K among intact, OVX, and OVX+E animals in either diet group, although the animals fed the 0.1% Ca diet had higher levels of calbindin9K than the animals fed the 0.5% Ca diet. In animals fed the 0.5% Ca diet, OVX resulted in elevated serum and duodenal levels of IGF-1, as compared with intact and OVX+E animals on the same diet. In animals fed the 0.1% Ca diet, there was no elevation of IGF-I in the OVX group relative to intact and OVX+E animals. These results lend additional support to the concept that alterations in duodenal active calcium transport that occur with alterations in ovarian hormones are not mediated by changes in serum levels of 1,25(OH)2D, but may be related to some factor related to growth, possibly IGF-I.


Testosterone alters duodenal calcium transport and longitudinal bone growth rate in parallel in the male rat.

Duodenal active calcium transport and longitudinal bone growth rate have been shown previously to be regulated in parallel by alteration of gonadal hormone status in sexually maturing female rats. The present study was designed to extend these observations to the sexually maturing male rat. Male rats were orchidectomized (ORX) and given Silastic implants containing either testosterone or estradiol at 6 weeks of age. At 9 weeks of age, duodenal active calcium transport was measured by the everted gut sac method and longitudinal bone growth rate was determined by tetracycline labeling. Decreases in body weight, longitudinal bone growth rate, duodenal calcium transport, and serum Ca and P were exhibited by ORX animals as compared with age-matched control animals. Testosterone administration to ORX animals resulted in an increase in body weight, longitudinal bone growth rate, duodenal calcium transport, and serum Ca and P as compared with ORX animals to a level not significantly different from that of age-matched control animals. Estradiol administration to ORX animals resulted in an additional decrease in body weight, although no significant effect on duodenal calcium transport, serum Ca, or P was noted as compared with ORX animals. There were no statistically significant alterations in the circulating levels of 1,25-dihydroxyvitamin D, parathyroid hormone, or osteocalcin in response to any of the experimental manipulations of gonadal status. These results indicate that, as in the female, gonadal hormone status affects intestinal calcium transport in sexually maturing male rats in parallel with changes in bone growth rate by mechanisms that are independent of circulating levels of 1,25-dihydroxyvitamin D.


Decentralization of the superior cervical ganglia and the immediate hypersensitivity response.

Bilateral decentralization of the superior cervical ganglia protects against pulmonary inflammation when measured 8 hr after induction of anaphylaxis in rats sensitized to the nematode, Nippostrongylus brasiliensis. Since anaphylactic shock produces immediate perturbations to the cardiovascular and respiratory systems, we examined whether bilateral decentralization of the superior cervical ganglia modified the responses of these two systems during the first 4 hr of the anaphylactic response. With the exception of the bronchioles, decentralization did not protect against anaphylaxis-associated increases in extravasation of albumin, and the small changes in respiratory function induced by anaphylaxis were unaffected by the denervation. Decentralization did not alter anaphylaxis-induced reductions in blood flow to the gastrointestinal tract; however, blood flow to the kidneys and spleen of decentralized rats was restored more rapidly to normal values. These results suggest that the protective effect of decentralization on the late phase pulmonary inflammation of anaphylaxis is unrelated to early changes in respiratory mechanics, although the protection may be facilitated by the more rapid re-establishment of normal cardiovascular homeostasis.


Phosphatidylserine suppresses antigen-specific IgM production by mice orally administered sheep red blood cells.

