Immuno-analysis of phospho-Akt in primary human breast cancers.

The aim of this study was to investigate the expression of the activated (phosphorylated) form of Akt (Ser473) in primary breast cancer and to correlate the results with clinicopathological and prognostic variables for clinically relevant associations. Phospho-Akt expression was studied using immunoblot analysis in 49 invasive breast carcinomas (median follow-up time 55 months, range 7-74 months). We assessed the level of phospho-Akt in different types of primary breast cancers and compared the use of autoradiograph X-ray film with a PVDF-DAB-staining system. Twelve percent of the tumours had no phospho-Akt protein, 25% had low phospho-Akt expression, 51% had intermediate expression and 12% had high phospho-Akt expression. No relationship was observed between phospho-Akt and tumour grade, tumour size or nodal status. A significant relationship was demonstrated between phospho-Akt score and oestrogen receptor status (P=0.014). Univariate analysis demonstrated that intermediate levels of phospho-Akt in breast tumour tissue are associated with a lower probability of developing recurrences (P=0.035), while in multivariate analyses, none of the phospho-Akt levels appeared to be independent predictors of disease recurrence or death. In addition, it has been clearly established that a suitable composition of reagents and components such as PVDF membranes treated with DAB substrate will enable the performing of sensitive immuno-analyses.

Inhibition of compensatory renal growth by the N-terminus of a sheep-derived peptide.

The N-terminal sequence of a novel sheep-derived peptide with growth inhibitory activity has been obtained. The N-terminal fragment was chemically synthesised and designated EPL001. The kidney was chosen as the first mammalian system in which to study EPL001 since kidney growth can be accurately quantified following a surgical reduction in renal mass. Cell proliferation was measured in mouse collecting duct kidney (MCDK) cells stimulated with insulin-like growth factor I (IGF-I). Compensatory renal growth (CRG) was induced in Wistar rats and either EPL001 or an EPL001 antibody delivered by continuous renal tissue infusion. Mouse monoclonal antibodies to EPL001 were generated for immunoneutralisation, rabbit polyclonal antibodies were generated for immunohistochemistry. EPL001 had no apparent effect on IGF-I stimulated cell proliferation in MCDK cells in vitro, yet provoked a dose-dependent inhibition of CRG in vivo. An EPL001 antibody potentiated CRG, in the absence of exogenous EPL001, consistent with an inhibitory role in kidney growth for an endogenous peptide containing the EPL001 sequence. Tubular staining for epitopes to the EPL001 sequence was detected in normal human kidney sections and enhanced in renal cell carcinoma. Results support the presence of growth inhibitory activity in the N-terminus of a sheep-derived peptide with evidence for both its presence and endogenous activity in the kidney. Attempts to further characterise its structure and activity are ongoing.

Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells.

To investigate the mechanisms underlying apoptosis in breast cancer cells, staurosporine was used as an apoptotic stimulus in the human breast cancer cell lines MCF-7 and T47D. Staurosporine induced dose and time dependent increases in DNA fragmentation which was abrogated by z-VAD-fmk. MCF-7 cells did not express caspase-3, suggesting that DNA fragmentation occurred in the absence of caspase-3 and that other caspases may be involved. Staurosporine induced DEVDase activity in T47D cells suggesting the involvement of caspase-3 and/or caspase-7, yet there was no DEVDase activity in MCF-7 cells, probably ruling out the involvement caspase-7. However, staurosporine induced the cleavage of pro-caspase-6 in MCF-7 cells, but not in T47D cells. Caspase dependent PARP cleavage was detected in MCF-7 cells at 3 h, whereas only partial PARP cleavage was detected in T47D cells and then only after 24 h. Moreover, staurosporine led to cytochrome c release at 2 h in MCF-7 cells and 6 h in T47D cells. In addition, a time dependent and caspase-independent reduction of the mitochondrial transmembrane potential was observed; which appeared to occur after the release of cytochrome c. Translocation of Bax from the cytosol to mitochondria was observed in both cell types, and this preceded cytochrome c release in both T47D and MCF-7 cells. Apoptotic events in both cell types differ temporally, involving activation of different caspases and mitochondrial changes.

Interleukin-1 beta induced synthesis of protein kinase C-delta and protein kinase C-epsilon in EL4 thymoma cells: possible involvement of phosphatidylinositol 3-kinase.

