Biomedical subjects

S H Young

Publications and source records attributed to S H Young.

At least 19 recordsLinked to original sources

PubMed2004

Phosphatidylinositol 3-kinase regulates Ca2+ signaling in pancreatic acinar cells through inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase.

Calcium is a key mediator of hormone-induced enzyme secretion in pancreatic acinar cells. At the same time, abnormal Ca(2+) responses are associated with pancreatitis. We have recently shown that inhibition of phosphatidylinositol 3-kinase (PI3-kinase) by LY-294002 and wortmannin, as well as genetic deletion of PI3-kinase-gamma, regulates Ca(2+) responses and the Ca(2+)-sensitive trypsinogen activation in pancreatic acinar cells. The present study sought to determine the mechanisms of PI3-kinase involvement in Ca(2+) responses induced in these cells by CCK and carbachol. The PI3-kinase inhibitors inhibited both Ca(2+) influx and mobilization from intracellular stores induced by stimulation of acini with physiological and pathological concentrations of CCK, as well as with carbachol. PI3-kinase inhibition facilitated the decay of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) oscillations observed in individual acinar cells. The PI3-kinase inhibitors decreased neither CCK-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] production nor Ins(1,4,5)P(3)-induced Ca(2+) mobilization, suggesting that the effect of PI3-kinase inhibition is not through Ins(1,4,5)P(3) or Ins(1,4,5)P(3) receptors. PI3-kinase inhibition did not affect Ca(2+) mobilization induced by thapsigargin, a specific inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Moreover, SERCA blockade with thapsigargin abolished the effects of pharmacological and genetic PI3-kinase inhibition on [Ca(2+)](i) signals, suggesting SERCA as a downstream target of PI3-kinase. Both pharmacological PI3-kinase inhibition and genetic deletion of PI3-kinase-gamma increased the amount of Ca(2+) in intracellular stores during CCK stimulation. Finally, addition of the PI3-kinase product phosphatidylinositol 3,4,5-trisphosphate to permeabilized acini significantly attenuated Ca(2+) reloading into the endoplasmic reticulum. The results indicate that PI3-kinase regulates Ca(2+) signaling in pancreatic acinar cells through its inhibitory effect on SERCA.

Animals↗

PubMed2003

Outsourcing and benchmarking in a rural public hospital: does economic theory provide the complete answer?

INTRODUCTION: The ideology and pronouncements of the Australian Government in introducing 'competitive neutrality' to the public sector has improved efficiency and resource usage. In the health sector, the Human Services Department directed that non-clinical and clinical areas be market tested through benchmarking services against the private sector, with the possibility of outsourcing. These services included car parking, computing, laundry, engineering, cleaning, catering, medical imaging (radiology), pathology, pharmacy, allied health and general practice. Managers, when they choose between outsourcing, and internal servicing and production, would thus ideally base their decision on economic principles. Williamson's transaction cost theory studies the governance mechanisms that can be used to achieve economic efficiency and proposes that the optimal organisation structure is that which minimises transaction costs or the costs of exchange. Williamson proposes that four variables will affect such costs, namely: (i) frequency of exchange; (ii) asset specificity; (iii) environmental uncertainty; and (iv) threat of opportunism. This paper provides evidence from a rural public hospital and examines whether Williamson's transaction cost theory is applicable. METHOD: Case study research operates within the interpretivism paradigm and is used in this research to uncover why the outsourcing decision was made. Such research aims to study real-life experiences by examining the way people think and act and, in contrast to positivism, allows the interviewer to participate to better understand the details and features of the experiences. In the present research, individual interviews were conducted with managers of the hospital and owners and staff of the vendor organisations using semi- and unstructured questions to ascertain the extent of, and processes used in, outsourcing specific functional areas, and areas that were not outsourced. RESULTS: Pathology, radiology, dental technician services and lawn mowing were outsourced while food services was retained internally. The outsourcing of radiology was due to the hospital being unable (or unwilling) to finance new equipment and the problematical relationship between the existing radiologists, and hospital management and staff. Outsourcing resulted in increased staff morale, upgraded capital equipment and improved services. The outsourcing of pathology and dental technical services aimed to increase labour flexibility, thereby decreasing costs. Additional drivers in pathology were the changing nature of the funding arrangements rendering it profitable for the private sector to move into the provision of pathology and the increasing power of the medical scientists' union. The outsourcing of lawn mowing was simply to reduce costs. Food services was not outsourced because there was a lack of evidence that costs could be reduced. In addition, the existing relationships with food services staff were regarded as important because they had previously made immense changes to work practices, reduced staff numbers and decreased costs. CONCLUSION: Transaction costs are important when analysing how managers make the outsourcing decision, but the evidence from this case is that not all transaction costs are included in the decision, and that such costs are more complex than can be included in the type of analysis often undertaken by decision-makers. Taking into account Williamson's variables, the research shows that the outsourcing of services did not comply solely with the levels of transaction frequency or the requirement of asset specificity. In addition, opportunistic behaviour was evident on the part of all parties and was used in some cases as a reason for outsourcing, and in others to sway the decision to the manager's predisposed choice. A variety of arrangements were used to reduce environmental uncertainty, such as the transfer of staff to the contractor and the use of long-term contracts. Indeed the case shows that relationships between the hospital, its staff and the vendor are an important consideration that may not always be factored into an analysis that relies solely on transaction costs.