Phosphatidylserine is an endogenous acidic phospholipid that has been shown to modulate nervous system function. In the immune system, phosphatidylserine has been shown to suppress T dependent and T independent immune responses after systemic administration of antigen and lipid. However, no studies on the possible regulation by phosphatidylserine on mucosal immunity have been undertaken. Therefore, we studied the action of phosphatidylserine on immunocompetence using orally immunized mice. Mice orally administered phosphatidylserine (25 mg/kg/day) and subsequently intubated intragastrically with sheep red blood cells showed a significant decrease in antigen-specific IgM production by splenic lymphocytes compared with controls. Furthermore, the response of splenic lymphocytes obtained from phosphatidylserine-treated, antigen-primed animals to antigen or pokeweed mitogen in proliferation assays was markedly suppressed, compared with splenic lymphocytes obtained from nontreated, antigen-primed mice. Similarly, splenic lymphocytes from phosphatidylserine-treated, antigen-primed animals cultured in the presence of antigen produced no measurable interleukin 4 and low levels of interleukin 2, whereas splenic lymphocytes from antigen-primed animals produced measurable levels of interleukin 4 and significantly higher levels of interleukin 2. By fluorescence-activated cell sorter analysis, brightly stained B lymphocytes (Ig+) take up a larger portion of phosphatidylserine than do brightly stained T lymphocytes (Thy 1.2+). Collectively, these results point to the immunosuppressive qualities of phosphatidylserine. Given that phosphatidylserine is released upon injury and destruction of eukaryotic cells, these results suggest that phosphatidylserine may be an endogenous anti-inflammatory molecule.


Papaverine effects on PGI2 and TXA2 release from the canine vascular wall.

Operative manipulation of blood vessels might lead to spasm, thereby destroying the endothelial cell function: the spasm can be prevented by the vasodilator papaverine. To study if this was mediated via the prostanoid pathway the following investigation was undertaken: canine jugular veins and carotid arteries were dissected with or without papaverine. Vessel segments were then perfused with Hank's balanced salt solution for five times 15 min. Prostacyclin was measured as the stable degradation product 6-keto-PGF1 alpha and thromboxane as TXB2, by radioimmunoassay. Control arterial segments' 6-keto-PGF1 alpha release was initially 129.5 + 20.1 pg/mm2/15 min, and 29.7 + 10.4 after 60 min (p less than 0.05 vs initial value) and responded to arachidonic acid (AA) with an increase to 139.2 +/- 23.1 pg/mm2/15 min (p less than 0.05). Segments treated with papaverine had the same release as the controls. In venous segments there was a lower initial release (p less than 0.05) from segments given papaverine than from controls, but this was more likely an effect of papaverine on the assay. There was no difference in release of prostacyclin from segments given papaverine in the perfusate compared to controls when using 125I tracer. When using 3H tracer including absorption of free antigen to dextran coated charcoal, papaverine displaced the free tracer giving artificially low values. There was no effect of papaverine given intraoperatively on the TXB2 release, neither from arteries nor from veins. In another experiment the vessel wall tension was examined and the cyclooxygenase inhibitor diclofenac did not inhibit the vasodilating effect of papaverine.(ABSTRACT TRUNCATED AT 250 WORDS)


Relevance of vasoactive mediators for the blood pressure effects of intravenous arachidonic acid injection in rats.

Vasopressor response and release of eicosanoids following intravenous injection of arachidonic acid (AA) were examined in normotensive rats. AA administration caused a rapid initial fall of arterial pressure followed by a brief rise and a subsequent prolonged fall in anesthetized rats. Immediately after AA injection the blood levels of TXB2 and 6-keto-PGF1 alpha, the stable metabolites of TXA2 and prostacyclin, rose, from 1.52 +/- 0.23 ng/ml to 176.4 +/- 42.6 ng/ml and from 4.05 +/- 0.67 ng/ml to 171.4 +/- 31.2 ng/ml, respectively. Blood pressure behaviour and eicosanoid blood level were influenced by different inhibitors and antagonists of vasoactive mediators. The cyclooxygenase inhibitor acetylsalicylic acid completely eliminated the second blood pressure depression after AA injection and simultaneously diminished TXB2 and 6-keto-PGF1 alpha formation in murine blood, whereas the TXA2 receptor antagonist BM 13.177 prevented the return of the blood pressure to preinjection level after the initial brief fall in arterial pressure. Although the TXA2 synthase inhibitor HOE 944 markedly inhibited TXB2 formation, no influence on AA-induced blood pressure changes could be registered. The receptor antagonist of platelet activating factor BN 52021 and the serotonin and histamine receptor antagonist cyproheptadine also reduced TXB2 amounts, in murine blood without any effects on blood pressure behaviour.


