The effects of caffeine on wound healing

The purine alkaloid caffeine is a major component of many beverages such as coffee and tea. Caffeine and its metabolites theobromine and xanthine have been shown to have antioxidant properties. Caffeine can also act as adenosine‐receptor antagonist. Although it has been shown that adenosine and antioxidants promote wound healing, the effect of caffeine on wound healing is currently unknown. To investigate the effects of caffeine on processes involved in epithelialisation, we used primary human keratinocytes, HaCaT cell line and ex vivo model of human skin. First, we tested the effects of caffeine on cell proliferation, differentiation, adhesion and migration, processes essential for normal wound epithelialisation and closure. We used 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) proliferation assay to test the effects of seven different caffeine doses ranging from 0·1 to 5 mM. We found that caffeine restricted cell proliferation of keratinocytes in a dose‐dependent manner. Furthermore, scratch wound assays performed on keratinocyte monolayers indicated dose‐dependent delays in cell migration. Interestingly, adhesion and differentiation remained unaffected in monolayer cultures treated with various doses of caffeine. Using a human ex vivo wound healing model, we tested topical application of caffeine and found that it impedes epithelialisation, confirming in vitro data. We conclude that caffeine, which is known to have antioxidant properties, impedes keratinocyte proliferation and migration, suggesting that it may have an inhibitory effect on wound healing and epithelialisation. Therefore, our findings are more in support of a role for caffeine as adenosine‐receptor antagonist that would negate the effect of adenosine in promoting wound healing.     

Acceleration of cutaneous wound healing by brassinosteroids

Brassinosteroids are plant growth hormones involved in cell growth, division, and differentiation. Their effects in animals are largely unknown, although recent studies showed that the anabolic properties of brassinosteroids are possibly mediated through the phosphoinositide 3‐kinase/protein kinase B signaling pathway. Here, we examined biological activity of homobrassinolide (HB) and its synthetic analogues in in vitro proliferation and migration assays in murine fibroblast and primary keratinocyte cell culture. HB stimulated fibroblast proliferation and migration and weakly induced keratinocyte proliferation in vitro. The effects of topical HB administration on progression of wound closure were further tested in the mouse model of cutaneous wound healing. C57BL/6J mice were given a full‐thickness dermal wound, and the rate of wound closure was assessed daily for 10 days, with adenosine receptor agonist CGS‐21680 as a positive control. Topical application of brassinosteroid significantly reduced wound size and accelerated wound healing in treated animals. mRNA levels of transforming growth factor beta and intercellular adhesion molecule 1 were significantly lower, while tumor necrosis factor alpha was nearly suppressed in the wounds from treated mice. Our data suggest that topical application of brassinosteroids accelerates wound healing by positively modulating inflammatory and reepithelialization phases of the wound repair process, in part by enhancing Akt signaling in the skin at the edges of the wound and enhancing migration of fibroblasts in the wounded area. Targeting this signaling pathway with brassinosteroids may represent a promising approach to the therapy of delayed wound healing.     

血管紧张素Ⅱ及其受体表达在创面愈合过程中的变化及作用

目的 观察创面愈合过程中血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)及其受体表达的动态变化,以及这种变化与创面愈合过程中细胞增殖和凋亡活动之间的关系,探讨AngⅡ在创面愈合过程中的可能作用.方法 建立小鼠背部全层皮肤缺损创面模型,于创面形成后第0、1、3、5、7、9、11、13、15天切取创面组织标本,用ELISA法检测创面局部组织AngⅡ产生的变化;采用BrdU及TUNEL染色检测创面细胞增殖和凋亡的变化;用免疫组织化学染色和RT-PCR检测创面局部组织AngⅡ受体AT1和AT2表达的组织细胞定位和mRNA水平的变化.结果 小鼠全层皮肤缺损创面局部组织AngⅡ、BrdU标记指数均在伤后逐渐增加,并于第7天达到峰值后即逐渐下降.TUNEL染色阳性细胞数在伤后即开始缓慢增加,并于创面完成上皮化后增加趋势更为明显.正常小鼠皮肤AT1和AT2受体在整个表皮层均有阳性表达,但在真皮层,AT1和AT2受体仅在微血管内皮细胞有阳性表达.AT1受体在角质形成细胞、成纤维细胞均有表达,阳性染色信号在伤后逐渐增加,在第7天最强,以后逐渐下降.AT2受体阳性染色信号也在伤后逐渐增加,7 d以后则逐渐下降.但当创面上皮化完成后,AT2受体阳性染色信号再次增加.RT-PCR结果 显示:AT1和AT2受体mRNA均有表达,AT1、AT2受体mRNA表达在伤后第7天均达到峰值,此后则逐渐下降,且AT2受体mRNA表达在创面上皮化完成后表达再次增加.结论 在创面愈合过程中,AngⅡ可能通过其产生及受体表达的变化调控创面的愈合及后期的塑形改建.AT1受体可能与细胞增殖活动密切相关;AT2受体可能与细胞凋亡及愈合过程中组织重建有关.