Journal Article↗

PubMed2001

Acute inflammation and recovery in rats after intratracheal instillation of a 1-->3-beta-glucan (zymosan A).

Although endotoxin is a known potent stimulant of inflammatory responses, the magnitude of pulmonary response following exposure to various organic dusts does not always correlate with endotoxin content of the dusts alone. Other components, such as 1-->3-beta-glucans, derived from the inner cell wall of yeasts and fungi, have been implicated in organic dust toxic syndrome. However, animal studies report conflicting results concerning the inflammatory potency of 1-->3-beta-glucan. In this experiment, the pulmonary reaction of rats to 1-->3-beta-glucan (zymosan A) exposure was assessed. Male Sprague-Dawley rats were exposed via intratracheal instillation (IT) to zymosan A (dose range 0-5 mg/kg body weight). Rats were sacrificed 1-7 d postexposure and the following pulmonary responses were monitored: (1) breathing frequency, (2) differential cell counts of hronchoalveolar lavage (BAL) cells, (3) chemiluminescence (CL) as a measure of alveolar macrophage activation, (4) nitric oxide production by alveolar macrophages, (5) albumin levels, and (6) lactate dehydrogenase (LDH) activity in the first acellular lavage fluid. Upon challenge with zymosan A, rats exhibited a dose-dependent pulmonary response at 1 d post IT that was significantly higher than the control level at a dose of 1-2.5 mg/kg body weight for each of these pulmonary parameters. Post-IT enhancement of breathing frequencies and polymorphonuclear leukocytes (PMN) obtained by BAL both correlated very well with zymosan A concentration (r = .95 and .99, respectively). Elevation of albumin levels and LDH activity of the acellular BAL fluid also correlated (r = .80) with the dose of zymosan. The recovery from a single intratracheal administration of zymosan A (2.5 mg/kg body weight) was monitored over 7 d. PMN and CL showed significant recovery from d 1 level by 3 d postexposure. Breathing frequencies and nitric oxide production showed significant recovery from d 1 level by 4 d postexposure. A good correlation (r2= .8) between recovery of PMN in BAL, CL, or nitric oxide production and the days postexposure was observed.

Animals↗

PubMed2001

Agonist-dependent immobilization of chimeric bombesin/GRP receptors: dependence on c-Src activity and dissociation from internalization.