The correlation between rises in intracellular calcium and PGI2 production in cultured vascular endothelial cells.

Elevation of intracellular calcium in response to trypsin, bradykinin, thrombin or histamine is associated with a proportional increase in PGI2 production in cultured human umbilical vein endothelial cells (HUVEC), bovine pulmonary artery endothelial cells (CPAE), and bovine aortic endothelial cells (BAEC). The major agonists that induce increases in intracellular calcium and PGI2 production are thrombin and trypsin in HUVEC, bradykinin in CPAE, and bradykinin and trypsin in BAEC. These results suggest that endothelial cells derived from different species or sites require different agonists to induce increases in intracellular calcium and PGI2 production and that only agonists which increase intracellular calcium can stimulate PGI2 production.


Production of platelet thromboxane A2 and arterial prostacyclin I2 from hypercholesterolemic rats.

The plasma cholesterol, plasma malonaldehyde (MDA), platelet thromboxane A2 (TXA2) and vascular prostacyclin (PGI2) were measured in male Sprague-Dawley rats fed diets supplemented with cholesterol (1%) and cholic acid (0.5%). For comparisons, measurements were made in rats fed normal diets. The concentration of cholesterol in the plasma of rats had reached a maximum in 1 week of feeding experimental diets. TXA2 production from collagen and thrombin stimulated platelets was significantly decreased in animals fed experimental diets for 1 week. The production of MDA in the plasma of animals fed experimental diets for 8 weeks was significantly lower compared to the animals fed normal diets. There was a small but significant reduction in the formation of PGI2 in rats fed experimental diets for 8 weeks. These data suggest that feeding cholesterol rich diets to rats alters the platelet membrane properties differently from human and rabbit. Furthermore, cholesterol feeding to rats had some damaging effect on the arterial PGI2 synthesis.


Expression of the cDNA for NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase as a catalytically active enzyme in Escherichia coli.

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme involved in the catabolism of the prostaglandins. The cDNA for human placental 15-PGDH has been expressed in Escherichia coli as a catalytically active protein. The polymerase chain reaction was used to introduce restriction endonuclease sites at each end of the 15-PGDH coding sequence. The 15-PGDH DNA was then inserted into the bacterial expression plasmids pUC-18 and pUC-19 which contain the isopropyl-l-thio-beta-D-galactopyranoside (IPTG) inducible lacZ promoter. Extracts from E. coli containing these expression plasmids exhibited 15-PGDH activity which was inducible with (IPTG). Crude extracts from E. coli expressing 15-PGDH activity were found to contain proteins of the predicted sizes in stained SDS-polyacrylamide gels and in Western blots using human placental 15-PGDH antiserum. The specific activity in E. coli extracts was several hundred-fold higher than that seen in extracts from human placenta.


Immunohistochemical localization of PGF2 alpha receptor in the rat ovary.

As a step towards understanding the role of prostaglandin F2 alpha (PGF2 alpha) in ovarian function, a rabbit antiserum against purified PGF2 alpha receptor (PGF2 alpha-R) was produced. This report details the use of this antiserum in immunohistochemical staining of ovaries of non-pregnant and pregnant rats to ascertain which cell types, in vivo, possess PGF2 alpha-R. In non-pregnant rats, three ovarian cell subpopulations contain immunoreactive PGF2 alpha-R. These include: a subpopulation of the cells found in corpora lutea, a subpopulation of the thecal cells surrounding secondary and mature (Graafian) follicles, and a subpopulation of primary and secondary interstitial cells. The ovarian tissues and cell types in which immunoreactive PGF2 alpha-R cannot be demonstrated include: the serosa overlying the ovary and its vessels, the coelomic epithelium and its underlying cortical stroma, medullary stroma and vessels, granulosa cells of primary, secondary and mature follicles, the oocyte, and the blood vessels and stroma within corpora lutea. PGF2 alpha-R immunohistochemical staining of corpora lutea from non-pregnant animals was examined both prior to the start of luteolysis and during luteolysis. During luteolysis, cells undergoing apoptosis stained for the presence of PGF2 alpha-R. PGF2 alpha-R immunohistochemical staining was also examined in corpora lutea during pregnancy and until 4 days postpartum. The major findings here were the apparent large increase in staining intensity of granulosa-lutein cells during pregnancy, and the loss of PGF2 alpha-R immunopositivity of the granulosa-lutein cells during the postpartum period. In summary, three ovarian cell subpopulations, all of which can secrete steroids, possess immunoreactive PGF2 alpha-R.


