BRCA1 Attenuates Progesterone Effects on Proliferation and NFκB Activation in Normal Human Mammary Epithelial Cells.

Germline mutations in the breast cancer susceptibility gene BRCA1, encoding a tumor suppressor protein, greatly enhance the risk of breast and ovarian cancer. This tissue-specificity implicates the role of ovarian hormones. Indeed, BRCA1 has been demonstrated to regulate the signalling axis of the hormone, progesterone, and its receptor, the progesterone receptor (PR), and progesterone action has been implicated in BRCA1-related tumorigenesis. BRCA1 also plays important roles in oxidative stress and activating nuclear factor kappaB (NFκB) signalling pathways. Like wildtype BRCA1 function, PR signalling has also been shown to inhibit NFκB activation. Although PR and BRCA1 networks are known to interact, their interaction at the level of NFκB activation in the human breast is not understood. This study investigates the effect of reduced BRCA1 expression on proliferation and NFκB activation in human breast cells, and the impact of progesterone on these effects. The major findings are that: 1) Reduced BRCA1 levels inhibit cell growth in normal human mammary cells and breast cancer cells; 2) Reduced BRCA1 levels stimulated inflammatory targets and NFκB activity in normal human mammary cells; 3) Wildtype BRCA1 inhibited the pro-proliferative effects of progesterone in normal mammary epithelial cells, and; 4) Progesterone attenuated BRCA1-mediated NFκB activation in normal human mammary cells. These data have important implications for our understanding of progesterone action in BRCA1 mutation carriers, and how inhibition of this action may potentially delay tumorigenesis or impart a more favourable prognosis.

Progesterone stimulates progenitor cells in normal human breast and breast cancer cells.

The epithelium of the human breast is made up of a branching ductal-lobular system, which is lined by a single layer of luminal cells surrounded by a contractile basal cell layer. The co-ordinated development of stem/progenitor cells into these luminal and basal cells is fundamentally important for breast morphogenesis. The ovarian steroid hormones, progesterone (P) and 17ß-estradiol, are critical in driving this normal breast development, yet ovarian activity has also been shown to be a major driver of breast cancer risk. We previously demonstrated that P treatment increases proliferation and augments the number of progenitor-like cells, and that the progesterone receptor (PR) is also expressed in the bipotent progenitor-enriched subfraction. Here we demonstrate that PR is expressed in a subset of CD10+ basal cells and that P stimulates this CD10+ cell compartment, which is enriched for bipotent progenitor activity. In addition, we have shown that P stimulates progenitor cells in human breast cancer cell lines and expands the cancer stem cell population via increasing the stem-like CD44+ population. As changes in cell type composition are one of the hallmark features of breast cancer progression, the demonstration that progenitor cells are stimulated by P in both normal breast and in breast cancer cells has critical implications in discerning the mechanisms of how P increases breast cancer risk.

Alanate anion, AlH4(-): photoelectron spectrum and computations.

The alanate anion, AlH4(-), was generated in the gas phase using a pulsed arc cluster ionization source. Its photoelectron spectrum was then measured with 193 nm photons. The spectrum consists of a broad feature, spanning electron binding energies from 3.8 eV to over 5.3 eV. This band reflects the photodetachment transitions between the ground state of the AlH4(-) anion and the ground state of its thermodynamically unstable neutral counterpart, AlH4. The vertical detachment energy (VDE) of AlH4(-) was measured to be 4.4 eV. Additionally, VDE values were also computed in a comprehensive theoretical study and compared both with the previously computed value and with our experimentally determined value.

Tamoxifen treatment promotes phosphorylation of the adhesion molecules, p130Cas/BCAR1, FAK and Src, via an adhesion-dependent pathway.