G-protein-coupled receptors (GPCRs) are membrane proteins that exhibit a decreased mobile fraction compared to a freely mobile plasma membrane protein. Recently, interest has focused on proteins other than heterotrimeric G-proteins that interact with GPCRs as scaffolding structures that affect receptor signal transduction. In order to investigate the physical state of receptors before and after agonist, we used fluorescence recovery after photobleaching of the bombesin/gastrin-releasing peptide (GRP) receptor fused to the intrinsically fluorescent green fluorescent protein (GFP-GRP receptor) expressed in KNRK cells to measure both the fraction of mobile receptors and their diffusion rate before and after agonist stimulation. In live cells at 37 degrees C, addition of GRP (100 nM) caused a rapid decrease in GFP-GRP receptor mobile fraction from 0.8 +/- 0.1 to 0.49 +/- 0.05, which was independent of endocytosis. Concurrently, the remaining mobile GFP-GRPreceptors showed an increase in the diffusion rate with the half-time of fluorescent recovery, tau(1/2) = 46 +/- 7 s for untreated cells, decreasing to tau(1/2) = 30 +/- 6 s for cells treated with GRP. Prior treatment with the Src-specific inhibitor PP-2 (10 microM) blocked GFP-GRP receptor immobilization while treatment with the inactive analog PP-3 (10 microM) did not affect receptor immobilization. These data suggest that agonist-bound GPCR have increased plasma membrane diffusion rates but an increased affinity for immobilization into a multiprotein complex that is mediated by Src activity.

Cell Membrane↗

PubMed2001

Rapid protein kinase D translocation in response to G protein-coupled receptor activation. Dependence on protein kinase C.

Protein kinase D (PKD)/protein kinase C (PKC) mu is a serine/threonine protein kinase that can be activated by physiological stimuli like growth factors, antigen-receptor engagement and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires PKC activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular translocation of a green fluorescent protein-tagged PKD was analyzed by real-time visualization in fibroblasts and epithelial cells stimulated with bombesin, a GPCR agonist. We found that bombesin induced a rapidly reversible plasma membrane translocation of green fluorescent protein-tagged PKD, an event that can be divided into two distinct mechanistic steps. The first step, which is exclusively mediated by the cysteine-rich domain in the N terminus of PKD, involved its translocation from the cytosol to the plasma membrane. The second step, i.e. the rapid reverse translocation of PKD from the plasma membrane to the cytosol, required its catalytic domain and surprisingly PKC activity. These findings provide evidence for a novel mechanism by which PKC coordinates the translocation and activation of PKD in response to bombesin-induced GPCR activation.

3T3 Cells↗

PubMed2001

Molecular mechanism of tumor necrosis factor-alpha production in 1-->3-beta-glucan (zymosan)-activated macrophages.

The molecular details of 1-->3-beta-glucans, a fungal cell wall component, induced inflammatory responses are not well understood. In the present study, we conducted a systematic analysis of the molecular events leading to tumor necrosis factor (TNF)-alpha production after glucan stimulation of macrophages. We demonstrated that activation of nuclear factor kappaB (NF-kappaB) is essential in zymosan A (a source of 1-->3-beta-glucans)-induced TNF-alpha production in macrophages (RAW264.7 cells). Zymosan A-induced TNF-alpha protein production was associated with an increase in the TNF-alpha gene promoter activity. Activation of the TNF-alpha gene promoter was dependent on activation of NF-kappaB. Time course studies indicated that DNA binding activity of NF-kappaB preceded TNF-alpha promoter activity. Inhibition of NF-kappaB activation led to a dramatic reduction in both TNF-alpha promoter activity and TNF-alpha protein production in the response to zymosan A. Mutation of a major NF-kappaB binding site (kappa3) in the gene promoter resulted in a significant decrease in the induction of the gene promoter by zymosan A, while mutation of Egr or CRE sites failed to inhibit the response to zymosan. Together, these results strongly suggest that NF-kappaB is involved in signal transduction of 1-->3-beta-glucans-induced TNF-alpha expression.

Animals↗

PubMed2001

Agonists of proteinase-activated receptor 1 induce plasma extravasation by a neurogenic mechanism.

