Human single-donor composite skin substitutes based on collagen and polycaprolactone copolymer.

The development and characterization of an enhanced composite skin substitute based on collagen and poly(epsilon-caprolactone) are reported. Considering the features of excellent biocompatibility, easy-manipulated property and exempt from cross-linking related toxicity observed in the 1:20 biocomposites, skin substitutes were developed by seeding human single-donor keratinocytes and fibroblasts alone on both sides of the 1:20 biocomposite to allow for separation of two cell types and preserving cell signals transmission via micro-pores with a porosity of 28.8 +/- 16.1 microm. The bi-layered skin substitute exhibited both differentiated epidermis and fibrous dermis in vitro. Less Keratinocyte Growth Factor production was measured in the co-cultured skin model compared to fibroblast alone condition indicating a favorable microenvironment for epidermal homeostasis. Moreover, fast wound closure, epidermal differentiation, and abundant dermal collagen deposition were observed in composite skin in vivo. In summary, the beneficial characteristics of the new skin substitutes exploited the potential for pharmaceutical screening and clinical application.

Cholecystokinin (CCK) receptor and CCK gene expression in human pituitary adenomas and in vitro effects of CCK peptides on GH and gonadotrophin secretion.

There is growing evidence that cholecystokinin (CCK) affects growth and differentiation of anterior pituitary cells, via the CCK-B receptor. The possibility of an autocrine / paracrine role for CCK to modulate hormone secretion in human pituitary tumour cells is demonstrated here by RT-PCR and direct sequencing. In support of this conclusion, a neutralising antibody against the CCK peptide exhibited a dose dependent inhibition of hormone secretion by functionless pituitary adenomas. Total RNA was extracted from human pituitary adenomas, reverse transcribed into cDNA and subjected to PCR using primers specific for the gene for CCK, CCK-A and CCK-B receptors. PCR bands of the predicted length were observed in all tumours using human CCK gene and CCK-B receptor primers. Restriction digestion and direct sequence analysis provided further evidence that they represented both the human CCK peptide along with the CCK-A and/B receptor mRNA. CCK-33 and CCK octapeptide sulphate (CCK-8s) both powerfully stimulated phosphatidylinositol hydrolysis, providing evidence for functional activity of the CCK-A and/B receptors. A direct stimulatory effect of CCK peptides on both LH and FSH secretion is reported for the first time, whereas stimulatory effects on GH were blocked by antagonists to CCK. These results may indicate an autocrine role for CCK in the functioning and perhaps development of human pituitary tumours.

Analysis of secretory, immunostaining and clinical characteristics of human "functionless" pituitary adenomas: transdifferentiation or gonadotropinomas?

In this study, the central technique of in vitro culture has been used to further investigate whether LH/FSH-expressing, but clinically "functionless" pituitary adenomas are gonadotropinomas or whether their hormone secretion is due to transdifferentiation events. 664 "functionless" pituitary adenomas were examined for hormone secretion by in vitro culture and for hormone content by immunostaining. The results were correlated with the clinical findings. 40 % of the tumours (n = 263) secreted at least one of the gonadotropins alone, 8 % (n = 53) exhibited various patterns of anterior pituitary hormones, whilst the remaining 52 % of tumours were not associated with any hormone. In the secretory tumours, immunostaining revealed only a few scattered hormone-containing cells (5 to 15 %). Mild hyperprolactinaemia was observed in some cases, presumably because of pressure effects of the tumours. The majority of the patients suffered clear cut hypopituitarism (p < 0.05). Pre-operatively, gonadotropin hypersecretion was observed in 3 cases, but only one of these secreted hormones in culture. Interestingly, a higher proportion of tumours removed from patients with hypopituitarism showed secretory activity in vitro than those tumours removed from patients showing no hormonal dysfunction or hyperprolactinaemia. We conclude that the term "gonadotropinoma" to describe functionless pituitary tumours associated with LH and/or FSH secretion is a misnomer, because the presence of LH and/or FSH confirmed by in vitro methods in the present series is a result of only a few scattered cells. We suggest that primary pituitary tumour cells differentiate into a secretory type (transdifferentiation), possibly in response to altered serum hormone levels such as decreased steroids. Further work is required to identify the factors which trigger the altered cells' characteristics.