We present evidence here that the proinflammatory cytokine, interleukin-1 beta (IL-1 beta) stimulates a significant increase in protein kinase C (PKC)-epsilon and PKC-delta protein levels and increases PKC-epsilon, but not PKC-delta, transcripts in EL4 thymoma cells. Incubation of EL4 cells with IL-1 beta induced protein synthesis of PKC-epsilon (6-fold increase) by 7 h and had a biphasic effect on PKC-delta levels with peaks at 4 h (2-fold increase) and 24 h (4-fold increase). At the level of mRNA, PKC-epsilon, but not PKC-delta levels, were induced after incubation of EL4 cells with IL-1 beta. The signalling mechanisms utilized by IL-1 beta to induce the synthesis of these PKC isoforms were investigated. Two phosphatidylinositol (PI) 3-kinase-specific inhibitors, wortmannin and LY294002, inhibited IL-1 beta-induced synthesis of PKC-epsilon. However, the PI 3-kinase inhibitors had little effect on the IL-1 beta-induced synthesis of PKC-delta in these cells. Our results indicate that IL-1 beta induced both PKC-delta and PKC-epsilon expression over different time periods. Furthermore, our evidence suggests that IL-1 beta induction of PKC-epsilon, but not PKC-delta, may occur via the PI 3-kinase pathway.

Possible role for protein kinase B in the anti-apoptotic effect of prolactin in rat Nb2 lymphoma cells.

Prolactin (PRL) is a mitogen for a number of cell types and its action as a survival factor has recently been demonstrated in Nb2 lymphoma cells. However, the intracellular signalling pathways by which PRL promotes the survival of Nb2 cells is unknown. In previous studies, we have shown that PRL caused the activation of phosphatidylinositol 3-kinase (PI3-kinase) and its association with tyrosine phosphorylated fyn. Protein kinase B (PKB), a serine/threonine kinase, is now known to be a downstream component of the PI3-kinase pathway. The aim of the present study was to examine the effect of PRL on the activation of PKB and to find out whether this has any role on the PRL-induced survival of Nb2 cells. Our studies have revealed the phosphorylation and activation of PKB in PRL-stimulated Nb2 cells. We have also observed, using confocal microscopy, translocation of PKB to the membrane of Nb2 cells in response to PRL. These effects were blocked by the PI3-kinase inhibitor, LY294002 (10 microgram/ml). Apoptosis was induced by the general protein kinase inhibitor, staurosporine (STS; 0.1-1 microM), the synthetic glucocorticoid, dexamethasone (Dex; 100 nM) or ionising radiation by exposing Nb2 cells to X-irradiation (IR; 10 Gy). PRL had no effect on STS-induced apoptosis. On the other hand, PRL (100 ng/ml) inhibited apoptosis induced by Dex or IR; this effect of PRL was reversed by the addition of LY294002 (10 microgram/ml). Furthermore, Western blot analysis using phosphospecific PKB antibody on lysates from PRL-treated Nb2 cells showed that phosphorylation of PKB in response to PRL was inhibited by STS (0.5 microM), but not by Dex (100 nM). These results suggest that the PI3-kinase/PKB pathway may mediate the anti-apoptotic effect of PRL in Nb2 cells.

Stimulation of endogenous GH and interleukin-6 receptors selectively activates different Jaks and Stats, with a Stat5 specific synergistic effect of dexamethasone.

The interaction of GH, interleukin (IL)-6 and glucocorticoids is likely to be important in regulating the GH-insulin-like growth factor (IGF)-I axis. The signalling cascades activated by GH and IL-6 appear to be very similar, as demonstrated by studies using overexpression of the receptor and other components of the Jak-Stat and mitogen-activated protein (MAP) kinase pathways. Here we show that the human embryonic kidney cell line 293 (HEK293) expresses GH and IL-6 receptors endogenously. To determine which specific pathways might be activated by the two cytokines, at physiological levels of all components, we studied GH and IL-6 mediated signal transduction both under basal conditions and in the presence of overexpressed receptors and Stat proteins. Our results suggest a receptor specificity of Jak2 for GH receptors, and Jak1 for IL-6 receptors. Stat activation in response to GH and IL-6 was determined by reporter gene induction. Both GH and IL-6 were able to induce the reporter gene containing the Stat5 responsive element (LHRE) but the IL-6 response appeared to be mediated mainly through Stat3 activation. In contrast, the reporter gene containing the Stat3 responsive element (SIE) was IL-6 specific. The levels of gene induction by GH and IL-6 were not altered by the co-stimulation with GH and IL-6, suggesting that there is little cross-talk at the Jak-Stat activation level between the two cytokines. Neither GH nor IL-6 activated the MAP-kinase responsive serum response element (SRE), unless GH receptors or gp130 were overexpressed. Transfection of Stat3 or Stat5 expression vectors enhanced the response to GH and IL-6. Stimulation with dexamethasone synergistically enhanced GH activation of the LHRE reporter gene but had no effect on the IL-6 activation of the same reporter or on the SIE reporter gene. Thus, our studies suggest that while each cytokine, GH and IL-6, may activate various members of the Jak-Stat pathway in overexpression studies, specific activation of Stat3 by IL-6 and of Jak2 and Stat5 by GH can be observed in HEK293 cells and that in this system the synergistic effect of dexamethasone appears specific for Stat5.

