Mirenas against malignancy: an alternative to operative management.

BACKGROUND: Complex atypical hyperplasia is a recognised precursor to endometrial cancer, the most common gynaecological cancer of the Western World, with hysterectomy being the first line of management. With the increasing elderly population size and BMI, optimal surgical management can be challenging in a sub-set of patients. Levonorgestrol releasing IUS system (Mirena coil) has been identified as a treatment for those unsuitable for surgical management. AIMS: To assess the efficacy of Mirena coils in the treatment of endometrial cancers and complex atypical hyperplasia in our patient population. METHOD: Retrospective analysis of all type 1 endometrial cancers and CAH diagnosed in Tayside from April 2011 to March 2016 (duration 5 years) managed by a Mirena coil. Primary outcome was resolution of malignancy on biopsy. Secondary outcome was continuation of treatment. RESULTS: Two hundred nineteen out of 245 eligible endometrial cancers and 38 of 41 CAH case notes were available for assessment. A Mirena coil was the primary mode of treatment in eight endometrial cancers (3.6%) and 18 cases of CAH (47.4%). Of the eight endometrial cancers treated, five (62%) continued with the Mirena coil as the primary mode of treatment. Two patients died during their follow-up due to unrelated illnesses and one patient proceeded to hysterectomy due to the absence of progestogenic effect on repeat Pipelle biopsy at six months (12.5%). Four patients have been followed for one year and of those; two showed no evidence of malignancy, one was unable to be biopsied and the last showed persistent disease but was too frail for intervention. At two-year follow-up, two patients remain with one showing disease recurrence. Thirteen patients (87%) have continued to use the Mirena as management of CAH out of 18 treatments. Of the five patients who did not continue with the IUS; two patients had progressive disease, despite the coil (11%), two patients lost enough weight to proceed to hysterectomy and one patient died unexpectedly. Medical co-morbidities and BMI were the main cited reasons for proceeding with the IUS as the primary treatment. Average BMI of endometrial cancer cases was 43.9 and 45.1 for CAH. CONCLUSIONS: Mirena coil is effective in treating or controlling the progression of endometrial malignancy in the subgroup of patients where there is an increased risk associated with definitive surgery. The treatment failure rate appears to be consistent at about one-tenth of the cohort of women with both endometrial cancers and complex atypical hyperplasia.

Caenorhabditis elegans RAC1/ced-10 mutants as a new animal model to study very early stages of Parkinson's disease.

Patients with Parkinson's disease (PD) display non-motor symptoms arising prior to the appearance of motor signs and before a clear diagnosis. Motor and non-motor symptoms correlate with progressive deposition of the protein alpha-synuclein (Asyn) both within and outside of the central nervous system, and its accumulation parallels neurodegeneration. The genome of Caenorhabditis elegans does not encode a homolog of Asyn, thus rendering this nematode an invaluable system with which to investigate PD-related mechanisms in the absence of interference from endogenous Asyn aggregation. CED-10 is the nematode homolog of human RAC1, a small GTPase needed to maintain the function and survival of dopaminergic neurons against human Asyn-induced toxicity in C. elegans. Here, we introduce C. elegans RAC1/ced-10 mutants as a predictive tool to investigate early PD symptoms before neurodegeneration occurs. Deep phenotyping of these animals reveals that, early in development, they displayed altered defecation cycles, GABAergic abnormalities and an increased oxidation index. Moreover, they exhibited altered lipid metabolism evidenced by the accumulation of lipid droplets. Lipidomic fingerprinting indicates that phosphatidylcholine and sphingomyelin, but not phosphatidylethanolamine or phosphatidylserine, were elevated in RAC1/ced-10 mutant nematodes. These collective characteristics reflect the non-motor dysfunction, GABAergic neurotransmission defects, upregulation of stress response mechanisms, and metabolic changes associated with early-onset PD. Thus, we put forward an easy-to-manipulate preclinical animal model to deepen our understanding of early-stage PD and accelerate the translational path for therapeutic target discovery.

An evaluation of a formative assessment process used on post take ward rounds.