A co-cultured skin model based on cell support membranes.

Tissue engineering of skin based on collagen:PCL biocomposites using a designed co-culture system is reported. The collagen:PCL biocomposites having collagen:PCL (w/w) ratios of 1:4, 1:8, and 1:20 have been proven to be biocompatible materials to support both adult normal human epidermal Keratinocyte (NHEK) and mouse 3T3 fibroblast growth in cell culture, respectively, by Dai, Coombes, et al. in 2004. Films of collagen:PCL biocomposites were prepared using non-crosslinking method by impregnation of lyophilized collagen mats with PCL/dichloromethane solutions followed by solvent evaporation. To mimic the dermal/epidermal structure of skin, the 1:20 collagen:PCL biocomposites were selected for a feasibility study of a designed co-culture technique that would subsequently be used for preparing fibroblast/biocomposite/keratinocyte skin models. A 55.3% increase in cell number was measured in the designed co-culture system when fibroblasts were seeded on both sides of a biocomposite film compared with cell culture on one surface of the biocomposite in the feasibility study. The co-culture of human keratinocytes and 3T3 fibroblasts on each side of the membrane was therefore studied using the same co-culture system by growing keratinocytes on the top surface of membrane for 3 days and 3T3 fibroblasts underneath the membrane for 6 days. Scanning electron microscopy (SEM) and immunohistochemistry assay revealed good cell attachment and proliferation of both human keratinocytes and 3T3 fibroblasts with these two types of cells isolated well on each side of the membrane. Using a modified co-culture technique, a co-cultured skin model presenting a confluent epidermal sheet on one side of the biocomposite film and fibroblasts populated on the other side of the film was developed successfully in co-culture system for 28 days under investigations by SEM and immunohistochemistry assay. Thus, the design of a co-culture system based on 1:20 (w/w) collagen:PCL biocomposite membranes for preparation of a bi-layered skin model with differentiated epidermal sheet was proven in principle. The approach to skin modeling reported here may find application in tissue engineering and screening of new pharmaceuticals.

Composite cell support membranes based on collagen and polycaprolactone for tissue engineering of skin.

The preparation and characterisation of collagen:PCL composites for manufacture of tissue engineered skin substitutes and models are reported. Films having collagen:PCL (w/w) ratios of 1:4, 1:8 and 1:20 were prepared by impregnation of lyophilised collagen mats by PCL solutions followed by solvent evaporation. In vitro assays of collagen release and residual collagen content revealed an expected inverse relationship between the collagen release rate and the content of synthetic polymer in the composite that may be exploited for controlled presentation and release of biopharmaceuticals such as growth factors. DSC analysis revealed the characteristic melting point of PCL at around 60 degrees C and a tendency for the collagen component, at high loading, to impede crystallinity development within the PCL phase. The preparation of fibroblast/composite constructs was investigated using cell culture as a first stage in mimicking the dermal/epidermal structure of skin. Fibroblasts were found to attach and proliferate on all the composites investigated reaching a maximum of 2 x 10(5)/cm(2) on 1:20 collagen:PCL materials at day 8 with cell numbers declining thereafter. Keratinocyte growth rates were similar on all types of collagen:PCL materials investigated reaching a maximum of 6.6 x 10(4)/cm(2) at day 6. The results revealed that composite films of collagen and PCL are favourable substrates for growth of fibroblasts and keratinocytes and may find utility for skin repair.

Clonal composition of human adamantinomatous craniopharyngiomas and somatic mutation analyses of the patched (PTCH), Gsalpha and Gi2alpha genes.

Craniopharyngioma is the most common childhood tumor and thought to arise from embryonic remnants of Rathke's pouch. The paucity of published data on the molecular basis of these tumors prompted us to examine 22 adamantinomatous craniopharyngiomas looking for genetic abnormalities. Using the X-linked polymorphic androgen receptor gene as a tool for X-chromosome inactivating analysis, we found that a subset of craniopharyngiomas are monoclonal and therefore are probably due to acquired somatic genetic defects. Thus, we investigated these tumours for mutations within three candidate genes, Gsalpha, Gi2alpha and patched (PTCH). Using single stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis and direct sequencing, the presence of somatic mutations in these genes could not be demonstrated in any tumor. Our data indicate that a subset of craniopharyngiomas are monoclonal and the mutations in the PTCH, Gsalpha, and Gi2alpha contribute little if any to craniopharyngioma development.