Prostaglandin D2 in cultured capillary and microvascular endothelium of human brain.

Production of prostaglandin D2 (PGD2) was investigated in cultured endothelial cells derived from capillaries and microvessels (small and large) of human brain using radioimmunoassays. Peptides, catecholamines, thrombin, protein kinase C-activating phorbol ester and calcium ionophore greatly stimulated the secretion of endothelial PGD2. Secretion of PGD2 induced by vasoconstricting peptides, angiotensin II and arginine-vasopressin, was almost completely abolished by their respective specific receptor antagonists [Sar1, Ala8]-Ang II and [1-6(beta-mercapto-beta,beta-cyclopentamethylene propionic acid) 2-O-methyltyrosine]. Thus, the augmented production of PGD2 by angiotensin II and arginine-vasopressin is a receptor-mediated event. It also indicates that the EC have specific angiotensin II and arginine-vasopressin (V1) receptors. This study represents the first demonstration of vasoactive agents modulating PGD2 production in capillary and microvascular endothelium of human brain.


Fatty acid changes during the induction of differentiation of human promyelocytic leukemia (HL-60) cells by phorbolmyristate acetate.

We studied fatty acid changes that are likely to occur during phorbolmyristate acetate (PMA)-induced differentiation of HL-60 cells. It was observed that PMA-induced differentiation is associated with increased uptake, but not synthesis, of fatty acids. Fatty acid analysis revealed that arachidonic acid (AA), 20:5 n-3 and 22:6 n-3 levels are reduced with a concomitant increase in 22:5 n-6 in the phospholipid fraction. In the FFA fraction there are increases in free AA, free 20:5 n-3, 22:5 n-3 and 22:6 n-3, and a fall in free 22:5 n-6 in PMA-treated cells. PMA-induced differentiation and nitroblue tetrazolium reduction by PMA-treated cells was only partially inhibited (about 20-30%) by indomethacin and nordihydroguiaretic acid (cyclooxygenase and lipoxygenase inhibitors respectively), but not by superoxide dismutase, catalase or mannitol. These results indicate that PMA-induced differentiation of HL-60 cells is accompanied by specific changes in the fatty acid composition of the cells.


Endogenous inhibitors of human placental prostaglandin dehydrogenase.

The presence of endogenous inhibitors of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) has been indicated by increasing total activity after the initial purification step of PGDH in human placenta. Based on this observation, we tried to characterize and analyze endogenous inhibitors of PGDH in human placenta in this study. The inhibitors were extracted from the supernatant by precipitation at pH 5.2 and partially purified by acetone precipitation and by thin layer chromatography. The inhibitors were stable to heating at 100 degrees C for 10 min, and to trypsin digestion. The pattern of inhibition was competitive with regard to PGE2 and uncompetitive with regard to NAD at pH 8.0. The Ki value for PGE2 was 18.9 microM. Analysis by gas chromatography and mass spectrometry indicated that the inhibitors consisted of fatty acids which were palmitic, stearic, oleic and linoleic acids. Myristic, palmitic and stearic acids were confirmed to exert an inhibitory action on PGDH and showed a competitive inhibition pattern. Stearic acid was less potent in inhibition than other fatty acids. These findings suggest that intracellular fatty acids may play a unique role in the control of PGDH activity.


Effects of different cyclooxygenase inhibitors on the segmental distribution of pulmonary vascular resistance in the dog.

