Diagnostic incisional biopsies in clinically indeterminate choroidal tumours.

Most intraocular tumours are reliably diagnosed by a careful clinical examination combined with one or more non-invasive diagnostic techniques. However, in a small percentage of tumours, typically small and clinically amelanotic, the features are insufficiently distinct for a confident clinical diagnosis and tissue is required for diagnosis. We used a 23-G vitreous cutter to access the biopsy site in 43 patients with clinically indeterminate tumours. After retinotomy, an incisional choroidal biopsy yielded a specimen of ∼1 mm(3). Obtained tissue was routinely processed for light microscopy including an immunohistochemical panel of monoclonal antibodies. Adequate tissue for diagnosis was provided in 41/43 (95%) patients. The sensitivity and specificity to detect malignant disease were 0.97 and 1.00, respectively. The positive predictive value was 1.00. Complications included progression of pre-existing retinal detachment in 5/43 (12%) patients and transient rise in intraocular pressure to >40 mm Hg in 6/43 (14%) patients; 4 of these 6 patients had a pre-existing retinal detachment. No patient with a pre-operatively attached retina had a retinal detachment. We conclude that an incisional transretinal choroidal biopsy yields abundant material and may adequately confirm or exclude malignancy in patients with clinically indeterminate tumours. The complication rate can be minimised when patients with pre-existing retinal detachment are excluded from biopsy.

A prospective randomised study on low-dose transpupillary thermotherapy versus photodynamic therapy for neovascular age-related macular degeneration.

AIM: To compare the efficacy of low-dose transpupillary thermotherapy (TTT) and verteporfin photodynamic therapy (PDT) in patients with occult neovascular age-related macular degeneration (AMD). METHODS: Patients were randomised to receive either low-dose TTT (136 mW/mm) (and sham PDT) (n = 52) or PDT (and sham TTT) (n = 46) with retreatment if leakage was documented by fluorescein angiography. At baseline and at every follow-up, best corrected visual acuity (BCVA) was measured with the Early Treatment Diabetic Retinopathy Study (ETDRS) chart, lesion size on fluorescein angiography and foveal thickness with optical coherence tomography. The primary outcome measure was the proportion of patients who lost <15 letters at 12 months' follow-up. Secondary outcome measures included the proportion of patients who gained >/=0 letters, the change in mean lesion size and the change in foveal thickness at 12 months' follow-up. RESULTS: The percent of patients losing fewer than 15 letters at 12 months was 75.0% in the TTT group and 73.9% in the PDT group (p>0.05). The percent of patients with preserved or improved BCVA was 36.5% in the TTT group versus 23.9% in the PDT group (p>0.05). The mean decrease in foveal thickness was 15% for TTT and 24% (p>0.05) for PDT-treated patients, and the mean increase in total lesion area was -0.7% and -1.1% (p>0.05), respectively. CONCLUSION: In this prospective, randomised trial low-dose TTT and PDT appeared to be equally efficient at stabilising visual acuity in patients with occult neovascular AMD. Low-dose TTT may be considered as an alternative to PDT in this set of patients and also as an adjuvant to pharmacotherapy.

The Swedish national survey of surgical excision for submacular choroidal neovascularization (CNV).

PURPOSE: To compare the visual outcome after surgical removal of subfoveal choroidal neovascularization (CNV) in patients younger and older than 50 years of age. METHODS: Patient records from all Swedish centers performing submacular CNV surgery were reviewed and 90 patients treated between 1992-1999 with a follow-up of 6 months or more were included. The results obtained in 49 patients aged 51-89 years (median=72 years) with neovascular disease caused by age-related macular degeneration (AMD) were compared with the outcome of 41 patients aged 6-49 years (median=36 years) with CNV secondary to non-AMD causes. The main outcome measure was the improvement or deterioration in visual acuity (standardized in logMAR units) at 6 months following surgery. Secondary endpoints were recurrent CNV and surgical complications. RESULTS: The level of preoperative visual acuity was not significantly different between younger patients with CNV associated with non-AMD and older patients with visual loss due to AMD (p=0.069). However, visual acuity at 6 months after surgery was better (p=0.0042) in younger patients (median improvement=0.19 logMAR) than in older patients (median improvement=0.0 log MAR). Marked visual improvement (>1 log MAR unit) was seen in 29% of non-AMD patients <50 years compared to 0% in the AMD group >50 years. CONCLUSION: Surgical removal of submacular CNV does not appear to improve visual acuity in patients > 50 years of age. However, it may be beneficial for younger patients where a substantial improvement of visual acuity is seen in a subset of these patients. Further studies are required to assess the long-term outcome.