The expression of the growth hormone secretagogue receptor ligand ghrelin in normal and abnormal human pituitary and other neuroendocrine tumors.

Ghrelin is a recently identified endogenous ligand of the GH secretagogue (GHS) receptor. It was originally isolated from the stomach, but has also been shown to be present in the rat hypothalamus. It is a 28-amino acid peptide with an unusual octanoylated serine 3 at the N-terminal end of the molecule, which is crucial for its biological activity. Synthetic GHSs stimulate GH release via both the hypothalamus and the pituitary, and the GHS receptor (GHS-R) has been shown by us and others to be present in the pituitary. We investigated whether ghrelin messenger ribonucleic acid (mRNA) and peptide are present in the normal human hypothalamus and in normal and adenomatous human pituitary. RNA was extracted from pituitary tissue removed at autopsy and transsphenoidal surgery (n = 62), and ghrelin and GHS-R type 1a and 1b mRNA levels were investigated using real-time RT-PCR. Both ghrelin and GHS-R mRNA were detected in all samples. Corticotroph tumors showed significantly less expression of ghrelin mRNA, whereas GHS-R mRNA levels were similar to those in normal pituitary tissue. Gonadotroph tumors showed a particularly low level of expression of GHS-R mRNA. Immunohistochemistry, using a polyclonal antibody against the C-terminal end of the ghrelin molecule, revealed positive staining in the homolog of the arcuate nucleus in the human hypothalamus and in both normal and abnormal human pituitary. Pituitary tumor ghrelin peptide content was demonstrated using two separate RIA reactions for the N-terminal and C-terminal ends of the molecule. Both forms were present in normal and abnormal pituitaries, with 5 +/- 2.5% octanoylated (active) ghrelin (mean +/- SD) present as a percentage of the total. We suggest that the presence of ghrelin mRNA and peptide in the pituitary implies that the locally synthesized hormone may have an autocrine/paracrine modulatory effect on pituitary hormone release.

A polymorphism in the growth hormone (GH)-releasing hormone (GHRH) receptor gene is associated with elevated response to GHRH by human pituitary somatotrophinomas in vitro.

A previous study has suggested that a G to A base change at position 169 of the GHRH-receptor gene in human somatotrophinomas is a mutation and confers hypersensitivity to GHRH. The alternative base converts codon 57 from GCG to AGC, resulting in replacement of alanine (Ala) with threonine (Thr). In the present study, two of five human GH-secreting somatotrophinomas were found to possess the codon 57 AGC sequence. The GCG allele was also detected, indicating heterozygosity. However, the patients' normal blood-derived DNA also yielded the same sequence pattern, indicating that the Ala --> Thr amino acid change is a normal polymorphism, and not a somatic mutation. Nevertheless, in vitro, the tumors possessing the Ala --> Thr amino acid change responded very strongly to GHRH in terms of cAMP formation, being increased 40- and 200-fold, in comparison to the 2-fold increases by tumors without the alternative GHRH-receptor sequence. Likewise, the in vitro response of GH secretion to GHRH was elevated. One of the two tumors with the alternative Thr residue, and the highest responder to GHRH, possessed a gsp mutation, despite the fact that these defects are thought to reduce responsiveness to GHRH. These results fail to confirm that the GCG --> AGC at codon 57 of the GHRH-receptor gene is a mutation, but do support the concept that the alternative form with Thr confers increased sensitivity to GHRH.

Interaction of the novel GH secretagogue hexarelin with GHRH in regulating the secretion of GH by cultured human pituitary somatotrophinomas in vitro.

The effects of the novel GH-releasing hexapeptide, Hexarelin, on the secretion of GH in cultured human pituitary somatotrophinomas was further investigated. Hexarelin (20 nmol/L) strongly stimulated GH secretion, which could be reduced by phloretin, but not by RP-cAMPS, an inhibitor of protein kinase A (PKA). (Ac-Tyr1,D-Arg2)-GRF(1-29)-NH2 failed to block the effects of Hexarelin but completely abolished the stimulation of GH secretion exerted by GHRH. When added alone to somatotrophinoma cell cultures, Hexarelin had no effect on cAMP levels, but it potentiated the stimulatory effects of GHRH. These results demonstrated that Hexarelin could directly stimulate GH secretion by human pituitary somatotrophs PKC-dependently, which might be contributed to the activation of the PI transduction system. In addition, Hexarelin could interact with GHRH on the adenylyl cyclase system.