Abstract：

Objective This study was undertaken to observe the change in the local level of angiotensin Ⅱ (Ang Ⅱ) and the expression of its corresponding receptors AT1 and AT2 during wound healing, and explore the possible role of Ang Ⅱ in wound healing . Methods A model of full-thickness cutaneous wound was developed on the back of C57/BL6 mice. Specimens were taken from the wound of each mouse on the day 0, 1, 3, 5, 7, 9, 11, 13 and 15 after wounding. The change in the generation of Ang Ⅱ in wounded tissue during the healing process was detected with ELISA. The proliferation and the apoptosis of cells were detected by bromodeoxyuridine (Brdu) and terminal deoxyuncleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) method in wounded skin during the healing process, respectively. The cellular localization and the mRNA level change of Ang Ⅱ receptors in wounded tissue during healing were detected with immunostaining and RT-PCR. Results Ang Ⅱ produced in wounded skin was increased in the first 7 days to reach the peak, and then gradually decreased during wound healing. BrdU labeling index was increased gradually in the first 7 days to reach the peak, and then gradually decreased during wound healing. The number of TUNEL-positive cells was increased slowly in the first 7 days after wounding. The increase in the number of TUNEL-positive cells was more markedly after epithelization of the wound. In normal mice, AT1 and AT2 receptor were found positively expressed in the whole epidermal layer, while positive expression was only found in the endothelial cells of the capillary vessels within the dermal layer, and positive expression was also found in appendages of the skin, i.e. hair follicle, sweat gland and sebaceous gland respectively. Positive staining signal of both AT1 and AT2 receptors were increased in the first 7 days to reach the peak, then gradually decreased. Expression of AT2R was increased again following the epithelization of wound. The result of RT-PCR showed that the expression of both AT1 and AT2 receptors was detectable, and AT1 receptor was increased in the first 7 days to the peak, and then gradually decreased during wound healing, while AT2 receptor expression reached its peak value on day 7, then gradually decreased, and increased again following the epithelization of wound. Conclusions These results indicate that Ang Ⅱ participate in wound repair and related to remolding in the late stage of wound healing through the change in production of angiotensin Ⅱ and expression of AT1 and AT2 receptors. AT1 receptor might be closely associated with cell proliferation,while AT2 receptor might play a role in cell apoptosis and remolding during wound healing.     

Inhibition of bone morphogenetic protein signalling promotes wound healing in a human ex vivo model

Background

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) play roles in embryonic development and postnatal remodelling of the skin. Many indications suggest that BMP signalling regulates keratinocyte proliferation and differentiation. Chronic wounds have been shown to exhibit high levels of BMP ligands; however, the effect of BMP pathway modulation on human skin healing remains undefined.

Methods

A human ex vivo skin wound healing model was used to analyse the expression of BMP signalling pathway components during healing and to investigate the effects of BMPs and the BMP antagonist Noggin on skin repair. Additionally, the effects of BMP signalling on keratinocyte proliferation, apoptosis and migration were tested using in vitro flow cytometry and ‘scratch’ migration assays, respectively.

Results

BMP receptor-1B (BMPR-1B) and downstream signalling protein phosphorylated-Smad-1/5/8 were highly expressed in healing epidermis. Treatment of human skin with exogenous BMPs impaired wound closure by reducing keratinocyte proliferation and increasing apoptosis. The BMP antagonist Noggin negated the inhibitory effects of BMP ligands, and when used alone, Noggin reduced keratinocyte apoptosis in the wound bed. In vitro, BMP ligands suppressed keratinocyte proliferation whilst Noggin stimulated proliferation. Keratinocyte migration was slowed following BMP treatment; in contrast, migration was significantly accelerated due to inhibition of BMP activity by either Noggin or BMPR-1B silencing.