Matrix metalloproteinase (MMP) expression in experimental choroidal neovascularization.

PURPOSE: Matrix metalloproteinases (MMP) are a family of proteolytic enzymes that degrade basement membrane and extracellular matrix proteins. To gain information on the possible role of MMPs in choroidal neovascularization (CNV), we have analyzed the mRNA expression of MMP-2 and MMP-9, two forms of MMPs implicated in ocular neovascularization, in a rat model. METHODS: Choroidal neovascularization was induced in pigmented rats by krypton laser photocoagulation of the fundus whereafter eyes were enucleated at 1, 3, 5, 7, 10 and 60 days. Antisense and sense riboprobes were generated using DNA complementary to MMP-2 and MMP-9, and mRNA expression was analyzed using in situ hybridization. RESULTS: In the untreated eyes MMP-2 mRNA expression was weakly detected in cells within the choroid. In laser-treated eyes MMP-2 mRNA expression was markedly increased and mainly localized to macrophage-like and retinal pigment epithelial (RPE)-like cells invading the choroid, subretinal space and inner retina. This increase in MMP-2 mRNA expression peaked at day 10 whereafter a decline was detected. MMP-9 mRNA expression was low in untreated eyes and did not increase following laser treatment. CONCLUSION: The results show that MMP-2 mRNA expression is increased in experimental CNV, and support of a role for MMP-2 in the development of CNV in age-related macular degeneration.

Expression of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) in inflammation-associated corneal neovascularization.

Matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) are all implicated in the development of neovascularization. To investigate the possible role of these factors in corneal neovascularization we have analysed the expression of MMP-2, MMP-9 and VEGF in a rat model of inflammation-associated corneal neovascularization. In this model, corneal neovascularization was induced in Long-Evans rats by krypton laser photocoagulation whereafter eyes were enucleated at 1, 4, 7, 10 and 20 days. Slit-lamp biomicroscopy and histologic analysis revealed a gradual development of corneal neovascularization that peaked 7-10 days after treatment when newly formed vessels could be seen throughout the corneal surface reaching deep into the stroma. Antisense and sense riboprobes were generated using DNA complementary to MMP-2, MMP-9 and VEGF, and mRNA expression was analysed using in situ hybridization. The expression of MMP-2 and MMP-9 in untreated corneas was low or absent whereas VEGF was weakly expressed in the corneal epithelium. MMP-2 expression was increased during corneal neovascularization and was mainly localized to the cells infiltrating areas of new vessel formation. Many of these cells appeared to be inflammatory cells. VEGF expression had a similar overall distribution to MMP-2 during neovascularization with the exception that its expression in the corneal epithelium remained and even increased slightly. MMP-9 was prominently expressed at the border of regenerating corneal epithelium in areas with epithelial wounding but was not detected in the vascularized stroma. Together, the results of the present study support a role for MMP-2 and VEGF in inflammation-associated corneal neovascularization whereas MMP-9 instead appears to be involved in corneal epithelial wound-healing.

Matrix metalloproteinases and metalloproteinase inhibitors in choroidal neovascular membranes.