Reports that the adhesion-associated molecule p130Cas/BCAR1 promotes resistance to tamoxifen suggested that adhesion-mediated signalling may be altered by tamoxifen treatment. We find that p130Cas/BCAR1 phosphorylation is enhanced in tamoxifen-treated estrogen receptor (ER)-positive MCF-7 breast cancer cells. The effects of estrogen and tamoxifen were assessed independently and in combination, and the results demonstrate that tamoxifen antagonizes estrogen regulation of p130Cas/BCAR1 phosphorylation. Phosphorylation correlates with tamoxifen ER antagonist effects, as phosphorylation effects are replicated by the pure antiestrogen ICI 182, 780. Correspondingly, phosphorylation is not changed in ER-negative cells exposed to tamoxifen. We show that deletion of the p130Cas/BCAR1 substrate domain substantially reduces tamoxifen-induced phosphorylation of p130Cas/BCAR1 and confers enhanced sensitivity to tamoxifen. P130Cas/BCAR1 forms a phosphorylation-dependent signalling complex with focal adhesion kinase (FAK) and Src kinase that promotes adhesion-mediated cell survival. Therefore, we examined the kinetics of p130Cas/BCAR1, Src and FAK phosphorylation over a 14-day time course and find sustained phosphorylation of these molecules after 7 days exposure to tamoxifen. Inhibition of Src kinase is shown to reduce tamoxifen-promoted p130Cas/BCAR1 phosphorylation and reduce cell viability. Stimulation of the Src/FAK/p130Cas/BCAR1 adhesion signalling pathway in tamoxifen-treated MCF-7 cells does not cause increased migration; however, there is Src-dependent phosphorylation of the cell survival molecule Akt. Correspondingly, Akt inhibition reduces cell viability in cells treated with tamoxifen. We propose that prolonged activation of adhesion-dependent signalling may confer a survival advantage in response to additional cellular insults or alternatively, may poise cells to develop a migratory phenotype in response to additional cellular cues.

Radiotherapy versus single-dose carboplatin in adjuvant treatment of stage I seminoma: a randomised trial.

BACKGROUND: Adjuvant radiotherapy is effective treatment for stage I seminoma, but is associated with a risk of late non-germ-cell cancer and cardiovascular events. After good results in initial studies with one injection of carboplatin, we undertook a large randomised trial to compare the approaches of radiotherapy with chemotherapy in seminoma treatment. METHODS: Between 1996 and 2001, 1477 patients from 70 hospitals in 14 countries were randomly assigned to receive radiotherapy (para-aortic strip or dog-leg field; n=904) or one injection of carboplatin (n=573; dose based on the formula 7x[glomerular filtration rate+25] mg), at two trial centres in the UK and Belgium. The primary outcome measure was the relapse-free rate, with the trial powered to exclude absolute differences in 2-year rates of more than 3%. Analysis was by intention to treat and per protocol. This trial has been assigned the International Standard Randomised Controlled Trial Number ISRCTN27163214. FINDINGS: 885 and 560 patients received radiotherapy and carboplatin, respectively. With a median follow-up of 4 years (IQR 3.0-4.9), relapse-free survival rates for radiotherapy and carboplatin were similar (96.7% [95% CI 95.3-97.7] vs 97.7% [96.0-98.6] at 2 years; 95.9% [94.4-97.1] vs 94.8% [92.5-96.4] at 3 years, respectively; hazard ratio 1.28 [90% CI 0.85-1.93], p=0.32). At 2 years' follow-up, the absolute differences in relapse-free rates (radiotherapy-chemotherapy) were -1.0% (90% CI -2.5 to 0.5) by direct comparison of proportions, and 0.9% (-0.5 to 3.0) by a hazard-ratio-based approach. Patients given carboplatin were less lethargic and less likely to take time off work than those given radiotherapy. New, second primary testicular germ-cell tumours were reported in ten patients allocated irradiation (all after para-aortic strip field) and two allocated carboplatin (5-year event rate 1.96% [95% CI 1.0-3.8] vs 0.54% [0.1-2.1], p=0.04). One seminoma-related death occurred after radiotherapy and none after carboplatin. INTERPRETATION: This trial has shown the non-inferiority of carboplatin to radiotherapy in the treatment of stage I seminoma. Although the absence of disease-related deaths and preliminary data indicating fewer second primary testicular germ-cell tumours favour carboplatin use, these findings need to be confirmed beyond 4 years' follow-up.

A randomised comparison of treosulfan and carboplatin in patients with ovarian cancer: a study by the Scottish Gynaecological Cancer Trials Group (SGCTG).

The management of older and unfit women with advanced ovarian cancer requires post-operative chemotherapy but many of these patients are not suitable for high-dose cisplatin-based regimes. Carboplatin has been an easier alternative and can be given in the ambulatory setting. Historical data suggests that oral alkylating agents to be just effective with similar efficacy. In this study we have compared platinum-based carboplatin to the alkylating agent treosulfan in a population unfit to receive high-dose cisplatin. The trial randomised patients to either intravenous carboplatin or treosulfan as single agent. The trial was stopped prematurely after the interim analysis showed improved survival and response rates in the carboplatin arm. We conclude that carboplatin is a safe and effective drug in a population that is unfit for high-dose cisplatin. Treosulfan showed limited activity but may be considered along with other oral drugs in limited circumstances. With the exception of myelosuppression, toxicity was mild in both arms. Carboplatin remains the gold standard in this older and less fit group of patients.