An increase in pulmonary vascular resistance (PVR) after cyclooxygenase inhibition (COI) is well documented in the dog, but the site of vasoconstriction to chemically distinct cyclooxygenase inhibitors is largely unknown. The purpose of the present study was to examine and compare equimolar concentrations of three chemically unrelated cyclooxygenase inhibitors, indomethacin (INDO; n = 6), meclofenamate (MECLO; n = 6) and ibuprofen (IBU; n = 5), upon the longitudinal distribution of PVR in the isolated canine lower left lung lobe perfused at constant flow with autologous blood. At successive increases in the blood concentration of each cyclooxygenase inhibitor, PVR was partitioned into upstream (arterial, Ra), middle (Rm) and downstream (venous, Rv) resistance by arterial and venous flow occlusion with capillary pressure estimated by a double flow occlusion technique. All three cyclooxygenase inhibitors produced significant pulmonary vasoconstriction with the largest increase in PVR after INDO (104 +/- 21%) and the smallest after IBU (69 +/- 10%). The PVR increase in the INDO and MECLO group was related to an elevation in both Ra (p less than 0.01) and Rv (p less than 0.01), whereas only Rv was increased by IBU (p less than 0.01). While none of the cyclooxygenase inhibitors increased Rm (p greater than 0.05), capillary pressure was increased from pretreatment levels by each cyclooxygenase inhibitor. Although each of the three chemically distinct cyclooxygenase inhibitors raised PVR, the segmental distribution of PVR and the magnitude of the capillary pressure increase varied at equimolar blood concentration.


Effects of methimazole on low-renal-mass hypertension: changes in blood pressure and pressor responsiveness to vasoconstrictors.

The administration of the antithyroid drug methimazole to rats via drinking water prevented the development of hypertension that usually accompanies subtotal nephrectomy and saline drinking (1% NaCl). In methimazole-treated rats, elevated blood pressure induced 5 weeks previously returned to normotensive levels. Pressor responsiveness to angiotensin, vasopressin and norepinephrine in unanesthetized rats was studied after prevention of hypertension in control, low-renal-mass hypertensive (LRM) and low-renal-mass methimazole-treated (LRM-M) rats, and in the reversion study in LRM and LRM-M rats. In LRM rats, responsiveness to vasoconstrictors was increased, whereas responsiveness to vasoconstriction was clearly reduced in LRM-M rats after prevention and reversion studies. These results suggest that (a) thyroid hormones are required in the early and established phases of LRM hypertension, and (b) the decreased pressor responsiveness to vasoconstrictors may play a role in the prevention and reversion of this type of hypertension following methimazole administration. However, the changes in pressor responsiveness may also be secondary to the reduction in blood pressure.


Muscarinic binding sites on bovine pulmonary arterial endothelial cells in culture.

We have investigated the presence and nature of muscarinic binding sites on membranes from cultured bovine pulmonary arterial endothelial cells (BPAE). BPAE were harvested and subcultured nonenzymatically; experiments were performed 3-5 days postconfluence and between 10 and 25 passage numbers. Utilizing radioligand binding techniques with the muscarinic receptor antagonists [3H]3-quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]MS) as probes, we identified a small population of atropine-sensitive muscarinic sites (1,800-2,000 sites/cell or 7-8 fmol/mg protein). Muscarinic binding sites on BPAE membranes resembled classical muscarinic receptors in that (a) the binding of 2 nM [3H]QNB was inhibited by muscarinic agonists and antagonists, (b) [3H]QNB binding was 30 times more sensitive to R(-)- than to S(+)-QNB, (c) binding of the muscarinic receptor agonist carbamylcholine involved high and low affinity components, (d) the stable GTP analog, Gpp(NH)p (100 microM) shifted agonist binding curves to the right by a factor of three, and (e) the high affinity binding of the agonist [3H]oxotremorine-M to muscarinic receptors was depressed by Gpp(NH)p. On the other hand, gallamine, which allosterically regulates muscarinic receptor binding in other tissues, did not affect the rates of dissociation of [3H]QNB, [3H]MS or [3H]oxotremorine-M from BPAE binding sites. We concluded that BPAE in culture exhibit muscarinic binding sites which possess many but not all of the properties associated with classical muscarinic receptors.