PURPOSE: Matrix metalloproteinases (MMP) are a family of extracellular matrix degrading enzymes associated with the development of neovascularization. To investigate the possible role of these enzymes in choroidal neovascularization, the mRNA expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) were analyzed in subfoveal fibrovascular membranes from patients with age-related macular degeneration (AMD). METHODS: Surgically removed subfoveal fibrovascular membranes from five eyes were analyzed for the expression of MMP and TIMP mRNA. In situ hybridization anti-sense and sense riboprobes were generated using DNA complementary to human collagenase (MMP-1), 72 kDa gelatinase (MMP-2), stromelysin (MMP-3), 92-kDa gelatinase (MMP-9), TIMP-1, TIMP-2, and TIMP-3. Vascular endothelial cells were detected using immunostaining for von Willebrand factor. RESULTS: MMP-2 and MMP-9 mRNA were detected in all specimens. Most of the membranes also expressed TIMP-1 and TIMP-3 mRNA, and two of the membranes expressed TIMP-2 mRNA. MMP-2, TIMP-1, and TIMP-2 mRNA had a similar overall distribution that was relatively uniform within the vascularized membrane stroma. MMP-2 expression appeared to be localized mainly to the vascular endothelial cells, whereas TIMP-1 and TIMP-3 were detected in other cell types such as fibroblastlike cells. MMP-9 expression was distinctly expressed by cells at the margins of the membranes and often in proximity to a thickened Bruch's membrane-like layer under the retinal pigment epithelial cells. TIMP-3 mRNA was strongly expressed within the retinal pigment epithelial cell layer and also in the stroma of one membrane. None of the membranes showed detectable MMP-1 or MMP-3 expression. CONCLUSIONS: The results support a role for MMPs in the development of choroidal neovascularization in AMD. The localization of MMP-2 and MMP-9 to the areas of new vessel formation and to the enveloping Bruch's-like membrane, respectively, suggests that MMP-2 and MMP-9 may be cooperatively involved in the progressive growth of choroidal neovascular membranes in AMD.

Localization of adenosine receptor messenger RNAs in the rat eye.

Adenosine is present in all cells and body fluids and has been suggested to play several roles in the physiology of ocular tissues. The present study was undertaken to determine which types of adenosine receptor mRNAs are present in the rat eye, and where they are expressed. RNA or deoxyoligodeoxynucleotides complementary to rat adenosine receptor subtypes A1, A2A, A2B and A3 were used to generate 35S labeled antisense and sense probes. The probes were then used for in situ hybridization on 10 microm cryosections of the rat eye including the cornea, iris, ciliary body, lens, retina, choroid and sclera. A1, A2A and A2B receptor mRNAs were demonstrated in the ciliary processes. A1 receptor mRNA was also expressed in the ganglion cell layer of the retina. The retina also showed A2A receptor mRNA expression, which was most prominent in the inner nuclear layer and less prominent in the ganglion cell layer and outer nuclear layer. Weak A2A expression was found in the retinal pigment epithelium and choriocapillaris. No significant expression of A3 receptor mRNA was found in the rat eye. In conclusion, using in situ hybridization, we have demonstrated expression of mRNA for A1, A2A and A2B adenosine receptors in the rat eye. The expression patterns support specific roles for adenosine in the ciliary process and retina.

Beta-amyloid protein protein precursor expression in lacrimal glands and tear fluid.

PURPOSE: Proteases in tear fluid play important roles in the regulation of corneal wound healing. Inhibitors of proteolytic activity are major modulators of the associated events. Although it is known that various enzyme inhibitors exist in tear fluid, it is not known whether certain isoforms of the beta-amyloid protein precursor (beta-APP), a potent inhibitor of serine proteases, are present in tear fluid. The purpose of this study was to investigate whether beta-APP can be detected in human tear fluid and, if so, to determine the isoform composition and cellular origin. METHODS: Tear fluid was collected from healthy volunteers. The beta-APP was identified and characterized by immunoblotting using antibodies specific for domains of the beta-APP. The protein was characterized further by ion exchange chromatography. Expression of the beta-APP gene was studied using in situ hybridization and RNA-RNA solution hybridization assay. RESULTS: beta-APP with protease inhibitory properties was identified in all samples of human tear fluid. Immunologic analysis revealed that it had been processed proteolytically before secretion. Gene expression studies showed that the beta-APP gene was expressed in lacrimal glands, particularly in acinar cells. The gene transcript almost exclusively corresponded to beta-APP containing the protease inhibitor insert. CONCLUSIONS: beta-APP is expressed in lacrimal glands and subsequently is secreted into tear fluid. Because the bulk of the beta-APP contained the protease inhibitor insert, the authors propose that beta-APP is an important regulator of proteolysis in tear fluid and that possibly it plays a role in the events associated with corneal wound healing. This suggests a novel physiological function of beta-APP in addition to those previously described-regulation of blood coagulation and cell growth.