Characterization of progesterone receptor A and B expression in human breast cancer.

The human progesterone receptor (PR) is a ligand-activated nuclear transcription factor that mediates progesterone action in target tissues. Two PR proteins, PR-A (M(r) 81,000-83,000) and PR-B (M(r) 116,000-120,000), have been described and different physiological activities ascribed to each on the basis of in vitro studies, suggesting that their ratio of expression may control progesterone responsiveness in target cells. Presence of PR in breast tumors is an important indicator of likely responsiveness to endocrine agents. However, the relative expression of PR-A and B in breast cancer has not been described, and its clinical significance has not been addressed. Expression of PR-A and B was measured by immunoblot analysis of 202 PR-positive human breast tumor cytosols. The ratio of expression of the two PR proteins (PR-A/B) ranged from 0.04 to 179.3. The median PR-A/B ratio was 1.26, and 61.4% of samples had PR-A/B ratios between 0 and 2. PR-A/B ratios deviated significantly from a normal log distribution; tumors containing a PR-A/B ratio greater than 4 were overrepresented in the group. Linear regression analysis revealed that high PR-A/B ratios, in general, derived from a low concentration of PR-B rather than high expression of PR-A. PR-A/B protein ratios were not correlated with the age of the patient or with total PR concentration. A third PR protein band (PR78kDa) was detected in a number of samples and comprised greater than 20% of total PR protein in 52 (25.7%) of the 202 tumor samples examined. The range or frequency distribution of PR-A/B ratios in samples containing PR78kDa was not different to the overall group. In summary, in PR-positive breast tumors, the ratio of expression of PR-A and B proteins is close to unity, as is seen in a number of other progestin target tissues. However, a significant proportion of tumors expressed very low levels of PR-B and a consequently high PR-A/B ratio. Although the clinical consequence of this observation is not known, the in vitro findings that PR-A may act as a repressor of PR-B suggest that tumors containing primarily PR-A may identify a subset of patients with low or aberrant response to endocrine agents.

Estrogen receptors alpha and beta: prevalence of estrogen receptor beta mRNA in human vascular smooth muscle and transcriptional effects.

BACKGROUND: Estrogens have vascular effects through the activation of estrogen receptors (ERs). In addition to ERalpha, the first ER to be cloned, a second subtype called ERbeta has recently been discovered. METHODS AND RESULTS: Using a reverse-transcriptase polymerase chain reaction assay that employs the same primer pair to simultaneously amplify ERalpha and ERbeta transcripts, we found that ERbeta is the ER form that is predominantly expressed in human vascular smooth muscle, particularly in women. The transcriptional effects of the 2 ERs in transfected HeLa cells differed. In response to 17beta-estradiol, ERalpha is a stronger transactivator than ERbeta at low receptor concentrations. However, at higher receptor concentrations, ERalpha activity self-squelches, and ERbeta is a stronger transactivator. Tamoxifen has partial agonist effects with ERalpha but not with ERbeta. CONCLUSIONS: The protective effects of estrogens in the cardiovascular system of women may be due to the genomic effects of ERbeta in vascular tissue.

Fractionated stereotactic external beam radiotherapy in the management of brain metastases.

24 patients with 28 brain metastases were treated with fractionated stereotactic radiotherapy (SRT). Doses ranged from 10 Gy in two fractions to 20 Gy in two fractions. 13 patients received SRT boost after whole brain radiotherapy (WBRT), 5 were treated with SRT alone and 6 were treated at the time of recurrence following WBRT. The median progression-free survival at the treated site was 18 months and the median survival was 18 months. All patients were treated without admission to hospital. Toxicity of fractionated SRT was minimal and patients treated without WBRT did not suffer significant alopecia. Fractionated SRT offers a non-toxic non-invasive alternative to excision surgery in patients with solitary brain metastases. The optimum fractionation schedule and the role of whole brain irradiation remain to be determined.

Expression of osteonectin mRNA in human breast tumours is inversely correlated with oestrogen receptor content.