The purpose of clinical training is to develop doctors capable of delivering professional, personal, effective, high quality, safe clinical care with Intelligent Kindness. The processes supporting training must promote development towards excellence. In 2004 a formative assessment process for use on medical post take ward rounds was introduced based on a model of a Driving Instructor and Learner Driver. This process has been evaluated in comparison with the Case based Discussion (CbD) and mini-Cex by 140 of 369 trainees, using online surveys. Ten trainees were interviewed in depth. The majority of trainees reported that this process had helped them more in their development as doctors than the CbD or mini-CEX. Trainees were able to describe positive effects in areas such as diagnosis, prescribing and confidence in their work. In the NHS the assessments are meant to be "trainee driven", however all but one of the trainees stated that they preferred the routine provision of an assessment to having to ask for an assessment. This evaluation of a truly formative assessment process shows that the trainees benefit in their progression towards clinical excellence. Effective formative feedback can be provided on an Acute Medical Unit even within the constraints of busy post take ward rounds. Within a team of Consultants one should be allowed time to develop an extended Clinical Supervisor role.

Clinically lean; "cutting the crap".

Proponents of Lean Philosophy believe that successful businesses must reduce waste in working time and resources to a minimum, and maximise their use in productive work. The productive work of the Acute Medical Unit is to provide effective clinical management to a daily cohort of acutely ill patients. Many Clinicians are cynical about Lean. In this article, Dr Caldwell discusses how many clinicians complain of too much crap in the workplace, which gets in the way of swift, safe high quality clinical care. He argues that "Cutting the Crap" in the Acute Medical Unit is entirely consistent with Lean approaches to management of complex systems.

The association between knee joint biomechanics and neuromuscular control and moderate knee osteoarthritis radiographic and pain severity.

OBJECTIVE: The objective of this study was to determine the association between biomechanical and neuromuscular factors of clinically diagnosed mild to moderate knee osteoarthritis (OA) with radiographic severity and pain severity separately. METHOD: Three-dimensional gait analysis and electromyography were performed on a group of 40 participants with clinically diagnosed mild to moderate medial knee OA. Associations between radiographic severity, defined using a visual analog radiographic score, and pain severity, defined with the pain subscale of the WOMAC osteoarthritis index, with knee joint kinematics and kinetics, electromyography patterns of periarticular knee muscles, BMI and gait speed were determined with correlation analyses. Multiple linear regression analyses of radiographic and pain severity were also explored. RESULTS: Statistically significant correlations between radiographic severity and the overall magnitude of the knee adduction moment during stance (r²=21.4%, P=0.003) and the magnitude of the knee flexion angle during the gait cycle (r²=11.4%, P=0.03) were found. Significant correlations between pain and gait speed (r²=28.2%, P<0.0001), the activation patterns of the lateral gastrocnemius (r²=16.6%, P=0.009) and the medial hamstring (r²=10.3%, P=0.04) during gait were found. The combination of the magnitude of the knee adduction moment during stance and BMI explained a significant portion of the variability in radiographic severity (R(2)=27.1%, P<0.0001). No multivariate model explained pain severity better than gait speed alone. CONCLUSIONS: This study suggests that some knee joint biomechanical variables are associated with structural knee OA severity measured from radiographs in clinically diagnosed mild to moderate levels of disease, but that pain severity is only reflected in gait speed and neuromuscular activation patterns. A combination of the knee adduction moment and BMI better explained structural knee OA severity than any individual factor alone.

Methylation profiling of rectal cancer identifies novel markers of early-stage disease.

BACKGROUND: Radical surgery is the de facto treatment for early rectal cancer. Conservative surgery with transanal endoscopic microsurgery can achieve high rates of cure but the histopathological measures of outcome used to select local treatment lack precision. Biomarkers associated with disease progression, particularly mesorectal nodal metastasis, are urgently required. The aim was to compare patterns of gene-specific hypermethylation in radically excised rectal cancers with histopathological stage. METHODS: Locus-specific hypermethylation of 24 tumour suppressor genes was measured in 105 rectal specimens (51 radically excised adenocarcinomas, 35 tissues adjacent to tumour and 19 normal controls) using the methylation-specific multiplex ligation-dependent probe assay (MS-MLPA). Methylation values were correlated with histopathological indices of disease progression and validated using bisulphite pyrosequencing. RESULTS: Five sites (ESR1, CDH13, CHFR, APC and RARB) were significantly hypermethylated in cancer compared with adjacent tissue and normal controls (P < 0·050). Methylation at these sites was higher in Dukes' A than Dukes' 'D' cancers (P = 0·013). Methylation at two sites (GSTP1 and RARB) was individually associated with localized disease (N0 and M0 respectively; P = 0·006 and P = 0·008). Hypermethylation of at least two of APC, RARB, TIMP3, CASP8 and GSTP1 was associated with early (N0 M0) disease (N0, P = 0·002; M0, P = 0·044). Methylation levels detected by MS-MLPA and pyrosequencing were concordant. CONCLUSION: Locus-specific hypermethylation was more prevalent in early- than late-stage disease. Hypermethylation of two or more of a panel of five tumour suppressor genes was associated with localized disease.