Thrombin, generated in the circulation during injury, cleaves proteinase-activated receptor 1 (PAR1) to stimulate plasma extravasation and granulocyte infiltration. However, the mechanism of thrombin-induced inflammation in intact tissues is unknown. We hypothesized that thrombin cleaves PAR1 on sensory nerves to release substance P (SP), which interacts with the neurokinin 1 receptor (NK1R) on endothelial cells to cause plasma extravasation. PAR1 was detected in small diameter neurons known to contain SP in rat dorsal root ganglia by immunohistochemistry and in situ hybridization. Thrombin and the PAR1 agonist TFLLR-NH(2) (TF-NH(2)) increased [Ca(2+)](i) >50% of cultured neurons (EC(50)s 24 mu ml(-1) and 1.9 microM, respectively), assessed using Fura-2 AM. The PAR1 agonist completely desensitized responses to thrombin, indicating that thrombin stimulates neurons through PAR1. Injection of TF-NH(2) into the rat paw stimulated a marked and sustained oedema. An NK1R antagonist and ablation of sensory nerves with capsaicin inhibited oedema by 44% at 1 h and completely by 5 h. In wild-type but not PAR1(-/-) mice, TF-NH(2) stimulated Evans blue extravasation in the bladder, oesophagus, stomach, intestine and pancreas by 2 - 8 fold. Extravasation in the bladder, oesophagus and stomach was abolished by an NK1R antagonist. Thus, thrombin cleaves PAR1 on primary spinal afferent neurons to release SP, which activates the NK1R on endothelial cells to stimulate gap formation, extravasation of plasma proteins, and oedema. In intact tissues, neurogenic mechanisms are predominantly responsible for PAR1-induced oedema.

Animals↗

PubMed2000

Activation of mitogen-activated protein kinase p38 and extracellular signal-regulated kinase is involved in glass fiber-induced tumor necrosis factor-alpha production in macrophages.

In a previous study, we demonstrated that the length of glass fibers was a critical determinant of fiber potency in induction of tumor necrosis factor (TNF)-alpha and that activation of NF-kappaB was an important factor in this response. In the present study, we analyzed the role of mitogen-activated protein (MAP) kinases in the induction of TNF-alpha by glass fibers. Glass fibers induced phosphorylation of MAP kinases, p38, and ERK in primary rat alveolar macrophages, and this phosphorylation was associated with TNF-alpha gene expression. Long fibers were more potent than short fibers in activation of MAP kinases. Results from mechanistic analysis support that MAP kinases activate transcription factor c-Jun. The activated c-Jun acts on the TNF-alpha gene promoter through two binding sites, the cyclic AMP response element and the activator protein 1-binding site. These results suggest that in addition to the NF-kappaB pathway for TNF-alpha production, glass fibers are able to activate c-Jun through MAP kinase pathways that lead to induction of TNF-alpha expression.

Animals↗

PubMed2000

Observation of a partially opened triple-helix conformation in 1-->3-beta-glucan by fluorescence resonance energy transfer spectroscopy.

This study used fluorescence resonance energy transfer (FRET) spectroscopy as an indirect method to investigate the effect of NaOH treatment on the conformation of a triple-helix (1-->3)-beta-D-glucan and then evaluated the effect of conformation on biological activity. Previous studies have suggested that treatment of the triple-helix glucans with NaOH produces single-helix conformers. FRET spectra of the triple-helix glucan, laminarin, doubly labeled with 1-aminopyrene as donor probe and fluorescein-5-isothiocyanate as acceptor probe attached at the reducing end, showed that a partially opened triple-helix conformer was formed on treatment with NaOH. Increasing degrees of strand opening was associated with increasing concentrations of NaOH. Based on these observations we propose that a partially opened triple-helix rather than a single helix, is formed by treating the triple-helix glucans with NaOH. After neutralizing the NaOH, changes in FRET indicated that the partially opened conformer gradually reverts to the triple-helix over 8 days. Laminarian was stabilized at different degrees of partial opening and its biological activity examined using the Limulus amebocyte lysate assay and nitric oxide production by alveolar macrophage. Both Limulus amebocyte lysate activity and nitric oxide production were related to the degree of opening of the triple-helix. Partially open conformers were more biologically active than the intact triple-helix.

Aniline Compounds↗

PubMed2000

Mechanical activation of dorsal root ganglion cells in vitro: comparison with capsaicin and modulation by kappa-opioids.