Conclusions

BMP signalling is inherently involved in wound healing. BMPs slow skin repair by suppressing keratinocyte proliferation and migration. Thus, modulation of BMP signalling using BMP inhibitors such as Noggin may serve as a new approach to promote cutaneous wound repair.Level of evidence: Not ratable.

     

Acute and chronic wound fluids influence keratinocyte function differently

Wound healing requires a proper functioning of keratinocytes that migrate, proliferate and lead to a competent wound closure. Impaired wound healing might be due to a disturbed keratinocyte function caused by the wound environment. Basically, chronic wound fluid (CWF) differs from acute wound fluid (AWF). The aim of this study was to analyse the effects of AWF and CWF on keratinocyte function. We therefore investigated keratinocyte migration and proliferation under the influence of AWF and CWF using MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] test and scratch assay. We further measured the gene expression by qRT‐PCR regarding growth factors and matrixmetalloproteinases (MMPs) involved in regeneration processes. AWF had a positive impact on keratinocyte proliferation over time, whereas CWF had an anti‐proliferative effect. Keratinocyte migration was significantly impaired by CWF in contrast to an undisturbed wound closure under the influence of AWF. MMP‐9 expression was strongly upregulated by CWF compared with AWF. Keratinocyte function was significantly impaired by CWF. An excessive induction of MMP‐9 by CWF might lead to a permanent degradation of extracellular matrix and thereby prevent wounds from healing.     

071TGF‐ß‐Mediated Paracrine Regulation of Keratinocyte Wound Closure

The importance of stromal‐epithelial interactions in wound healing is well established. These interactions likely involve autocrine and paracrine action of multiple growth factors, including members of the TGF‐ß family. TGF‐ß1, ß2 and ß3 isoforms signal by sequentially binding to the TGF‐ß type II and type I receptors, respectively. We address the role of TGF‐ß signaling in dermal fibroblasts using a conditional fibroblastic TGF‐ß type II receptor knockout mouse model (termed FßKO). We found that the loss of TGF‐ß signaling in the dermal fibroblasts results in accelerated excision‐wound closure compared with similar wounds in wild type mice. The mechanism of the altered rate of re‐epitheliaization in the FßKO mice was examined with regard to keratiocyte motility and proliferation. The migration of keratinocytes through collagen I coated 8 μm pore filters in the presence or absence of fibroblast‐conditioned media was tested. These experiments showed increased keratinocyte migration when incubated with FßKO dermal fibroblast conditioned media compared to media conditioned in wild type fibroblasts. Immuno‐histochemical staining of paraffin embedded intact skin indicated both wild type and FßKO mice had similar low levels of keratinocyte proliferation, based on Ki67 staining. In healing wounds, only the distal wound edges of wild type mice were proliferative. In contrast, the FßKO mice exhibited elevated proliferation across the length of the wound, including the leading edge of epithelial closure. Together our results suggest TGF‐ß signaling by the dermal fibroblasts suppresses re‐epithelialization of excision wounds by regulating keratinocyte motility and proliferation through paracrine mechanisms.
Funding: DOD BC99184 and NIH CA85492.     

Organic light emitting diode improves diabetic cutaneous wound healing in rats

A major complication for diabetic patients is chronic wounds due to impaired wound healing. It is well documented that visible red wavelengths can accelerate wound healing in diabetic animal models and patients. In vitro and in vivo diabetic models were used to investigate the effects of organic light emitting diode (OLED) irradiation on cellular function and cutaneous wound healing. Human dermal fibroblasts were cultured in hyperglycemic medium (glucose concentration 180 mM) and irradiated with an OLED (623 nm wavelength peak, range from 560 to 770 nm, power density 7 or 10 mW/cm² at 0.2, 1, or 5 J/cm²). The OLED significantly increased total adenosine triphosphate concentration, metabolic activity, and cell proliferation compared with untreated controls in most parameters tested. For the in vivo experiment, OLED and laser (635 ± 5 nm wavelength) treatments (10 mW/cm², 5 J/cm² daily for a total of seven consecutive days) for cutaneous wound healing were compared using a genetic, diabetic rat model. Both treatments had significantly higher percentage of wound closure on day 6 postinjury and higher total histological scores on day 13 postinjury compared with control. No statistical difference was found between the two treatments. OLED irradiation significantly increased fibroblast growth factor‐2 expression at 36‐hour postinjury and enhanced macrophage activation during initial stages of wound healing. In conclusion, the OLED and laser had comparative effects on enhancing diabetic wound healing.     