Clinical correlates in acromegalic patients with pituitary tumors expressing GSP oncogenes.

We herein review published findings on the clinical characteristics of acromegalic patients harboring pituitary somatotrophinomas expressing adenylyl cyclase activating gsp mutations and present an update of our own data on a large series of 176 patients with and without these oncogenes. Gsp oncogenes are the result of point mutations in either codon 201 or 227 of the Gs-alpha subunit of the Gs-protein which controls adenylyl cyclase. They result ultimately in increased intracellular cAMP levels and thus in excessive growth hormone (GH) secretion. Our large series has allowed us to characterise patients with mutations in codon 201 and the far rarer group possessing codon 227 defects. Both groups were compared with patients without gsp oncogenes. In accordance with previous findings, there was no statistically significant difference in age of the patients belonging to each group, the overall average tumor diameter nor in pre-operative serum GH levels, although the latter showed a tendency to be lower in patients with gsp oncogenes. The distribution of different types of response during an oral glucose tolerance test (no change, paradoxical rise or greater than 50% decrease in serum GH levels) did not differ between the 3 groups. However, the incidence of microadenomas was higher in acromegalics expressing gsp oncogenes in patients possessing mutations in codon 227. Additionally, the incidence of invasiveness was much lower (10% v. 33%) in those tumors with mutations in codon 227. Finally, previous in-vitro data indicating that gsp oncogene-expressing tumors may respond more efficiently to the somatostatin analogue, octreotide, have been confirmed by subsequent in-vivo studies showing a better reduction in serum GH levels in patients with gsp oncogenes. These latter findings suggest that presence of gsp oncogenes may be a marker for good reponsiveness to octreotide. Assessment of gsp oncogene status of surgically removed pituitary somatotrophinomas may thus be helpful in designing optimal medical therapies in those acromegalics requiring further post-operative management of the disease.

Expression and growth stimulatory effect of fibroblast growth factor 9 in human brain tumors.

OBJECTIVE: Fibroblast growth factor 9 (FGF-9) is a relatively new member of the FGF family isolated from the conditioned medium of a human glioblastoma cell line as a secreting type factor that exhibits a growth-stimulating effect on primary glial cells. To elucidate the roles of FGF-9 in human brain tumors, the expression and biological activities of FGF-9 were studied using culture cells and surgically obtained tumor specimens. METHODS: Measurement of FGF-9 and basic FGF in conditioned media of cell cultures was performed by using a sandwich enzyme immunoassay. The mitogenic effect of FGF-9 was evaluated by cell growth studies. FGF-9 expression in vivo was demonstrated by immunohistochemistry. RESULTS: One of 4 glioma cell lines and 4 of 16 human meningiomas examined actually secreted detectable amounts of FGF-9 proteins. In comparison, basic FGF production was detected from 3 of 4 glioma cell lines and 11 of 16 human meningiomas. Similarly to basic FGF, recombinant human FGF-9 significantly stimulated the in vitro cell proliferation in three of four glioma cell lines investigated in a dose-dependent manner. A time course growth study using U87 MG cells revealed an accelerated growth stimulation by FGF-9 after Day 4. The growth stimulatory activity was also shown in three of four human meningiomas studied. Moderate to strong immunoreactivity for FGF-9 was observed in 40 (82%) of 49 human brain tumors examined irrespective of origin, tumor type, grade of malignancy, or whether initial or recurrent. In contrast, strong immunostaining was localized in neurons in the normal human cerebral cortex. CONCLUSION: The present findings suggest that FGF-9 may be involved in the biology of human brain tumors with a possible importance in tumor cell growth. Whether the growth factor is more generally involved in oncogenesis of human tumors awaits further investigation.

Comparative genomic hybridization analysis of nonfunctioning pituitary tumors.