The aim of this study was to characterize plasma membrane pathways involved in the intracellular calcium ([Ca(2+)](i)) response of small DRG neurons to mechanical stimulation and the modulation of these pathways by kappa-opioids. [Ca(2+)](i) responses were measured by fluorescence video microscopy of Fura-2 labeled lumbosacral DRG neurons obtained from adult rats in short-term primary culture. Transient focal mechanical stimulation of the soma, or brief superfusion with 300 nM capsaicin, resulted to [Ca(2+)](i) increases which were abolished in Ca(2+)-free solution, but unaffected by lanthanum (25 microM) or tetrodotoxin (10(-6) M). 156 out of 465 neurons tested (34%) showed mechanosensitivity while 55 out of 118 neurons (47%) were capsaicin-sensitive. Ninty percent of capsaicin-sensitive neurons were mechanosensitive. Gadolinium (Gd(3+); 250 microM) and amiloride (100 microM) abolished the [Ca(2+)](i) transient in response to mechanical stimulation, but had no effect on capsaicin-induced [Ca(2+)](i) transients. The kappa-opioid agonists U50,488 and fedotozine showed a dose-dependent inhibition of mechanically stimulated [Ca(2+)](i) transients but had little effect on capsaicin-induced [Ca(2+)](i) transients. The inhibitory effect of U50,488 was abolished by the kappa-opioid antagonist nor-Binaltorphimine dihydrochloride (nor-BNI; 100 nM), and by high concentrations of naloxone (30-100 nM), but not by low concentrations of naloxone (3 nM). We conclude that mechanically induced [Ca(2+)](i) transients in small diameter DRG somas are mediated by influx of Ca(2+) through a Gd(3+)- and amiloride-sensitive plasma membrane pathway that is co-expressed with capsaicin-sensitive channels. Mechanical-, but not capsaicin-mediated, Ca(2+) transients are sensitive to kappa-opioid agonists.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed2000

Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism.

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 and, by unknown mechanisms, induce widespread inflammation. We found that a large proportion of primary spinal afferent neurons, which express proteinase-activated receptor 2, also contain the proinflammatory neuropeptides calcitonin gene-related peptide and substance P. Trypsin and tryptase directly signal to neurons to stimulate release of these neuropeptides, which mediate inflammatory edema induced by agonists of proteinase-activated receptor 2. This new mechanism of protease-induced neurogenic inflammation may contribute to the proinflammatory effects of mast cells in human disease. Thus, tryptase inhibitors and antagonists of proteinase-activated receptor 2 may be useful anti-inflammatory agents.

Animals↗

PubMed2000

Dorsal ulnar cutaneous nerve conduction studies in an asymptomatic population.

OBJECTIVES: To establish the average distal sensory latency and amplitude of the dorsal ulnar cutaneous nerve under controlled temperature and settings. DESIGN: Dorsal ulnar cutaneous nerve conduction studies were performed with a bar electrode between the fourth and fifth metacarpals. The nerve was stimulated at 8 and 10cm from the active electrode. The temperature of the limbs under study was kept at 32 degrees C to 35 degrees C. PARTICIPANTS: Fifty-four arms of 27 subjects were studied. RESULTS: Results showed that distal sensory latencies were normally distributed. The average distal sensory latencies at 8cm and 10cm were 1.84 +/- .20msec and 2.09 +/- .21msec, respectively, with average amplitudes of 26.5 +/- 9.7microV and 23.5 +/- 8.8microV. Comparison with a previously reported study in which the limb temperature was not controlled showed a statistically significant difference. CONCLUSION: It is important to use standardized technique and to measure and maintain optimal temperature of the arm under study to prevent erroneous results and misdiagnosis.

Adult↗

PubMed1999

Chemical signaling from colonic smooth muscle cells to DRG neurons in culture.