Caspase-3-like activity is necessary but not sufficient for daunorubicin-induced apoptosis in Jurkat human lymphoblastic leukemia cells.

In the present study we have shown that the cancer therapeutic drug, daunorubicin, induces apoptosis in the human lymphoblastic leukemia cell line Jurkat E6.1. This effect was both dose-and time-dependent with nuclear fragmentation detectable by 8 h. Caspases have been implicated in pro-apoptotic events. By utilizing synthetic fluorochrome-linked substrates of the caspases, we observed that a caspase-3-like enzyme had dramatically increased activity (3340 130% with respect to basal levels) in response to daunorubicin treatment. Furthermore, by using an inhibitor to caspase-3, Ac-DEVD-CHO, we have shown that activation of a caspase-3-like enzyme appears to be necessary for nuclear fragmentation and apoptotic body formation, but is not required for chromatin condensation. In contrast, a general caspase inhibitor, Z-VAD-fmk, inhibited all apoptotic parameters measured. Ceramide has been implicated in daunorubicin-induced apoptosis in human myeloid leukemia cells. However, in Jurkat cells, caspase activation does not appear to be a consequence of ceramide generation since, although ceramide levels were elevated through the action of ceramide synthase in response to daunorubicin treatment, this occurred with slower kinetics than either nuclear fragmentation or caspase activation. In contrast, caspase inhibitors abrogated ceramide elevation induced by DNR treatment, suggesting that ceramide synthase may be a downstream target for caspase action. Therefore, daunorubicin-induced apoptosis does not appear to be mediated by ceramide in the lymphoblastic leukemia cell line, Jurkat E6.1. Instead, caspase 3 activity appears to be necessary, but not sufficient for this process.

The role of caspase 3 and BclxL in the action of interleukin 7 (IL-7): a survival factor in activated human T cells.

The effects of interleukin 7 (IL-7) on apoptosis in interleukin 2 (IL-2)-dependent, activated, primary, human T lymphocytes (hT cells) was examined. IL-7 (like IL-2) rescued cells from apoptosis, as measured by their cellular DNA profile and fragmentation. IL-2 also acted as a mitogen in these T cells. Both cytokines abrogated the dexamethasone-induced stimulation of Caspase 3 and prevented the cleavage of poly (ADP-ribose) polymerase (PARP), a substrate for the Caspase 3. IL-7 upregulated the expression of Bc1xL and counteracted the downregulation of this anti-apoptotic protein by the synthetic glucocorticoid, dexamethasone. Bcl-2 protein expression was uupregulated by IL-7 with or without dexamethasone, but Bc1-2 was expressed at a much lower level than BclxL in these cells. Levels of Bax did not markedly change on either cytokine stimulation or dexamethasone treatment. An unidentified 23-kDa band, which was recognized by the anti-Bc1-2 antibody, was induced by dexamthasone and suppressed by IL-7 and IL-2. This protein was subject to independent regulation as compared to the p26 Bc1-2 protein, suggesting that it may be a novel factor, possibly involved in the regulation of apoptosis. A clear role for IL-7 as a survival factor for cytokine withdrawal and glucocorticoid induced apoptosis in activated primary hT cells is implicated. In addition, regulation of BclxL and downstream inhibition of Caspase 3 activity may mediate this rescue signal.

Interleukin-1beta-induced expression of protein kinase C (PKC)-delta and epsilon in NIH 3T3 cells.