Clinically nonfunctioning pituitary adenomas constitute about one third of pituitary neoplasms and are considered monoclonal tumors. The molecular mechanisms of tumorigenesis in these neoplasms are poorly understood, as evidenced by the paucity of reported somatic genetic alterations. Furthermore, the somatic mutations detected to date were primarily ascribed to candidate genes or chromosomal regions: gsp, ras, p53 mutations, and allelic losses of 11q and 13q. To gain insight into which chromosomal regions bear genes involved in nonfunctioning pituitary tumorigenesis, we examined 23 such tumors by comparative genomic hybridization. Four tumors showed no genetic abnormality, and the rest (17 of 23, 74%) exhibited at least one chromosomal region of abnormality. Gains and losses affected all chromosomes (except for chromosome 14). Notably, 8 of 23 tumors (34.7%) displayed sex chromosome and chromosome 18 aberrations (amplifications or deletions). Nonrandom DNA amplification of sub-chromosomal regions on 4q, 5q (5q13-->5q23), 9p (9p21-->9pter), 13q (13q21-->13q32), and 17q were detected in 10-30% of the tumors. Noteworthy, no tumor displayed deletion of 11q, the MEN1 gene locus. These findings suggest that genes localized to previously undescribed chromosomal regions play a role in the tumorigenesis of nonfunctioning pituitary adenomas.

Presence of growth hormone secretagogue receptor messenger ribonucleic acid in human pituitary tumors and rat GH3 cells.

A novel G11-protein-coupled receptor specific for synthetic GH-releasing peptides (GHRPs) has recently been cloned and sequenced. Two forms exist, types 1a and 1b, the latter of which is biologically inactive. Using RT-PCR, we looked for the presence in tumorous pituitary cells of messenger ribonucleic acid (mRNA) for this novel GH secretagogue receptor (GHS-R). Both subtypes of GHS-R mRNA were detected in all six human pituitary somatotropinomas removed from patients with acromegaly. In culture, four of the tumors exhibited strong responses to GHRP-2 in terms of both phosphatidylinositol (PI) hydrolysis and GH secretion, but two were resistant. There was no apparent difference in the type 1a and type 1b expression pattern, as judged by RT-PCR, between responsive and nonresponsive tumors. Similarly, the rat pituitary tumor cell line, GH3, was found to express GHS-R mRNA, although these cells also did not respond to GHRPs. RT-PCR failed to detect GHS-R mRNA in eight functionless human pituitary tumors. In contrast, prolactinomas were found to express the receptor and, in culture, significant stimulation of PRL secretion and PI hydrolysis occurred in two of three tumors tested. These results demonstrate that tumorous somatotrophs express the GHS-R gene and that the occasionally observed nonresponsiveness of somatotropinomas to GHRPs is not due to the absence of the biologically active type 1a receptor. Additionally, human pituitary prolactinomas also express GHS-R and are able to respond to GHRPs in terms of PI hydrolysis and PRL secretion. In contrast, GHS-R gene expression does not appear to be associated with human functionless pituitary tumors.

Presence of GHRH mRNA in human pituitary somatotrophinomas and its relationship to in vitro effect of a GHRH-antagonist on GH secretion and cAMP production.

Several earlier studies have shown that some human pituitary GH-secreting somatotrophinomas are able to synthesise and release hypothalamic GHRH and it has been proposed that a positive autocrine feedback loop involving this tumor-derived GHRH may participate in tumorigenesis. We have used in-vitro cell culture and exploited an antagonist to GHRH, (Ac-Tyr1,D-Arg2)-GHRH (1-29)-amide (GHRH-A), to further investigate whether an autocrine loop involving somatotrophinoma-derived GHRH may exist. In situ hybridization demonstrated presence of GHRH transcripts in 5 of 9 human somatotrophinomas. In culture, GHRH-A failed to inhibit basal release of GH or production of cAMP irrespective of presence or absence of GHRH transcripts. However, GHRH-A was able to completely or partially abolish the stimulatory effects of exogenously added GHRH peptide. Additionally, the average stimulatory effect of exogenous GHRH on in vitro GH secretion by somatotrophinomas possessing GHRH mRNA was identical to that shown by tumors not expressing the GHRH gene. Whilst confirming that many human pituitary somatotrophinomas are able to express the GHRH gene, the failure of GHRH-A to inhibit basal GH secretion argues against the concept of the existence of an autocrine stimulatory loop involving secreted GHRH peptide.