Infection of human endothelium in vitro by cytomegalovirus causes enhanced expression of purinergic receptors: a potential virus escape mechanism?

BACKGROUND: Human cytomegalovirus (CMV) uses different strategies to escape from human host defense reactions. Previously we have observed that infection of endothelial cells with CMV in vitro leads to enhanced activity of endothelial ectonucleotidases. These ectoenzymes are responsible for hydrolysis of extracellular adenine nucleotides, resulting in the formation of adenosine. Infection with CMV in vivo therefore may result in local increase of adenosine production, providing an anti-inflammatory and antiaggregatory microenvironment, which may facilitate entry of the virus into the target cell. METHODS: The present study focuses on the expression of P2 type purinergic receptors on endothelial cells after infection with CMV. Human endothelial cells were infected with CMV and compared with either uninfected cells or endothelial cells infected with other herpesviruses (herpes simplex virus [HSV] 1 or 2) for the expression of P2 receptors such as P2Y1, P2Y2, or P2X7. For comparison, cells stimulated with nonspecific agents were also studied. RESULTS: A strong upregulation of the P2 receptors tested was shown, exclusively in CMV-infected cells. Stimulation with either HSV-1 or HSV2, nonspecific stimulants, or various cytokines did not affect the expression of these P2 receptors significantly. CONCLUSION: Infection of endothelium with CMV causes significant upregulation of the P2 receptors studied. As these receptors may potentially be able to concentrate nucleotides along the ectonucleotidases of the endothelial cell membrane, rapid local hydrolysis of adenosine triphosphate and adenosine diphosphate may be facilitated by enhanced P2 receptor expression. Such a CMV induced mechanism might enable the virus to escape from an important host defense response, such as local microthrombus formation.     

Purinergic modulation of mesangial extracellular matrix production: role in diabetic and other glomerular diseases

BACKGROUND: Extracellular adenosine triphosphate (ATP) (eATP) mediates several biologic activities via purinergic P2 receptors (P2Rs). This study aimed at (1) evaluating the role of the purinergic system in modulating mesangial extracellular matrix (ECM) and transforming growth factor-beta (TGF-beta) production and (2) its contribution to diabetes-induced mesangial ECM accumulation. METHODS: Rat mesangial cells were grown in normal glucose (5.5 mmol/L) or high glucose (30 mmol/L) containing media and probed with purinergic agonists and antagonists for the assessment of the expression pattern and function of P2Rs; release of ATP and activity of ectoATPases; and changes in ECM and TGF-beta expression. RESULTS: Cells cultured in normal glucose and high glucose expressed similar amounts of functional P2Rs of the P2X(2), P2X(3), P2X(4), P2X(5), P2X(7), P2Y(1), P2Y(2), P2Y(4), and P2Y(6) subtypes. Levels of eATP were higher in high glucose vs. normal glucose, with unchanged ectoATPase activity. The ATP-hydrolyzing enzymes hexokinase or apyrase reduced ECM and TGF-beta production from cells grown in high glucose, but not normal glucose. Under both normal glucose and high glucose conditions, ATP and the P2X(7) agonist benzoylbenzoylATP increased dose-dependently ECM and TGF-beta production, whereas the P2Y agonist uridine triphosphate (UTP) produced the opposite effect. The P2X(7) inhibitor oxidized ATP attenuated the ECM and TGF-beta up-regulation induced by ATP and, to a lesser extent, that caused by high glucose. A TGF-beta neutralizing antibody also prevented ATP-induced ECM up-regulation. CONCLUSION: These data indicate a role for eATP in regulating ECM production via TGF-beta and suggest that P2XRs and P2YRs differentially modulate this process. An increased ATP release induced by hyperglycemia might contribute to mesangial matrix expansion occurring in diabetes.     