Effect of phorone and allopurinol on ischemia-reperfusion injury in gastrointestinal mucosa of the rat.

We studied the effect of inhibition of oxyradical formation and of endogenous glutathione (GSH) depletion on lesion formation in the gastrointestinal tract in a modified rat hemorrhagic shock model (1 h hypotension and 1 h reperfusion). Allopurinol, an inhibitor of xanthine oxidase, did not protect against lesion formation. This suggests that oxygen radicals generated from xanthine oxidase may not be the major cause of injury under these conditions of prolonged 'ischemia'-reperfusion. Phorone (diisopropylideneacetone), a GSH depletor, decreased mucosal GSH levels in the corpus, duodenum and small intestine, and also significantly reduced lesion formation histologically in the corpus, antrum, duodenum and small intestine. However, there was no significant differences in mucosal blood flow (as estimated by changes in mucosal hemoglobin concentrations and oxygen saturation of mucosal hemoglobin) in the corpus, antrum, duodenum and small intestine between phorone-pretreated and control rats. We conclude that phorone decreased mucosal GSH concentrations and exerted a protective effect against hemorrhagic shock-induced gastrointestinal mucosal lesions. The protective effect appears to be independent of mucosal blood flow.


Probucol attenuates a reduction in serum immunoreactive insulin levels by interleukin in adrenalectomized rats.

We determined a protective action of probucol (4,4'-[isopropylidenedithio]bis[2,6-di-tert-butylphenol]) on the reduction of serum immunoreactive insulin (IRI) levels by recombinant human interleukin-1 beta (IL-1) in adrenalectomized rats. IL-1 reduced the serum IRI levels 4 h after injection in rats fed a standard diet. However, IL-1 failed to inhibit serum IRI levels in rats fed a diet containing 1% probucol. In contrast, IL-1 decreased plasma glucose levels in both groups. Although IL-1 reduced plasma prostaglandin (PG) E2 levels in standard diet-fed rats, IL-1 did not affect plasma PGE2 levels in the animals fed the probucol-containing diet. The present study suggested that the free radical scavenger action of probucol can block the IL-1 effect on serum IRI levels.


Dose-dependent actions of spironolactone on the inner and outer zones of the guinea pig adrenal cortex.

The results of prior in vitro studies indicated that spironolactone (SL) caused far greater degradation of cytochromes P-450 in the outer (zona glomerulosa plus zona fasciculata) than inner (zona reticularis) zone of the guinea pig adrenal cortex and selectively decreased microsomal 17 alpha-hydroxylase activity. Studies were done to determine if the effects of SL in vivo were similarly zone and/or enzyme selective. Administration of high doses of SL (100 mg/kg) to guinea pigs altered the gross appearance of the adrenal glands and caused declines in 17 alpha- and 21-hydroxylase activities in both inner and outer zone microsomal preparations. The losses in enzyme activities were accompanied by decreases in microsomal cytochrome P-450, cytochrome b5 and heme concentrations, and in mitochondrial P-450 levels in both zones. Microsomal P-450(17 alpha) apoprotein levels were also decreased in both zones. A lower dose (25 mg/kg) of SL did not affect adrenal morphology, but decreased microsomal P-450 levels in both zones. Neither mitochondrial P-450 nor microsomal b5 concentrations were affected in either zone. 17 alpha-Hydroxylase activities and P-450(17 alpha) apoprotein concentrations in both zones were decreased by the lower dose of SL, but 21-hydroxylase activity declined in the inner zone only. The results indicate that very high doses of SL have a variety of nonspecific effects on the adrenals which may be the consequence of drug toxicity. Nontoxic doses exert more selective effects on microsomal cytochromes P-450 in both adrenal zones, more closely mimicking the in vitro actions of the drug.


Defect of the potassium transport process in the kidney of spontaneously hypertensive rats.