Expression of vascular endothelial growth factor (VEGF) in retinoblastoma but not in posterior uveal melanoma.

All solid tumors must acquire a vascular stroma to grow beyond a minimal size. Vascular endothelial growth factor (VEGF) is a potent and specific angiogenic growth factor both in vitro and in vivo that may participate in the formation of the vascular tumor stroma. In the present study, we examined the expression of VEGF in the paraffin sections of 20 eyes harboring retinoblastoma or posterior uveal melanoma, but also in corresponding tumor cellines. By using in situ hybridization, we found that all but one of the retinoblastomas expressed VEGF mRNA. Particularly high expression was detected in areas of loosely packed tumor cells with prominent chromatin. By contrast, none of the posterior uveal melanomas expressed significant amounts of VEGF mRNA. Immunostaining with an antibody against VEGF confirmed that retinoblastomas, but not posterior uveal melanomas, also contained detectable VEGF protein. To further study the expression of VEGF in these tumor cells we performed Northern blotting on a retinoblastoma celline, Y79, and on an uveal melanoma celline, OM431. Both of these cellines expressed low levels of VEGF mRNA under normal culture conditions. However, when the cells were cultured under hypoxic conditions, a strong increase in VEGF mRNA could be seen in Y79 cells but not in OM431 cells. By using a bioassay, we also found that hypoxia stimulated the secretion of VEGF protein into the culture medium of Y79 cells. In conclusion, we have shown that VEGF mRNA and protein are expressed in retinoblastomas but not in posterior uveal melanomas. Moreover we have shown that VEGF is hypoxia-inducible in retinoblastoma cells. These results suggest that focal hypoxia may act as a stimulus for VEGF production in retinoblastomas, that in turn may contribute to tumor growth by stimulating the formation of a vascular stroma.

Subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor.

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent and specific angiogenic growth factor, in vitro and in vivo, that may be associated with the development of intraocular neovascularization. In the current study, the authors analyze the expression of VEGF in subfoveal fibrovascular membranes from patients with age-related macular degeneration. METHODS: Surgically removed subfoveal fibrovascular membranes from 18 eyes were analyzed for the expression of VEGF mRNA and protein using in situ hybridization and immunohistochemistry, respectively. RESULTS: Most specimens expressed both VEGF mRNA and protein. The VEGF mRNA expression was particularly high in areas with a marked inflammatory response, in which the expression was concentrated to cells resembling fibroblasts and to surrounding inflammatory cells. VEGF protein expression was seen in fibrovascular parts of the membranes and was predominantly localized to the cytoplasm of fibroblastlike cells. In some of these membranes, strong VEGF protein immunoreactivity also was concentrated to extracellular matrix foci within the fibrovascular stroma. CONCLUSIONS: Results indicate that VEGF may be of pathogenetic importance for the development of the choroidal neovascularization (age-related macular degeneration) and also may implicate a role of fibroblasts of presumable choroidal origin in this process.

Expression and regulation of vascular endothelial growth factor in choroidal fibroblasts.

Subretinal neovascularization is a severe sight-threatening complication into age-related macular degeneration. Previous immunohistochemical studies on surgically removed neovascular membranes have revealed that these membranes, in addition to the neovascular stroma, are comprised of several different cell types such as retinal pigment epithelial (RPE) cells, choroidal fibroblasts and vascular endothelial cells. Since vascular endothelial growth factor (VEGF) potently and specifically induces angiogenesis it was investigated whether VEGF is expressed and/or inducible in choroidal fibroblasts and RPE cells. Choroidal fibroblasts and RPE cells were isolated from human adult post-mortem eyes and expression of VEGF mRNA and protein was measured. By using Northern blotting, both choroidal fibroblasts and RPE cells were found to express VEGF mRNA at low levels. In order to examine whether this VEGF expression was further inducible, the intracellular effector enzyme protein kinase C was activated by phorbol esters. This activation resulted in a prominent increase in VEGF mRNA in choroidal fibroblasts, but not in RPE cells, with a maximal increase detected after 6 h. Elevation of intracellular cyclic AMP levels by forskolin had no clear effect on VEGF mRNA in either cell type. Stimulation with interleukin-1, transforming growth factor beta, tumour necrosis factor alpha and platelet derived growth factor was tested to see if VEGF expression is cytokine inducible. Both interleukin-1 and transforming growth factor beta induced VEGF expression in choroidal fibroblasts although with different time courses. Whereas the transforming growth factor beta effect was transient the interleukin-1 effect was sustained for at least 48 h. None of the cytokines tested affected VEGF expression in RPE cells. By using Western blotting, it was further found that stimulation with interleukin-1 induced VEGF protein expression in choroidal fibroblasts but not in RPE cells. In conclusion, choroidal fibroblasts respond by elevated VEGF mRNA levels after phorbol ester, interleukin-1 and transforming growth factor beta stimulation and elevated VEGF protein levels after phorbol ester and interleukin-1 stimulation suggesting that choroidal fibroblasts may be target cells for increased VEGF synthesis secondary to paracrine cytokine production.