Overexpression of stimulatory G protein alpha-subunit is a hallmark of most human somatotrophic pituitary tumours and is associated with resistance to GH-releasing hormone.

The heterotrimeric Gs protein-adenylyl cyclase (AC) cascade plays a pivotal role in controlling hormone secretion by endocrine glands. Consequently, deficiency of the alpha-subunit of Gs leads to endocrine hypofunction and hypoplasia in the affected cells whereas AC hyperactivity results from activating point mutations within the Gs-alpha gene. The latter, termed gsp oncogenes, are found primarily in a subset of growth hormone (GH)-secreting human pituitary tumours (somatotrophinomas) and are thus associated with excessive GH secretion. We present here evidence that another type of defect in human somatotrophinomas may be overexpression of the Gs-alpha subunit. Immunohistochemistry using an antibody against recombinant human Gs-alpha revealed high levels of expression in 25 of 39 somatotrophinomas but weak staining in normal human pituitary cells. These results were confirmed by Western blot analysis. Additionally, cholera toxin-mediated ADP-ribosylation in the presence of 32P-labelled NAD+ resulted in an autoradiographic signal intensity which correlated directly with magnitude of immunostaining and amount of antigen shown by Western blot analysis, providing evidence for overexpression of functionally active subunit. Finally, reconstitution assays were applied and directly demonstrated the increased activity of overexpressed Gs-alpha. In vivo, the effect of Gs-alpha on AC activity may be partially counterregulated by high levels of inhibitory G protein that also occurred in these tumours. In culture, GH-releasing hormone (GHRH) had markedly reduced effects on GH secretion by somatotrophinomas exhibiting Gs-alpha overexpression, whereas powerful stimulation occurred in weakly staining tumours. In contrast to these observations with Gs-alpha, immunostaining for the phospholipase C-coupled G11-alpha subunit was relatively weak in all somatotrophinomas studied and synthetic GH-releasing peptide, which acts via a specific G11-coupled receptor, led to powerful and consistent stimulation of GH secretion by different tumours. These results indicate that Gs-alpha overexpression is associated with dysfunction in hormone secretion by some somatotrophinomas.

Progesterone receptor gene expression in craniopharyngiomas and evidence for biological activity.

OBJECTIVE: Previous studies have demonstrated the presence of estrogen receptors in human craniopharyngiomas, raising the possibility that these lesions can be influenced by steroids. To complement these earlier findings, we examined for the presence of progesterone receptor (PR) messenger RNA in surgically removed craniopharyngiomas and performed some studies to determine whether progestogens can exert biological effects on these tumors in vitro. METHODS: Total RNA was extracted from fresh surgically removed craniopharyngiomas and reverse-transcribed into cDNA. The polymerase chain reaction was applied to this craniopharyngioma-derived cDNA using amplimers complementary to exons 4 and 7 of the PR gene. Additionally, craniopharyngioma cell cultures were established, and the in vitro effects of progesterone and 6 alpha-methyl-17 alpha-hydroxyprogesterone acetate on [3H]thymidine uptake and 17 beta-estradiol oxidoreductase activity were determined. RESULTS: Reversed-transcribed polymerase chain reaction of craniopharyngioma-derived RNA yielded bands of predicted size (389 base pairs) in six of seven tumors studied. Hinfl digestion and direct sequencing of the bands confirmed that the polymerase chain reaction DNA was representative of PR messenger RNA. Treatment of craniopharyngioma cell cultures with progesterone resulted in reduced [3H]thymidine uptake. Both progesterone and 6 alpha-methyl-17 alpha-hydroxyprogesterone acetate powerfully increased oxidative 17 beta-estradiol oxidoreductase activity. CONCLUSION: These results provide evidence that PR messenger RNA can be produced by at least some human craniopharyngiomas and indirectly show that this is translated into biologically active receptor protein.

Human meningiomas possess muscarinic acetylcholine receptors: stimulation of phosphatidylinositol turnover by carbachol.