Unlocking the Potential of Purinergic Signaling in Transplantation

Purinergic signaling has been recognized as playing an important role in inflammation, angiogenesis, malignancy, diabetes and neural transmission. Activation of signaling pathways downstream from purinergic receptors may also be implicated in transplantation and related vascular injury. Following transplantation, the proinflammatory “danger signal” adenosine triphosphate (ATP) is released from damaged cells and promotes proliferation and activation of a variety of immune cells. Targeting purinergic signaling pathways may promote immunosuppression and ameliorate inflammation. Under pathophysiological conditions, nucleotide‐scavenging ectonucleotidases CD39 and CD73 hydrolyze ATP, ultimately, to the anti‐inflammatory mediator adenosine. Adenosine suppresses proinflammatory cytokine production and is associated with improved graft survival and decreased severity of graft‐versus‐host disease. Furthermore, purinergic signaling is involved both directly and indirectly in the mechanism of action of several existing immunosuppressive drugs, such as calcineurin inhibitors and mammalian target of rapamycin inhibitors. Targeting of purinergic receptor pathways, particularly in the setting of combination therapies, could become a valuable immunosuppressive strategy in transplantation. This review focuses on the role of the purinergic signaling pathway in transplantation and immunosuppression and explores possible future applications in clinical practice.     

Chronic wounds – is cellular ‘reception’ at fault? Examining integrins and intracellular signalling

As with all physiologic processes, chronic wounds are associated with unique intracellular and cellular/extracellular matrix (ECM) receptor types and signalling messages. These cellular receptors mediate responses of the epidermis to provisional wound matrix and change in form and number in cases of impaired wound healing. Integrins are the major cell‐surface receptors for cell adhesion and migration and epidermal keratinocytes express several integrins that bind ECM ligands in provisional wound ECM. Integrin receptors and more particularly integrin clusters and focal adhesion points appear to influence epidermal and dermal cell matrix interactions, cell motility, cell phenotype and ultimate healing trajectory. In chronic wounds, a variety of changes in receptors have been identified: decreased integrin α5β1 receptors affect the integration of fibronectin and subsequent keratinocyte migration; integrin αvβ6 stimulate transforming growth factor (TGF)‐β and may increase the susceptibility to ulceration and fibrosis; however, TGF‐β signal receptors have been found to be dysfunctional in many chronic wounds; additionally receptor interactions result in increased senescent cells including fibroblasts, myofibroblasts and even keratinocytes – this produces a degradative ECM and wound bed and corrosive chronic wound fluid. The activation or inhibition of integrin receptors by various agents may provide an excellent means of influencing wound healing. This process offers an earlier intervention into the wound healing cascade promoting intrinsic healing and elaboration of growth factors and ECM proteins, which may be more cost effective than the traditional attempts at extrinsic addition of these agents.     

Mice lacking β6 integrin in skin show accelerated wound repair in dexamethasone impaired wound healing model

Integrin αvβ6 is an epithelial-specific receptor that is absent from the healthy epidermis but synthesized de novo during wound repair. However, its function in wound repair is unknown. Integrin-mediated transforming growth factor-β1 (TGF-β1) activation is the main activation mechanism of this key cytokine in vivo. Impaired wound healing caused by glucocorticoids is a major clinical problem and is associated with a disturbed balance of TGF-β1 activity. Therefore, αvβ6 integrin-mediated regulation of TGF-β1 activity may be involved in this process. To determine the function of αvβ6 integrin in glucocorticoid-induced impaired wound healing, both β6 integrin-deficient (β6−/−) and wild-type mice were exposed to dexamethasone treatment. Multiple wound parameters, keratinocyte proliferation, inflammation, and TGF-β1 activation were assessed. Wound healing was significantly accelerated in the dexamethasone-treated β6−/− mice compared with the corresponding wild-type mice. The dexamethasone-treated β6−/− mice showed enhanced keratinocyte proliferation in both wound epithelium and hair follicles while the production of proinflammatory cytokines and TGF-β1 activation were reduced. Accelerated wound repair in the dexamethasone-treated β6−/− mice might be associated with the reduced antiproliferative and proinflammatory effects of TGF-β1. Inhibition of αvβ6 integrin may provide a future target for treatment of impaired wound healing.     

Vasoactivity of diadenosine polyphosphates in human small renal resistance arteries.