[Vascular endothelial growth factor. A vascular growth factor of high specificity]. / Vascular endothelial growth factor. En vaskulär tillväxtfaktor med hög specificitet.

Angiogenesis, the formation of new blood vessels, is regulated by angiogenic growth factor. Vascular endothelial growth factor (VEGF) is a recently discovered growth factor that, in contrast to other growth factors with angiogenic activity, acts specifically on the vascular endothelium. VEGF is involved in the induction of new blood vessel formation in various physiological and pathological processes associated with increased angiogenesis.

Expression and secretion of transforming growth factor-beta in transformed and nontransformed retinal pigment epithelial cells.

The expression and secretion of different isoforms of transforming growth factor-beta (TGF beta) were examined in cultured transformed and nontransformed human retinal pigment epithelial (RPE) cells. Transformed RPE cells were found to express high levels of TGF beta 1 mRNA, low levels of TGF beta 3 mRNA but no detectable TGF beta 2 mRNA. If the cells were grown under serum-free conditions the expression of TGF beta increased. The mRNA expression was accompanied by secretion of TGF beta 1 (but not TGF beta 2) protein into the culture media. By comparison, nontransformed RPE cells were found to secrete similar amounts of TGF beta as transformed cells but predominantly secreted TGF beta 2. The secretion of TGF beta from both transformed and nontransformed RPE cells increased if the cells were grown without serum. In conclusion, the results show that TGF beta is expressed and secreted by transformed and nontransformed human RPE cells and that this expression and secretion are regulated by the presence or absence of exogenous factors.

Synergistic effects between protein kinase C and cAMP on activator protein-1 activity and differentiation of PC-12 pheochromocytoma cells.

In rat pheochromocytoma cells (PC-12) cells, we have studied the effect of protein kinase C (PKC) and cAMP on the activity of the nuclear transcription factor activator protein-1 (AP-1) and on differentiation of the cells into sympathetic nerve-like phenotype. By using mobility gel-shift assays, we found that both PKC and cAMP activation led to an increase in the binding of AP-1 to its consensus nucleotide sequence (TRE). When the PKC and cAMP pathways were activated simultaneously, a clear-cut synergistic effect was seen on the binding of AP-1 to TRE. Both PKC and cAMP activation were furthermore able to increase the AP-1 transcriptional activity in PC-12 cells transiently transfected with TRE-expressing plasmids. In agreement with the mobility gel-shift results, simultaneous activation of PKC and cAMP synergistically increased the AP-1 transcriptional activity. We next analyzed the effect of PKC and cAMP stimulation on differentiation and proliferation of PC-12 cells. Whereas PKC activation had no effect on the morphology of PC-12 cells, elevation of the intracellular cAMP level resulted in a marked increase in the number of neurite-bearing cells. This effect was paralleled by a strong inhibition of PC-12 cell proliferation. Interestingly, when PKC and cAMP activation were combined, the differentiation was further pronounced and growth further inhibited. These results show that both PKC and cAMP increase the AP-1 activity in PC-12 cells, and that these effects are synergistic. Moreover, we show that cAMP induces differentiation and inhibits growth of PC-12 cells, and that PKC activation acts synergistically with cAMP on these effects. The possible role of AP-1 in PC-12 cell differentiation is discussed.

Activation of protein kinase C and elevation of cAMP interact synergistically to raise c-Fos and AP-1 activity in Jurkat cells.