Wnt signalling in adenomas of familial adenomatous polyposis patients.

BACKGROUND: Epigenetic silencing of Wnt antagonists and expression changes in genes associated with Wnt response pathways occur in early sporadic colorectal tumourigenesis, indicating that tumour cells are more sensitive to Wnt growth factors and respond differently. In this study, we have investigated whether similar changes occur in key markers of the Wnt response pathways in the genetic form of the disease, familial adenomatous polyposis (FAP). METHODS: We investigated epigenetic and expression changes using pyrosequencing and real-time RT-PCR in samples from seven patients without neoplasia, and matched normal and tumour tissues from 22 sporadic adenoma and 14 FAP patients. RESULTS: We found that 17 out of 24 (71%) FAP adenomas were hypermethylated at sFRP1, compared with 20 out of 22 (91%) of sporadic cases. This was reflected at the level of sFRP1 transcription, where 73% of FAP and 100% of sporadic cases were down-regulated. Increased expression levels of c-myc and FZD3 were less common in FAP (35 and 46% respectively) than sporadic tumours (78 and 67% respectively). CONCLUSION: Overall, the changes in expression and methylation were comparable, although the degree of change was generally lower in the FAP adenomas. Molecular heterogeneity between multiple adenomas from individual FAP patients may reflect different developmental fates for these premalignant tumours.

Reorganisation of Wnt-response pathways in colorectal tumorigenesis.

In most colorectal tumours, APC mutation stabilises beta-catenin and mimics elements of Wnt growth factor signalling, but the high frequency of epigenetic loss of Wnt antagonists indicates an additional role for ligand-mediated Wnt signalling. Here, we have investigated the expression of key components of beta-catenin-independent Wnt response pathways to determine whether their profiles change during the transition from normal mucosa to colorectal adenomas. Transcription of the Wnt/planar cell polarity pathway determinant NKD1 (naked cuticle homologue 1) was induced in adenomas by a median 135-fold and in cancers by 7.4-fold. While some Frizzleds (FZDs) were downregulated in adenomas, the Wnt/Ca(2+) receptors FZD3 and FZD6 were induced by a median factor of 6.5 and 4.6, respectively. Naked cuticle homologue 1, FZD3 and FZD6 expression were coordinated in pre-malignant disease, but this relationship was lost in invasive cancers, where FZD induction was seen less frequently. Naked cuticle homologue 1 expression was associated with nuclear localisation of phospho-c-Jun in adenomas. In cultured cells, NKD1 transcription was induced by lithium chloride but FZD3 expression required Wnt growth factor treatment. These data show that Wnt responses are consistently directed towards both beta-catenin-independent routes in early colorectal tumorigenesis and elements of this are retained in more advanced cancers. These beta-catenin-independent Wnt signalling pathways may provide novel targets for chemoprevention of early colorectal tumours.

Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone.

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[S-farnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.

Ultrasonic characterization of cancellous bone using apparent integrated backscatter.

Apparent integrated backscatter (AIB) is a measure of the frequency-averaged (integrated) backscattered power contained in some portion of a backscattered ultrasonic signal. AIB has been used extensively to study soft tissues, but its usefulness as a tissue characterization technique for cancellous bone has not been demonstrated. To address this, we performed measurements on 17 specimens of cancellous bone over two different frequency ranges using a 1 MHz and 5 MHz broadband ultrasonic transducer. Specimens were obtained from bovine tibiae and prepared in the shape of cubes (15 mm side length) with faces oriented along transverse (anterior, posterior, medial and lateral) and longitudinal (superior and inferior) principal anatomic directions. A mechanical scanning system was used to acquire multiple backscatter signals from each direction for each cube. AIB demonstrated highly significant linear correlations with bone mineral density (BMD) for both the transverse (R2 = 0.817) and longitudinal (R2 = 0.488) directions using the 5 MHz transducer. In contrast, the correlations with density were much weaker for the 1 MHz transducer (R2 = 0.007 transverse, R2 = 0.228 longitudinal). In all cases where a significant correlation was observed, AIB was found to decrease with increasing BMD.