We investigated the natriuretic and kaliuretic effect of distal tubular diuretics in saline-loaded spontaneously hypertensive Wistar rats (SHR) from three different sources and normotensive Wistar rats (NWR). Orally administered early distal tubular diuretics (hydrochlorothiazide, chlorthalidone, metolazone, indapamide and cicletanine) caused much less potassium excretion in SHR than in NWR, whereas the magnitude of concurrent natriuresis was similar in both NWR and SHR. The intriguing renal handling of potassium excretion was exemplified by hydrochlorothiazide, for which enhanced kaliuresis was dose dependent in NWR but not in SHR. The doses tested ranged from 1 to 100 mg/kg, p.o. Amiloride, a late distal tubular diuretic, was also evaluated for its effect on sodium and potassium excretion in NWR and SHR. Amiloride produced potassium retention more effectively in NWR than in SHR, although the magnitude of natriuresis was similar. The difference between SHR and NWR with regard to potassium-retaining activity of amiloride was consistent at all doses tested (1-30 mg/kg, p.o.). In conclusion, it was suggested that SHR appear to have a genetic defect in potassium transport in the distal nephron.


A beta-carboline derivative (ZK 93426) counteracts the cardiorespiratory depressant effects of intravenous midazolam.

The purpose of this study was to test the effects of the new beta-carboline ZK 93426 on midazolam-induced cardiorespiratory depression. Seven pentobarbital-anesthetized (35 mg/kg i.p.) cats were treated with intravenous midazolam (2 mg/kg) while monitoring the respiratory minute volume (VE), tidal volume, respiratory rate, blood pressure, heart rate and expired CO2. Midazolam caused significant decreases in VE (p less than 0.05) and blood pressure (p less than 0.05). ZK 93426 (5 mg/kg i.v.) antagonized these effects and produced significant increases in VE and blood pressure that resulted in the return of these variables to premidazolam control values. In 4 animals with morphine-induced respiratory depression, intravenous ZK 93426 failed to antagonize the respiratory effects of morphine. Administration of intravenous ZK 93426 alone to 4 pentobarbital-anesthetized animals also failed to produce significant changes in cardiorespiratory activity. We conclude that ZK 93426 is effective in counteracting the cardiorespiratory depressant effects of midazolam and that these effects appear to be specific. The present data suggest that this compound may be useful for the treatment of benzodiazepine oversedation and overdose.


Effects of haloperidol, bromocriptine and amphetamine on the development of Ehrlich ascites carcinoma in mice.

We determined the effect of 13 days of treatment with 2.0 mg/kg haloperidol, 4.0 mg/kg bromocriptine or 2.0 mg/kg amphetamine on the number of tumor cells of mice bearing Ehrlich ascites carcinoma. The dopaminergic blocker significantly reduced the number of tumor cells of experimental mice, but the two dopamine-mimetic drugs used did not significantly affect tumor development. These results suggest that although neuroleptic drugs to inhibit Ehrlich ascites tumors, this effect does not seem to be related to changes in dopaminergic neuronal transmission.


Effects of potassium channel blockers on the negative inotropic responses induced by cromakalim and pinacidil in guinea pig atrium.

The K+ channel openers cromakalim and pinacidil induced a concentration-dependent reduction in atrial contraction force with EC50 values of 25 +/- 2 and 37 +/- 2 mumol/l, respectively. This depressant effect was antagonised by 50 mumol/l tacrine which displaced the concentration-response curves of cromakalim and pinacidil to the right. The respective DR50 values were 3.8 and 2.3. Increasing the tacrine concentration (100 and 500 mumol/l) produced no additional effect on the concentration-response relationships. Addition of 1 mumol/l atropine enhanced the antagonism due to tacrine by increasing the DR50 value from 3.8 to 6.5 for cromakalim and from 2.3 to 5.2 for pinacidil. Glibenclamide, an ATP-sensitive K+ channel blocker, competitively inhibited the negative inotropic effects of cromakalim and pinacidil. The respective dissociation constants for glibenclamide against cromakalim and pinacidil were 0.57 and 0.35 mumol/l. Neither apamin nor variation in external Ca2+ concentration affected the negative inotropic effects of the K+ channel openers. It was suggested that the mechanical effects of cromakalim and pinacidil are mediated through the ATP-sensitive K+ channels in the heart.