Transduction mechanisms between target cells within the intestinal wall and peripheral terminals of extrinsic primary afferent neurons are poorly understood. The purpose of this study was to characterize the interactions between smooth muscle cells from the rat distal colon and lumbar dorsal root ganglion (DRG) neurons in coculture. DRG neurons visually appeared to make contact with several myocytes. We show that brief mechanical stimulation of these myocytes resulted in intracellular Ca2+ concentration ([Ca2+]i) transients that propagated into 57% of the contacting neurites. Direct mechanical stimulation of DRG neurites cultured without smooth muscle had no effect. We also show that colonic smooth muscle cells express multiple connexin mRNAs and that these connexins formed functional gap junctions, as evidenced by the intercellular transfer of Lucifer yellow. Furthermore, thapsigargin pretreatment and neuronal heparin injection abolished the increase in neurite [Ca2+]i, indicating that the neuronal Ca2+ signal was triggered by inositol 1,4, 5-trisphosphate-mediated Ca2+ release from intracellular stores. Our results provide evidence for intercellular chemical communication between DRG neurites and intestinal smooth muscle cells that mediates the exchange of second messenger molecules between different cell types.

Animals↗

PubMed1999

Calcium waves in colonic myocytes produced by mechanical and receptor-mediated stimulation.

The mechanisms underlying intracellular Ca2+ waves induced by either mechanical or receptor-mediated stimulation of myocytes isolated from the longitudinal muscle layer of the rabbit distal colon were compared using fura 2 and fluorescence videomicroscopy. Light focal mechanical deformation of the plasma membrane or focal application of substance P resulted in localized intracellular Ca2+ concentration ([Ca2+]i) transients that propagated throughout the cell. In both cases, the Ca2+ response consisted of a transient peak response followed by a delayed-phase response. Substance P-mediated [Ca2+]i responses involved generation of inositol 1,4, 5-trisphosphate and release of Ca2+ from thapsigargin-sensitive stores, whereas mechanically induced responses were partially (29%) dependent on La3+-sensitive influx of extracellular Ca2+ and partially on release of intracellular Ca2+ from thapsigargin-insensitive stores gated by ryanodine receptors. The delayed-phase response in both cases was dependent on extracellular Ca2+. However, although the response to substance P was sensitive to La3+, that after mechanical stimulation was not. In the later case, the underlying mechanism may involve capacitative Ca2+ entry channels that are activated after mechanical stimulation but not by substance P.

Animals↗

PubMed1998

Apolipoprotein B-100: conservation of lipid-associating amphipathic secondary structural motifs in nine species of vertebrates.

Development of a computer program called LOCATE allowed us to show that human apolipoprotein B-100 is composed of five domains, NH2-alpha1-beta1-alpha2-beta2-alpha3-COOH, enriched, alternately, in amphipathic alpha helixes and amphipathic beta strands. Using updated versions of this program, here we compare the complete sequence of human apolipoprotein B-100 with partial sequences from eight additional species of vertebrates (chicken, frog, hamster, monkey, mouse, pig, rat, and rabbit). The lipid-associating amphipathic alpha helixes cluster in domains alpha2 (between residues 2075 +/- 25 and 2575 +/- 25) and alpha3 (between residues 4100 +/- 100 and 4550 +/- 50) in all species for which those regions have been sequenced but with little conservation of individual helixes. Lipid-associating amphipathic beta strands cluster in domains beta1 (approximately residues 827-2000) and beta2 (approximately residue 2571 to residue 4000 +/- 50) in all species for which these regions have been sequenced, with conservation of several individual amphipathic beta strands. Hydrophobic segments are present in apolipoprotein B-100 sequences of all nine species but the frequency of occurrence is no greater than generally found in beta sheet-containing proteins. We conclude that four alternating lipid-associating domains, -beta1-alpha2-beta2-alpha3-COOH, are common supramolecular features of apolipoprotein B-100 in nine vertebrate species.

Amino Acid Sequence↗

PubMed1997

Mechanotransduction in colonic smooth muscle cells.