Metabolism and excretion of the antiepileptic/antimigraine drug, Topiramate in animals and humans.

The metabolism and excretion of 2,3:4,5-bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate (TOPAMAX, topiramate, TPM) have been investigated in animals and humans. Radiolabeled [14C] TPM was orally administered to mice, rats, rabbits, dogs and humans. Plasma, urine and fecal samples were collected and analyzed. TPM and a total of 12 metabolites were isolated and identified in these samples. Metabolites were formed by hydroxylation at the 7- or 8-methyl of an isopropylidene of TPM followed by rearrangement, hydroxylation at the 10-methyl of the other isopropylidene, hydrolysis at the 2,3-O-isopropylidene, hydrolysis at the 4,5-O-isopropylidene, cleavage at the sulfamate group, glucuronide conjugation and sulfate conjugation. A large percentage of unchanged TPM was recovered in animal and human urine. The most dominant metabolite of TPM in mice, male rats, rabbits and dogs appeared to be formed by the hydrolysis of the 2,3-O-isopropylidene group.

A bacterial metabolite induces glutathione-tractable proteostatic damage, proteasomal disturbances, and PINK1-dependent autophagy in C. elegans.

Gene-by-environment interactions are thought to underlie the majority of idiopathic cases of neurodegenerative disease. Recently, we reported that an environmental metabolite extracted from Streptomyces venezuelae increases ROS and damages mitochondria, leading to eventual neurodegeneration of C. elegans dopaminergic neurons. Here we link those data to idiopathic disease models that predict loss of protein handling as a component of disorder progression. We demonstrate that the bacterial metabolite leads to proteostatic disruption in multiple protein-misfolding models and has the potential to synergistically enhance the toxicity of aggregate-prone proteins. Genetically, this metabolite is epistatically regulated by loss-of-function to pink-1, the C. elegans PARK6 homolog responsible for mitochondrial maintenance and autophagy in other animal systems. In addition, the metabolite works through a genetic pathway analogous to loss-of-function in the ubiquitin proteasome system (UPS), which we find is also epistatically regulated by loss of PINK-1 homeostasis. To determine remitting counter agents, we investigated several established antioxidants and found that glutathione (GSH) can significantly protect against metabolite-induced proteostasis disruption. In addition, GSH protects against the toxicity of MG132 and can compensate for the combined loss of both pink-1 and the E3 ligase pdr-1, a Parkin homolog. In assessing the impact of this metabolite on mitochondrial maintenance, we observe that it causes fragmentation of mitochondria that is attenuated by GSH and an initial surge in PINK-1-dependent autophagy. These studies mechanistically advance our understanding of a putative environmental contributor to neurodegeneration and factors influencing in vivo neurotoxicity.

Relationship between pituitary and other target organ responsiveness in hypothyroid patients receiving thyroxine replacement.

This study was undertaken to compare the sensitivity of the thyrotrophs to that of other tissues to T4 treatment in hypothyroid patients. To do so, we measured serum total and free thyroid hormones and TSH, in addition to several serum markers of peripheral tissue response to thyroid status, in 21 hypothyroid patients treated with 50-micrograms increments of T4 to a maximum of 200 micrograms daily (group I) and in 104 clinically euthyroid patients receiving a long term constant replacement dose (group II). In group I patients, dose-dependent increases (P less than 0.05) in serum glutathione S-transferase, sex hormone-binding globulin, and angiotensin-converting enzyme occurred, whereas serum T4-binding globulin, creatine kinase, and creatinine levels decreased (P less than 0.05). In both patient groups, abnormally high levels of glutathione S-transferase, sex hormone-binding globulin, angiotensin-converting enzyme, alanine aminotransferase, and gamma-glutamyl transferase were found in some patients during treatment. One or more of these biochemical abnormalities suggestive of hyperthyroidism occurred in 15 (71%) group I patients and 27 (26%) group II patients. These were associated with an undetectable serum TSH (less than 0.1 microU/ml) and raised free T4 concentrations in 13, and raised free T3, T4, and T3 concentrations in only 8, 6, and 1 group I patients, respectively. In group II patients, they were more closely associated with an undetectable TSH (67%) or raised free T4 (85%) level than with raised concentrations of free T3 (33%), T4 (26%), or T3 (0%). The use of high sensitivity TSH assays will permit more accurate adjustment of T4 replacement and minimize abnormalities in peripheral tissue biochemistry indicative of overtreatment.