NIH 3T3 cells express the alpha, delta, epsilon and zeta isoenzymes of protein kinase C(PKC). Following stimulation of cells (24 h) with the pro-inflammatory cytokine, interleukin 1beta (IL-1beta), we observed, by Western blotting, a dose-dependent effect on the levels of PKC-epsilon and delta, but not on alpha or zeta. Moreover, time course analysis revealed that the isoenzymes, PKC-delta and epsilon were induced by IL-1beta after 7 h. Again, no change in PKC-alpha or zeta levels after IL-1beta treatment were detected. Incubation with selective PKC inhibitor peptides blocked the PKC-alpha, delta, epsilon and zeta antibodies binding to their respective isoenzyme bands. We also observed that the addition of the tumour-promoting phorbol ester, Phorbol 12-myristate 13-acetate (PMA), downregulated PKC-alpha, delta and epsilon by 7 h in NIH 3T3 cells. PMA did not affect constitutively produced PKC-zeta protein levels even after 24-h treatment. In summary, these results demonstrate that IL-1beta induces protein synthesis of the Ca2+-independent PKC-delta and epsilon isoforms in NIH 3T3 cells. The differences observed here between PKC isoenzymes in response to IL-1beta suggest that each isoenzyme may have a unique role in the signal transduction pathways of IL-1beta and that such isoenzyme may have a unique role in the signal transduction pathways of IL-1beta and that such selective expression may influence the action of agents which require PKC for signal transduction acting in concert with IL-1.

A 70-kDa protein facilitates interleukin-4 signal transduction in the absence of the common gamma receptor chain.

Interleukin-4 signal transduction (and activation of STAT 6) is known to be mediated via its binding to a p140 receptor chain and the common gamma chain (gamma c). In non-activated monocytes, neither the gamma c nor its associated signal transducing molecule, Jak3, is expressed. We nevertheless show that IL-4 can initiate the tyrosine phosphorylation and DNA binding of STAT 6 in these cells. We present evidence for an additional 70 kDa IL-4 receptor chain which mediates the tyrosine phosphorylation of STAT 6 via Jak2, and suggest that this is the means by which IL-4 can signal in cells lacking the gamma c.

A short isoform of the human growth hormone receptor functions as a dominant negative inhibitor of the full-length receptor and generates large amounts of binding protein.

The GH receptor (GHR) is a member of the cytokine receptor family. Short isoforms resulting from alternative splicing have been reported for a number of proteins in this family. RT-PCR experiments, in human liver and cultured IM-9 cells, using primers in exon 7 and 10 of the GHR, revealed three bands reflecting alternative splicing of GHR mRNA: the predicted product at 453 bp and two other products at 427 and 383 bp. The 427-bp product (GHR1-279) utilized an alternative 3'-acceptor splice site 26 bp downstream in exon 9; the predicted C-terminal residues are six frameshifted exon 9 codons ending in an inframe stop codon. The 383-bp product (GHR1-277) resulted from skipping of exon 9; the predicted C-terminal residues are three frame-shifted exon 10 codons ending in an in-frame stop codon. RNase protection experiments confirmed the presence of the GHR1-279 variant in IM-9 cells and human liver. The proportion of alternative splice to full length was 1-10% for GHR1-279 and less than 1% for GHR1-277. The function of GHR1-279 was examined after subcloning in an expression vector and transient transfection in 293 cells. Scatchard analysis of competition curves for [125l]-hGH bound to cells transfected either with GHR full length (GHRfl) or GHR1-279 revealed a 2-fold reduced affinity and 6-fold increased number of binding sites for GHR1-279. The increased expression of GHR1-279 was confirmed by cross-linking studies. The media of cells transfected with GHR1-279 contained 20-fold more GH-binding protein (GHBP) than that found in the media of cells transfected with the full-length receptor. Immunoprecipitation and Western blotting experiments, using a combination of antibodies directed against extracellular and intracellular GHR epitopes, demonstrated that GHRfl and GHR1-279 can form heterodimers and that the two forms also generate a 60-kDa GHBP similar in size to the GHBP in human serum. Functional tests using a reporter gene, containing Stat5-binding elements, confirmed that while the variant form was inactive by itself, it could inhibit the function of the full-length receptor. We have demonstrated the presence of a splice variant of the GHR in human liver encoding a short form of the receptor similar in size to a protein previously identified in human liver and choroid plexus. Expression studies in 293 cells support the hypothesis that while the expression of the splice variant accounts for only a small proportion of the total GHR transcript, it produces a short isoform that modulates the function of the full-length receptor, inhibits signaling, and generates large amounts of GHBP. The differential expression of GHR receptor short forms may regulate the production of GHBP, and truncated receptors may act as transport proteins or negative regulators of GHR signaling.