We have earlier found that in Jurkat cells activation of protein kinase C (PKC) enhances the cyclic adenosine monophosphate (cAMP) accumulation induced by adenosine receptor stimulation or activation of Gs. Here we have therefore examined the effect of the phorbol ester PMA (phorbol 12-myristate 13-acetate) which stimulates PKC and a combination of the adenosine receptor agonist NECA (5'-(N-ethyl)-carboxamido adenosine) and forskolin to raise cAMP, on the levels of c-Fos and Jun and on the binding and transcriptional activity of the transcription factor, activator protein-1 (AP-1). PMA treatment caused a concentration- and time-dependent increase in both c-Fos and Jun immunoreactivity in contrast to cAMP elevation that had only a slight effect. Both PMA and the combination of NECA and forskolin acted together either to increase (c-Fos) or decrease (Jun) protein levels as well as increasing AP-1 binding, as judged by gel-shift assay, and AP-1 transcriptional activity. Furthermore there was a clear-cut synergy between the PKC stimulator and the cAMP elevating agents. The results demonstrate that the simultaneous activation of PKC and elevation of cAMP leads to an enhanced AP-1 transcriptional activity in a T-leukemia cell line, suggesting that the previously observed interaction between the parallel signal transduction pathways may have functional consequences at the level of gene transcription.

Mitogen stimulation of T-cells increases c-Fos and c-Jun protein levels, AP-1 binding and AP-1 transcriptional activity.

We have analysed the effect of mitogenic lectins on c-Fos and c-Jun protein levels as well as on activator protein-1 (AP-1) binding and enhancer activity in Jurkat T-cells. Both c-Fos and c-Jun protein levels were increased after Con A and PHA stimulation. Since T-cell stimulation increases both intracellular Ca2+ and cAMP levels and activates protein kinase C (PKC), the possible involvement of these intracellular messengers in c-Fos and c-Jun induction was tested. PMA, which directly activates PKC, mimicked the effect of the lectins on c-Fos and c-Jun, but elevation of either intracellular Ca2+ or cAMP levels had little or no effect. The mitogen-induced increase of c-Fos and c-Jun immunoreactivity was inhibited by H-7, a kinase inhibitor with relatively high specificity for PKC, and less efficiently by H-8, a structurally related kinase inhibitor less active on PKC, but more active on cyclic nucleotide-dependent kinases. Con A stimulation was found to increase both binding of AP-1 to the AP-1 consensus sequence, TRE, and AP-1 enhancer activity, in Jurkat cells. PMA was also found to increase the AP-1 enhancer activity, whereas elevation of Ca2+ or cAMP had only minor effects. We conclude that stimulation with mitogenic lectins is sufficient to increase both c-Fos and c-Jun protein levels, AP-1 binding and AP-1 enhancer activity in Jurkat cells and that they act via mechanisms that could involve the activation of PKC.

Further evidence that propentofylline (HWA 285) influences both adenosine receptors and adenosine transport.

We have examined the actions of a novel xanthine derivative, propentofylline (HWA 285), that has been shown to protect against ischemic brain damage in rats and gerbils, on adenosine receptors (A1 and A2), and on adenosine transporters using several techniques, cells and tissues. Propentofylline and its hydroxylated metabolite A 72 0287 were about 20 times less potent than theophylline in displacing A1-agonist binding to membranes from rat cortex, and A1-antagonist binding to whole DDT, MF-2 smooth muscle cells. A1-agonist binding to adenosine A1-receptors in several brain structures was inhibited in a concentration-dependent manner by A 72 0287 and propentofylline as judged by quantitative autoradiography (IC50-values 300-600 microM in eg striatum and in cortex layer IV). In two functional assays, A1-receptor mediated effects were blocked by propentofylline. A1-receptor-mediated inhibition of cyclic AMP accumulation was virtually abolished by 100 microM propentofylline. The A1-receptor-mediated inhibition of evoked acetylcholine release was also reduced by propentofylline, but in this case the effect is not due exclusively to adenosine receptor antagonism but also to another action since the presynaptic inhibitory effect of carbachol was also inhibited. Adenosine A2-receptors were also antagonized by propentofylline as judged by a concentration-dependent antagonism of A2-agonist-induced cAMP accumulation in human T-leukemia cells (possessing putative A2b-receptors; pA2-value 180 microM compared to 0.26 microM for 8-cpt), and in PC-12 cells (possessing putative A2a-receptors, Ki-value 365 microM). Finally, adenosine transporters were affected by propentofylline and A 72 0287. Thus, [3H]-nitrobenzylthioinosine-binding to guinea-pig cardiac membranes was blocked by propentofylline or A 72 0287 (Ki 270 microM). The present results show that propentofylline and its hydroxylated metabolite can influence adenosine mechanisms in a multitude of ways. How these different actions may contribute to the ability of propentofylline to reduce the magnitude of ischemic damage is discussed.