Metabolism profiling, and cytochrome P450 inhibition & induction in drug discovery.

To reduce the high attrition rates of NCEs in preclinical and clinical development uncovering pharmacokinetics, toxicokinetics, drug metabolism, and drug-drug interactions early in drug discovery would be highly valuable. There have been many in vitro screens developed for these areas that have higher sample throughput, which is consistent with the iterative cycle of a typical drug discovery research project. We have presented the present status and given detailed descriptions of biotransformation, metabolic stability assays, identification of drug metabolizing P450 enzymes, prediction of pharmacokinetic parameters from in vitro metabolism data, structure elucidation of metabolites, CYP450 inhibition assays and CYP450 induction assays from a drug discovery perspective. Strategies for the proper sequencing of primary and secondary assays employedfor drug metabolism and CYP450 inhibition & induction is discussed.

The new pre-preclinical paradigm: compound optimization in early and late phase drug discovery.

The attrition rates of new chemical entities (NCEs) in preclinical and clinical development are staggeringly high. NCEs are abandoned due to insufficient efficacy, safety issues, and economic reasons. Uncovering drug defects that produce these failures as early as possible in drug discovery would be highly effective in lowing the cost and time of developing therapeutically useful drugs. Unfortunately, there is no single factor that can account for these NCE failures in preclinical and clinical development since factors, such as solubility, pKa, absorption, metabolism, formulation, pharmacokinetics, toxicity and efficacy, to name a few, are all interrelated. In addition, there are many problems in scaling-up drug candidates from the laboratory bench scale to the pilot plant scale. To address the problem of attrition rates of NCEs in preclinical and clinical development and drug scale-up issues, pharmaceutical companies need to reorganize their preclinical departments from a traditional linear approach to a parallel approach. In this review, a strategy is put forth to integrate certain aspects of drug metabolism/pharmacokinetics, toxicology functions and process chemistry into drug discovery. Compound optimization in early and late phase drug discovery occurs by relating factors such as physicochemical properties, in vitro absorption, in vitro metabolism, in vivo pharmacokinetics and drug scale-up issues to efficacy optimization. This pre-preclinical paradigm will improve the success rate of drug candidates entering development.

The use of on-line and off-line chromatographic extraction techniques coupled with mass spectrometry for support of in vivo and in vitro assays in drug discovery.

We have investigated various sample chromatographic extraction and sample preparation methods for liquid chromatography mass spectrometry analysis in order to increase the throughput of various in vivo and in vitro assays in support of drug discovery. The results indicated that direct plasma injection, although certainly faster than conventional protein precipitation for sample preparation, had problems associated with column longevity and overall robustness. Frequently a single study could not be completed without column replacement. On-line solid phase extraction, on the other hand, compared well with off-line solid phase extraction, using our LC extraction column design, as contamination of the extraction column was minimized by back flushing using the Gilson syringe pump. Finally, on-line solid phase extraction for support of Caco-2 permeability studies worked very well for both single components and mixtures as the matrix was much simpler, presenting fewer contamination problems.

Retinal blood flow following a tyramine-induced increase in blood pressure.

The effect of increasing systemic blood pressure on retinal blood flow was investigated in anaesthetised miniature pigs. Blood pressure was increased by the infusion of the sympathomimetic amine, tyramine. Volume flow was determined from axial erythrocyte velocity, measured by laser Doppler velocimetry, and vessel diameter, measured from monochromatic retinal photographs. Measurements were taken when mean arterial pressures were elevated by a mean of 22 +/- 3% and 50 +/- 8% above resting values, which represented increases of 31 +/- 2% and 74 +/- 16% in ocular perfusion pressures. Retinal blood flow increased by 8.5 +/- 8% at the lower infusion rate and by 57 +/- 19% at the higher infusion rate. We conclude that tyramine infusion is a suitable method for the study of retinal autoregulation and that the upper limit of retinal autoregulation in miniature pigs lies between 22-50% above resting mean arterial pressure.