BACKGROUND: We examined for the first time the vascular effects of purinergic agents that contribute to the regulation of peripheral vascular resistance in human small renal resistance arteries (hRRAs). METHODS AND RESULTS: Diadenosine polyphosphates (ApnAs, n = 3-6) and ATP, mounted in a microvessel myograph, caused vasoconstriction in hRRAs (rank order of potency: Ap5A > Ap6A = Ap4A > Ap3A = ATP). ADP, AMP and adenosine had less contractile potency than ApnA, suggesting that the observed effects were not induced by ApnA degradation products. The ApnA agent, Ap5A, but not Ap4A, induced vasoconstrictions that were inhibited by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; a P2X purinoceptor antagonist), but not by ADP3'5' (a P2Y purinoceptor antagonist). In pre-contracted hRRAs, all of the ApnA agents caused vasorelaxation, and the potencies did not differ from each other. The ApnA degradation products had less vasorelaxing potencies than ApnA, suggesting that the vasorelaxation was caused by the ApnA agents themselves. Ap4A-induced vasorelaxation was inhibited by ADP3'5' and PPADS. In contrast, Ap5A-induced vasorelaxation was not antagonized by ADP3'5', but was antagonized more strongly by PPADS than was Ap4A. CONCLUSIONS: We found that the tone of resistance arteries in human kidneys can be considerably influenced by these purinergic agonists, and most potently by ApnAs. Ap5A-induced vasoconstriction appeared to be mediated by P2X purinoceptors, whereas constriction due to Ap4A was caused by a different purinoceptor. Vasorelaxation due to Ap4A, but not Ap5A, appeared to be mediated by P2Y purinoceptors.     

Extracellular matrix-derived tripeptide proline-glycine-proline inhibits keratinocyte proliferation and migration

Keratinocytes are the predominant cell type in epidermis, and are primarily responsible for the epithelialization phase of wound healing. Previous studies by our group showed a positive correlation between IL‐8 concentration and delayed healing of porcine cutaneous partial‐thickness wounds. Interleukin‐8 and collagen‐breakdown product N‐acetyl‐Pro‐Gly‐Pro (PGP) are known as chemoattractant molecules for neutrophils during inflammation. The activity of both molecules is dependent on chemokine receptors CXCR1 and CXCR2. In addition to neutrophils, keratinocytes also express CXCR1 and CXCR2. Here we investigated the effects of IL‐8 and PGP on keratinocyte proliferation and migration. Our results showed that IL‐8 up to 100 ng/mL does not have any significant impact on keratinocyte proliferation or migration. ECM‐derived tripeptide PGP chemotactically attracts neutrophils but not keratinocytes. PGP strongly inhibits keratinocyte proliferation and migration in a cell‐type specific manner. Thus, collagen breakdown product PGP plays a key role in modulating both the inflammatory and epithelialization phases of wound healing.     

Changed responsiveness of the detrusor in rabbits with alloxan induced hyperglycemia: possible role of 5-hydroxytryptamine for diabetic bladder dysfunction

PURPOSE: We assessed whether the responsiveness of the detrusor is changed in rabbits with alloxan induced hyperglycemia. MATERIALS AND METHODS: Hyperglycemia was induced by a bolus intravenous injection of alloxan (60 mg./kg.) in Japanese White male rabbits. At 16 weeks after alloxan detrusor muscle strips prepared from age matched normoglycemic and hyperglycemic rabbits were mounted in organ chambers. Contractile responses to KCl, carbachol, adenosine triphosphate, 5-hydroxytryptamine and electrical field stimulation were compared in the 2 groups. The effect of sarpogrelate as a selective antagonist of 5-hydroxytryptamine 2A receptor on the contractile response to 5-hydroxytryptamine was also compared. RESULTS: The current experiments demonstrated that hyperglycemia caused significant decreases in neurogenic and carbachol induced contractions accompanied by unchanged adenosine triphosphate and KCl induced contractions. Neurogenic bladder contraction in the hyperglycemic rabbit was significantly potentiated by exogenously applied 5-hydroxytryptamine. Potentiation was detectable even after the desensitization of purinoceptors but undetectable in the presence of atropine. Hyperglycemia resulted in enhancement of the 5-hydroxytryptamine induced bladder contraction. Sarpogrelate tended to normalize the enhanced contraction. CONCLUSIONS: The decrease in neurogenic bladder contraction possibly accompanied by the decreased density of muscarinic receptors, the potentiation of neurogenic bladder contraction with 5-hydroxytryptamine probably due to facilitated cholinergic transmission and the enhanced contractility to 5-hydroxytryptamine would be at least in part involved in bladder dysfunction associated with hyperglycemia.     