Role of a pertussis toxin sensitive G-protein in mediating the effects of phorbol esters on receptor activated cyclic AMP accumulation in Jurkat cells.

In the human T-cell line, Jurkat, the accumulation of cyclic AMP induced by adenosine is enhanced by tumor-promoting phorbol esters, whereas prostaglandin E2 receptor-stimulated cAMP accumulation is antagonized (Nordstedt et al. 1989). In the present study we examine the involvement of pertussis toxin sensitive guanine nucleotide binding proteins (G-proteins) in producing the phorbol ester effects. Pertussis toxin pretreatment of the Jurkat cells invariably caused an ADP ribosylation of two G-proteins that inhibit adenylyl cyclase, tentatively identified as Gi2 and Gi3, using Western blots. Pertussis toxin treatment had little effect on basal cAMP accumulation, but sometimes inhibited, sometimes stimulated agonist and cholera toxin induced cAMP accumulation. The latter effect was not mimicked by the B-oligomer. Irrespective of whether pertussis toxin stimulated or inhibited NECA and cholera toxin-induced cAMP accumulation it could not block the effect of phorbol-12,13-dibutyrate (PDBu). The inhibitory effect of PDBu on prostaglandin E2-induced cAMP accumulation was, however, invariably eliminated by pertussis toxin treatment. In conclusion, activation of protein kinase C by phorbol esters reveals a Gi-mediated prostaglandin E receptor-induced inhibition of adenylate cyclase in addition to the prostaglandin E receptor-mediated stimulation of cAMP accumulation in Jurkat cells. The enhancement of adenosine A2 receptor stimulated cAMP accumulation by PDBu, on the other hand, does not involve a PTX sensitive Gi-protein.

CD3-dependent increase in cyclic AMP in human T-cells following stimulation of the CD2 receptor.

We have previously shown that stimulation of the Ti/CD3 receptor complex on human T-cells potentiates adenylate cyclase activation by adenosine or forskolin. Anti-CD2 receptor antibodies shared with anti-CD3 antibodies the ability to potentiate dose dependently the adenosine- and forskolin-stimulated cyclic adenosine monophosphate (cAMP) accumulation, whereas stimulation of the CD45 receptor had no effect on cyclase activity. Modulation of the CD3 complex with anti-CD3 antibodies was found to decrease the CD2 receptor effect on adenylate cyclase activity greatly. The possible involvement of CD3-stimulated phospholipase C (PLC) activation on the cAMP potentiation was examined using HPB-ALL cells that express a CD3 complex with a defect coupling to PLC. Stimulation of the CD3 complex on HPB-ALL cells had only slight effects on adenosine-stimulated cAMP formation, whereas the effect on forskolin-stimulated cAMP was virtually unchanged. The CD3 effect was further analyzed in Jurkat cell membranes. In contrast to the results obtained after stimulation of intact cells, it was found that OKT3 stimulation of membranes did not potentiate the forskolin response. Finally, we tested whether inhibition of endogenous adenylate cyclase agonist production affected the CD3 effect. Inhibition of adenosine production or adenosine breakdown with 8-p-sulphophenyl theophylline (8-PST) or adenosine deaminase (ADA), respectively, did not alter the CD3 effects. Indometacin, which inhibits prostaglandin production, also had no effect. Together, these data show that stimulation of the CD2 receptor potentiates adenylate cyclase responses by a mechanism that is dependent on CD3 expression. Furthermore, the CD3 effect on cAMP appears to be mediated by two different mechanisms, one which is, and one which is not dependent on PLC. Finally, this effect is not due to an endogenous production of adenylate cyclase agonists.