Protein structure determination using a combination of comparative modeling and NMR spectroscopy. Application to the response regulator protein, Spo0F.

A practical combination of comparative modeling and NMR spectroscopy was used to generate a three-dimensional structure of the response regulator protein, Spo0F. The backbone structure obtained compares to the Spo0F Y13S mutant X-ray structure with an rmsd of 2.0 A. We provide results which suggest that structures obtained by this method are suitable for drug discovery. The results of the GRID and DOCK methods as applied to the model and X-ray structures of Spo0F are remarkably similar and tend to suggest the same design conclusions. This trend is illustrated by these same techniques applied to two experimentally derived structures of the analogous protein, CheY, which exhibit a pairwise rmsdBB on the same order as that found for the two Spo0F structures.

Retinal blood flow during hyperglycemia. A laser Doppler velocimetry study.

The effect of different rates of glucose infusion on the retinal circulation was studied in Gottingen breed minipigs. Seven minipigs were made hyperglycemic rapidly with an intravenous bolus injection of 50% dextrose, after which a slow dextrose infusion maintained hyperglycemia for 60 minutes. Seven minipigs were more gradually made hyperglycemic over 60 minutes with a slow intravenous infusion of 50% dextrose, and a further seven had a control infusion of urea of equal volume and osmolality over 60 minutes. Retinal blood flow (RBF) was determined from the maximum (centerline) velocity of the blood (Vmax) (determined by bidirectional laser doppler velocimetry) and the vessel diameter (D) (determined from monochromatic fundus photographs). Measurements were made in a single temporal retinal vein of each animal at baseline, during, and after each of the infusions. Plasma glucose rose from 6.1 +/- 0.5-25.3 +/- 1.5 mM (mean +/- standard error) during the bolus infusion and from 6.4 +/- 0.7-22.0 +/- 0.7 mM during the slow infusion. The bolus and the slow glucose infusions both produced large increases in RBF (63% and 62%, respectively) which were mainly attributable to increases in Vmax. The urea infusion had no significant effect on RBF, Vmax, or D. The ocular perfusion pressure rose slowly and was significantly elevated after 60 minutes of slow glucose infusion but not after the urea infusion.

Intracellular mechanisms involved in the stimulation of growth hormone release from rat anterior pituitary cells by Russell's viper venom.

We have previously shown that a heat-stable component of Russell's viper venom (RVV) releases GH in a dose-dependent manner from cultured rat anterior pituitary cells. We have now investigated the intracellular mechanisms involved in RVV-stimulated GH release by concomitant administration of RVV with known intracellular mediators in rat pituitary cells. 3-Isobutyl-1-methylxanthine (IBMX; 0.5 mmol/l), added to cultured rat anterior pituitary cells simultaneously with RVV, at concentrations up to a maximally effective dose of 10 micrograms/ml, increased GH release (3.7-fold, 4.0-fold and 2.0-fold; P less than 0.001) compared with the effect of venom alone. These effects were additive, indicating that RVV and IBMX stimulate through different intracellular messengers. RVV failed to increase the formation of basal or IBMX-stimulated intracellular cyclic AMP (cAMP), confirming that RVV affects GH release through a cAMP-independent pathway. 12-0-Tetradecanoylphorbol-13-acetate (TPA; 0.1 mumol/l), added simultaneously with various doses of RVV (0.1-10 micrograms/ml), did not increase GH release beyond the maximal effect of RVV. This result indicates that RVV might be stimulating GH release through a similar mechanism to that of TPA (by activating protein kinase C). When pituitary cells were perifused with Ca(2+)-free medium or verapamil (50 mumol/l), RVV-stimulated GH release was inhibited by 65 and 42% respectively. This reflects the recognized requirement of Ca2+ for secretory processes. However, RVV (10 micrograms/ml) had no significant effect on intracellular free Ca2+ concentrations as measured using the fluorescent Ca2+ probe quin-2.(ABSTRACT TRUNCATED AT 250 WORDS)