The effect of carbachol, an acetylcholine receptor agonist, on rate of phosphatidylinositol (PI) turnover in cultured human meningioma cells was investigated. Exposure of meningioma cells for 2 h to carbachol (3.12-200 mumol/L) resulted in a dose-dependent stimulation of PI turnover to a maximum of 5.5-fold over basal controls. A time course study showed stimulation of IP3 formation after 30 s followed by increases in IP1 and IP2. The stimulatory effect of carbachol on PI turnover was completely abolished by the muscarinic receptor antagonist, atropine, but was unaltered by the nicotinic antagonist, hexamethonium. Reverse-transcription of meningioma-derived RNA into cDNA followed by amplification by the polymerase chain reaction using specific primers revealed presence of ml type muscarinic receptor mRNA. These results provide evidence that human meningioma cells possess muscarinic acetylcholine receptors the activation of which leads to PI hydrolysis.

Relationship between invasiveness of pituitary somatotrophinomas and structural abnormalities of protein kinase C gene in human.

The potential role of the protein kinase C (PKC) transduction system in controlling proliferation of human pituitary somatotrophinomas was investigated. Twenty somatotrophinomas were studied using PCR and direct sequencing methods. No point mutation within the alpha PKC gene, previously thought to be associated with invasive pituitary tumors, was found in any of the 20 somatotrophinomas. It is concluded that PKC transduction system may play an important role in controlling pituitary somatotrophinoma proliferation, but there is no correlation between invasiveness and the previously reported alpha PKC gene mutation.

Relationship between GHRP-6 and TPA in the regulation of growth hormone secretion by human pituitary somatotrophinomas.

Growth hormone releasing peptide (GHRP-6) is a synthetic hexapeptide which specifically stimulates secretion of growth hormone (GH) by pituitary somatotrophs. Phorbel ester, 1, 2 tetradecanoylphorbol 13 acetate (TPA) can also stimulate releasing of GH. The precise intracellular mechanism has not been entirely deciphered. We used cell cultures of human pituitary somatotrophinomas to investigate the relation between GHRP-6 and TPA on membrane phosphatidylinositol (PI) turnover and GH secretion. The results showed that the working mechanisms of GHRP-6 and TPA are not identical, although they all can stimulate GH secretion in human pituitary somatotrophinomas. This indicates that PI-PKC signal transduction system may play a crucial role in the regulation of GH secretion.

Relationship between protein kinase C and adenylyl cyclase activity in the regulation of growth hormone secretion by human pituitary somatotrophinomas.

OBJECTIVE: To determine the potential role of protein kinase C (PKC) and its relationship to adenylyl cyclase activity in controlling growth hormone (GH) secretion by human pituitary somatotrophinomas. METHODS: Twenty-eight somatotrophinomas were placed into cell culture, and the in vitro effects of the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) and the PKC inhibitor staurosporine on basal and GH-releasing hormone (GHRH)-stimulated GH secretion were examined. In addition, the influence of chronic exposure of cultured somatotrophinoma cells to TPA on the rate of inositol 1,4,5-trisphosphate production was determined. Each tumor was assessed for the presence of gsp oncogenes, and thus constitutive adenylyl cyclase activity, by direct sequence analysis of polymerase chain reaction-generated deoxyribonucleic acid. GH secretory responses of tumors with and without these oncogenes were compared. RESULTS: TPA consistently stimulated GH secretion by cultured somatotrophinoma cells. There was no difference in response between somatotrophinomas with and without gsp oncogenes, and the effects did not correlate with the variable stimulation exerted by GHRH. Tumors in which GHRH had no significant effect nevertheless responded to TPA. In combination, TPA and GHRH exerted additive stimulation. TPA treatment of cultured somatotrophinoma cells eventually resulted in suppression of inositol 1,4,5-trisphosphate production, probably reflecting down-regulation of membrane phosphatidylinositol hydrolysis, a second messenger system that also generates the endogenous PKC activator diacylglycerol. GHRH had no effect on phosphatidylinositol hydrolysis. In contrast to the effects of TPA, the PKC inhibitor staurosporine tended to reduce GH secretion, although this effect was not observed in all tumors examined. As with TPA, the effects of staurosporine did not correlate with presence or absence of gsp oncogenes. Furthermore, staurosporine did not reduce the stimulatory effects exerted by GHRH on GH secretion. CONCLUSION: These results demonstrate a role for the phosphatidylinositol-PKC second messenger cascade in controlling GH secretion by human pituitary somatotrophinomas. The results also show that the system operates relatively independent of intracellular adenylyl cyclase and, thus, protein kinase A.