Prolactin induced tyrosine phosphorylation of p59fyn may mediate phosphatidylinositol 3-kinase activation in Nb2 cells.

Our previous studies indicated that PI3-kinase is involved in prolactin (PRL) signalling. We have now examined the involvement of the src tyrosine kinase, fyn, in PRL-induced the activation of PI3-kinase in the rat lymphoma cell line, Nb2. Cells were stimulated with increasing doses of PRL, lysed and immunoprecipitated with anti-fyn specific antibody. Then PI3-kinase activity was measured as the increase in the phosphorylation of phosphatidylinositol to phosphatidylinositol 3-phosphate separated by TLC. Our data indicated that, in PRL treated cells, co-precipitation of PI3-kinase with anti-fyn antiserum led to time and dose-dependent activation of PI3-kinase in vitro and that this activation was blocked by the addition of LY294002. However, LY294002 appeared to have no effect on fyn autophosphorylation. Furthermore, the physical association of PI3-kinase with fyn was confirmed by Western blot analysis employing the same specific antisera. These data provide evidence that PRL-induced activation of PI3-kinase may be mediated by the tyrosine phosphorylation of fyn in Nb2 cells.

Activation of phosphatidylinositol 3-kinase by prolactin in Nb2 cells.

In the present studies, using anti-phosphotyrosine (PY20) and PI3-kinase (p85) antibodies, we have shown that PRL causes activation of phosphatidyl inositol 3-kinase (PI3-kinase) in vitro in a dose- and time-dependent manner in Nb2 cells. PRL activated PI3-kinase was completely inhibited by LY294002 (1 microgram/ml). Stimulation of the cells with PRL also increased tyrosine phosphorylation of the 85-kDa regulatory subunit. Moreover, in vitro kinase assay followed by SDS-PAGE protein separation demonstrated the phosphorylation of several other proteins besides the p85. However, no direct association between p85 and JAK2 tyrosine kinase was observed. These results indicate, for the first time, the involvement of PI3-kinase in PRL-stimulated Nb2 cell growth.

Interleukin-1 beta induces the synthesis of adenylyl cyclase in Swiss 3T3 fibroblasts and MG-63 osteosarcoma cells.

In this study, the longer term (> or = 6 hours) effect of interleukin-1 beta on adenylyl cyclase activity was investigated in Swiss 3T3 and MG-63 cells. No change was evident after 6 hours but after 1, 7 or 15 day incubations a significant increase in basal (1.5- 2 fold) and NaF-stimulated (2-4 fold) adenylyl cyclase activity was observed in interleukin-1 beta pre-treated cell membranes compared with non pre-treated controls. The response to forskolin, a direct stimulus of adenylyl cyclase, was also significantly enhanced, indicating that the effect of interleukin-1 beta was targeted to the enzyme itself. This action of interleukin-1 beta was blocked by co-incubation with cycloheximide, an inhibitor of protein synthesis, which demonstrated that de novo protein synthesis was required. It is concluded that interleukin-1 induces the expression of adenylyl cyclase in 3T3 and MG-63 cells, leading to enhanced activation by NaF and forskolin.

The mitogenic action of recombinant basic FGF in Swiss 3T3 cells is independent of early diradylglycerol production and downregulatable protein kinase C activity.

In this study we have investigated the requirement for phosphoinositide metabolism, diradylglycerol (DG) production and protein kinase C (PKC) activation in recombinant basic fibroblast growth factor (rbFGF)-mediated reinitiation of DNA synthesis in Swiss 3T3 cells. We have assessed the involvement of PKC activation in rbFGF-induced DNA synthesis by two approaches; enzymic inhibition by H7 and down-regulation by prolonged phorbol-ester treatment. In both conditions we observed that rbFGF was able to sustain a significant component of its mitogenic response, therefore denying an exclusive role for the activation of downregulatable and H7-sensitive PKC isoforms in rbFGF-induced reinitiation of DNA synthesis. Moreover, we have found no evidence for diacylglycerol accumulation in response to rbFGF by 3T3 cells. In previous studies, we observed that rbFGF caused a moderate and slow accumulation of total inositol phosphates. This effect was significant only after a 60 min incubation. It is our contention that rbFGF, in our culture system, does not exert a direct effect on phosphoinositide metabolism.

Stimulation of arachidonic-acid release from Swiss 3T3 cells by recombinant basic fibroblast growth factor: independence from phosphoinositide turnover.