We evaluated mechanisms which mediate alterations in intracellular biochemical events in response to transient mechanical stimulation of colonic smooth muscle cells. Cultured myocytes from the circular muscle layer of the rabbit distal colon responded to brief focal mechanical deformation of the plasma membrane with a transient increase in intracellular calcium concentration ([Ca2+]i) with peak of 422.7 +/- 43.8 nm above an average resting [Ca2+]i of 104.8 +/- 10.9 nM (n = 57) followed by both rapid and prolonged recovery phases. The peak [Ca2+]i increase was reduced by 50% in the absence of extracellular Ca2+, while the prolonged [Ca2+]i recovery was either abolished or reduced to less than or = 15% of control values. In contrast, no significant effect of gadolinium chloride (100 microM) or lanthanum chloride (25 microM) on either peak transient or prolonged [Ca2+]i recovery was observed. Pretreatment of cells with thapsigargin (1 microM) resulted in a 25% reduction of the mechanically induced peak [Ca2+]i response, while the phospholipase C inhibitor U-73122 had no effect on the [Ca2+]i transient peak. [Ca2+]i transients were abolished when cells previously treated with thapsigargin were mechanically stimulated in Ca2+-free solution, or when Ca2+ stores were depleted by thapsigargin in Ca2+-free solution. Pretreatment with the microfilament disrupting drug cytochalasin D (10 microM) or microinjection of myocytes with an intracellular saline resulted in complete inhibition of the transient. The effect of cytochalasin D was reversible and did not prevent the [Ca2+]i increases in response to thapsigargin. These results suggest a communication, which may be mediated by direct mechanical link via actin filaments, between the plasma membrane and an internal Ca2+ store.

Actin Cytoskeleton↗

PubMed1997

Intercellular communication between dorsal root ganglion cells and colonic smooth muscle cells in vitro.

The mechanism(s) by which intestinal smooth muscle tension is signaled to extrinsic primary afferent neurons is poorly understood. In order to characterize myocyte-neuron communication, we developed a coculture system using rat dorsal root ganglion (DRG) neurons and myocytes obtained from the circular muscle layer of the rat distal colon. Both cell types maintained their phenotype in culture, as demonstrated by positive immunocytochemical staining for neuron-specific enolase and smooth muscle actin. Myocytes showed mechanosensitivity in the form of increases in [Ca2+]i in response to light mechanical touch of the plasma membrane. This increase in [Ca2+]i was independent of extracellular Ca2+ and passed as a propagated wave from muscle cells into adjacent DRG neurites. The inhibitory effect of octanol on this intercellular propagation suggests propagation of [Ca2+]i gradients via heterologous gap junctions. This preparation may serve a useful model system for the study of the interaction of visceral afferents and their target cells.

1-Octanol↗

PubMed1996

Propagation of calcium waves between colonic smooth muscle cells in culture.

Intercellular propagation of a diffusible substance through direct cytoplasmic communication between multiple cells could represent an important mechanism for mutual multiple cell signaling between cells in a tissue. The current study was aimed at characterizing the mechanism(s) underlying the intercellular propagation of calcium concentration ([Ca2+]i) transients between colonic smooth muscle cells. Changes in [Ca2+]i in smooth muscle cells from the rabbit distal colon in primary cultures were monitored using videomicroscopy with the fluorescent dye Fura-2. Myocytes responded to light mechanical deformation of the plasma membrane with a localized increase in [Ca2+]i which spread in a wave-like fashion through up to 5 adjacent cells, with little change in wave amplitude. Dye coupling between cells was demonstrated by Lucifer Yellow, and intercellular wave propagation was abolished by octanol, suggesting propagation of Ca2+ waves via gap junctions. Wave propagation was not dependent on extracellular [Ca2+]i suggesting regenerative release of Ca2+ from intracellular stores. Propagation of Ca2+ waves through silent cells suggested a diffusible messenger other than Ca2+. Wave propagation and kinetics were unaffected by ryanodine (50 microM) or caffeine (10 mM), but abolished by depletion of thapsigargin-sensitive Ca2+ stores and by the phospholipase C inhibitor U-73122 (10 microM), implicating inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-sensitive stores as the major Ca2+ source for propagated Ca2+ transients. These results indicate that, in a connected complex of colonic smooth muscle cells in culture, multiple cells can monitor the mechanical status of a single cell through diffusion of Ins(1,4,5)P3, Ca2+, or another intercellular messenger.

Animals↗