Salutary roles of CD39 in transplantation

Transplantation exposes vascularized grafts to several potential injuries, including ischemia-reperfusion injury and rejection. These processes are associated with, at least in part, extracellular nucleotide-triggered (type 2 purinergic receptor) inflammatory responses that if left unchecked might compromise the long-term function and survival of the graft. Modulation of type 2 purinergic receptor–mediated signaling occurs by ectoenzymes that hydrolyze nucleotides to adenosine, which in turn activate type-1 purinergic receptors, and play a critical role in limiting inflammation and thrombosis.CD39 is the prototype nucleoside triphosphate diphosphohydrolase and is highly expressed on the endothelium. Different levels of CD39 expression by the vasculature influence the fate of the transplanted organ in ischemia-reperfusion injury and transplant rejection models. Additional roles for extracellular nucleotides/adenosine have also been defined within the immune system. Moreover, CD39 is a phenotypic marker of regulatory T cells, and through the generation of adenosine, contributes to the suppressive capabilities of these cells in vitro and in vivo.Our experimental models suggest that purinergic mechanisms may provide an integration point for the vascular and immunological responses in transplantation. CD39, the dominant ectonucleotidase, receptors for nucleotide mediators and adenosine are expressed in a regulated manner on both vascular and immune cells. We predict that administration of soluble CD39 and adenosine agonists or the targeted expression of CD39 through genetically modified organs or cells may have future therapeutic application in transplant-associated and other vascular diseases.     

Mechanisms of the inhibition of epithelial Na(+) channels by CFTR and purinergic stimulation

The epithelial Na+ channel ENaC is inhibited when the cystic fibrosis transmembrane conductance regulator (CFTR) coexpressed in the same cell is activated by the cyclic adenosine monophosphate (cAMP)-dependent pathway. Regulation of ENaC by CFTR has been studied in detail in epithelial tissues from intestine and trachea and is also detected in renal cells. In the kidney, regulation of other membrane conductances might be the predominant function of CFTR. A similar inhibition of ENaC takes place when luminal purinergic receptors are activated by 5'-adenosine triphosphate (ATP) or uridine triphosphate (UTP). Because both stimulation of purinergic receptors and activation of CFTR induce a Cl(-) conductance, it is likely that Cl(-) ions control ENaC activity.     

ATP induces Ca(2+) signaling in human chondrons cultured in three-dimensional agarose films

OBJECTIVE: In vivo, chondrocytes are surrounded by an extracellular matrix, preventing direct cell-to-cell contact. Consequently, intercellular communication through gap junctions is unlikely. However, signaling at a distance is possible through extracellular messengers such as nitric oxide (NO) and nucleotides and nucleosides, adenosine triphosphate (ATP), uridine triphosphate (UTP), or adenosine diphosphate (ADP). We hypothesized that chondrons, chondrocytes surrounded by their native pericellular matrix, increase their intracellular calcium concentration ([Ca(2+)]ic) in response to ATP and other signaling molecules and that the source of Ca(2+) is from intracellular stores. The objectives of this study were to determine if chondrons in a 3-D gel respond to ATP by increasing [Ca(2+)]ic through a purinoceptor mechanism and to test whether chondrons in whole tissue samples would respond to ATP in a similar fashion. DESIGN: Human chondrons, cultured in a three-dimensional agarose gel or in whole cartilage loaded with Fura-2AM, a calcium sensitive dye, were stimulated with 1, 5 and 10 microM ATP. A ratio-imaging fluorescence technique was used to quantitate the [Ca(2+)]ic. RESULTS: ATP-stimulated chondrons increased their [Ca(2+)]ic from a basal level of 60 nM to over 1000 nM. Chondrons incubated in calcium-free medium also increased their [Ca(2+)]ic in response to ATP, indicating the source of Ca(2+) was not extracellular. ATP-induced calcium signaling was inhibited in chondrons pre-treated with suramin, a generic purinoceptor blocker. In addition, UTP and adenosine 5'-O-(3-thiotriphosphate) (ATPgammas) induced a calcium response, but 2-methylthio-ATP (2-MeSATP), ADP, and adenosine did not induce a significant increase in [Ca(2+)]ic, substantiating that the P2Y2 purinoceptor was dominant. Chondrons in whole cartilage increased [Ca(2+)]ic in response to ATP. CONCLUSIONS: We conclude that chondrons in 3-D culture respond to ATP by increasing [Ca(2+)]ic via P2Y2 receptor activation. Thus, ATP can pass through the agarose gel and the pericellular matrix, bind purinoceptors and increase intracellular Ca(2+) in a signaling response.