In this study we have attempted to characterize the mechanism of recombinant bovine basic fibroblast growth factor (rbFGF)-induced release of arachidonic acid from prelabelled Swiss 3T3 fibroblasts. Recombinant bFGF caused the release of [3H]arachidonic acid from metabolically labelled cells in a dose- and time-dependent manner. This effect was maximal with 10 ng rbFGF/ml and became significant after a 30-min incubation. Although rbFGF was able to cause a modest increase in total inositol phosphate accumulation, an examination of the time-course of the latter effect revealed that enhanced [3H]arachidonic-acid release could not have been derived from phosphoinositide metabolism. Evidence suggesting that rbFGF-induced release of [3H]arachidonic acid was being mediated via a PLA2 pathway was obtained by pharmacological antagonism using mepacrine, a putative PLA2 inhibitor. Moreover, treatment of cells with neomycin failed to attenuate rbFGF-mediated release of [3H]arachidonic acid. Chelation of extracellular calcium by EGTA was found to abrogate rbFGF-induced liberation of [3H]arachidonic add. Down-regulation of protein kinase C (PKC) by prolonged treatment of cells with the phorbol ester, PMA, was observed to have no effect on the action of rbFGF on [3H]arachidonic add release from Swiss 3T3 fibroblasts. While rbFGF was found to cause the indomethacin-sensitive production of prostaglandin E2 (PGE2) in a dose-dependent manner, this effect was independent of rbFGF-induced reinitiation of DNA synthesis. Clearly, the effect of rbFGF on cellular DNA synthesis was being mediated independently of PGE2 biosynthesis. We discuss the potential importance of the PLA2-signalling pathway in the mechanism of action of fibroblast growth factors.

Bradykinin stimulates the production of prostaglandin E2 and interleukin-6 in human osteoblast-like cells.

The effect of bradykinin (BK) on proteinase activity, prostaglandin synthesis, and the production of interleukin-6 (IL-6) was investigated in cultures of human osteoblast-like cells. Bradykinin had no effect on stromelysin activity and plasminogen activator activity produced by human osteoblast-like cells. However, BK stimulated the production of prostaglandin E2, an effect that was markedly enhanced by pre-incubation with recombinant interleukin-1 alpha (rhIL-1 alpha), but was apparently unaffected by BK receptor antagonists types 1 and 2. Bradykinin stimulated the intracellular accumulation of total inositol phosphates suggesting that its effects were mediated by stimulation of phosphoinositide metabolism. Bradykinin within the dose range of 10(-11)-10(-5) M also significantly stimulated the production of IL-6. Bradykinin may, therefore, mediate a variety of responses in bone under both physiological and pathological conditions.

Effect of the GABAA agonist muscimol on prolactin secretion from human prolactin-secreting adenomas and GH3 rat pituitary tumour cells.

The effect of muscimol, a specific potent GABAA receptor agonist, on prolactin release from human prolactin-secreting tissue was investigated using a perifusion system. Perifusion studies on normal rat anterior pituitary tissue, which has identical GABA receptors to those found in normal human pituitary glands, show that muscimol has a specific biphasic effect on prolactin release. This is characterized by an initial transient stimulation (222.3 +/- 21.6% of basal) lasting for 5-10 min followed by a more prolonged inhibitory phase (63.9 +/- 3.1% inhibition of basal). Five human prolactin-secreting adenomas were studied, and in none of the tumours could a biphasic response be demonstrated. One of the prolactin-secreting adenomas had a blunted inhibitory response, but the other 4 showed no inhibitory effect of muscimol on prolactin release. Muscimol had no significant effect on basal or thyrotropin-releasing-hormone (TRH)-stimulated prolactin secretion from GH3 rat pituitary tumour cells. These studies suggest that the GABAergic effect on prolactin secretion is absent or altered in both rat and human prolactin-secreting tumour cells.

The regulation of connective tissue metabolism by vasoactive intestinal polypeptide.

Immunohistochemical studies have confirmed the innervation of bone with neuropeptidergic neurons containing vasoactive intestinal polypeptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP). In this study, we report effects of VIP on connective tissue cell metabolism. VIP stimulated PGE2 production in human articular chondrocytes, human osteoblast-like cells and human synovial cells, however, stromelysin production was unaffected. VIP also stimulated cAMP production in human osteoblast-like cells, but not in human articular chondrocytes or synovial cells. These findings are suggestive of a role of VIP in connective tissue cell metabolism which may contribute to the inflammatory processes of arthritis.