Osteonectin is a secreted glycoprotein which is detected in a number of normal and neoplastic human tissues in vivo. It is an extracellular matrix (ECM)-associated protein which is postulated to regulate cell migration, adhesion, proliferation and matrix mineralisation and previous reports suggest that it may be modulated by steroid hormones in target tissues. The aim of this study was to measure osteonectin mRNA and protein expression in breast tumour biopsies and compare these with oestrogen (ER) and progesterone receptor (PR) levels in the same tumours. An inverse correlation was seen between osteonectin mRNA expression and ER level. Samples with low ER protein expression had a mean osteonectin mRNA level which was almost 4-fold greater than the mean level of expression observed in tumours containing high concentrations of ER protein. This inverse correlation was statistically significant. Despite the strong inverse relationship between osteonectin mRNA levels and tumour ER content, no correlation was seen when osteonectin protein concentration was measured in tumour cytosols on immunoblots and compared to ER and PR levels in the same tumours. However, since it is a secreted protein, osteonectin protein expression may not reflect cellular osteonectin levels in breast tumours. In summary, these data suggest that ER-mediated suppression of osteonectin gene expression may contribute to the less aggressive characteristics associated with receptor-positive tumours and that loss of ER expression may lead to over-expression of osteonectin and contribute to a poorer differentiated, more invasive phenotype.

Regulation of the expression and activity by progestins of a member of the SOX gene family of transcriptional modulators.

The mammalian testis-determining gene Sry and the related Sox genes define a family of transcriptional regulators widely expressed during embryogenesis. Tightly controlled temporal profiles of expression are a feature of the Sox gene family and may be required for initiation of a cascade of gene expression, yet the molecular mechanisms that control Sox gene expression are unknown. We now show that human SOX4 is expressed in the normal breast and in breast cancer cells. In these cells SOX4 is a progesterone-regulated gene, the expression of which is increased by progestins, leading to a marked increase in SOX-mediated transcriptional activity. Treatment of T-47D breast cancer cells with the synthetic progestin ORG 2058 directly increased SOX4 transcription, resulting in a 4-fold increase in SOX4 mRNA levels within 4 h of treatment. No effect of ORG 2058 was noted on other SOX genes measured, nor were other hormone-regulated HMG box proteins detected in this system, suggesting that the observed ability of progestin to increase SOX mRNA expression was confined to SOX4. The increase in SOX4 transcription was reflected in increased SOX4 protein expression, as progestin treatment of T-47D cells transfected with a SOX-responsive reporter resulted in a marked increase in reporter gene expression. Progesterone is essential for normal development and differentiation of the female reproductive system, plays an essential role in regulating growth and differentiation of the mammary gland and is required for opposing the proliferative effects of estrogen in specific cell types. The detection of SOX4 expression in the normal and malignant breast and the demonstration that SOX4 expression is under progesterone control suggests that changes in SOX4 gene expression may play a role in commitment to the differentiated phenotype in the normal and malignant mammary gland.

Cytoskeletal responsiveness to progestins is dependent on progesterone receptor A levels.

Changes in the cell cytoskeleton occur in cell transformation and recent data suggest the involvement of ovarian hormones, which are implicated in cancer development and progression. In human breast and endometrial tumors, there is disrupted expression of progesterone receptor (PR) isoforms and predominance of one isoform, usually PRA. PRA predominance is an early event in carcinogenesis, and in cancers is associated with poor clinical features. Overexpression of PRA in vitro causes altered progestin regulation of cell morphology, suggesting that PRA overexpression may provoke deleterious changes in cell functioning. This study aimed to identify pathways of cytoskeleton regulation responsive to progestins and to determine whether these are perturbed when PRA is overexpressed to the levels seen in cancers. Progestin treatment of PR-positive breast cancer cells caused increased cell surface area whereas after induction of a stably integrated PRA construct, cells became rounded and the cell surface was decreased. The effect of PRA induction on cell rounding was reversed by the anti-progestin RU38486. Altered tropomyosin (Tm) isoforms were implicated in these morphological differences, as there was a PRA-mediated alteration in Tm5 isoform levels, and transfection of Tm5a mimicked progestin-mediated cell rounding in PRA-overexpressing cells. Ezrin was redistributed from the membrane to cytoplasmic locations in the presence of progestin, and discrete focal localization was evident in cells with PRA predominance. Progestin effects on the cytoskeleton in PRA-overexpressing cells provide evidence for novel endocrine regulation of aspects of actin microfilament composition, suggesting that changes in the cytoskeleton known to be associated with cancer development and progression may be regulated in part by altered PRA expression which develops early in carcinogenesis.

Whole body doses from linear accelerator-based stereotactic radiotherapy.

PURPOSE: The objective of this work was to measure whole body radiation doses in a humanoid phantom from linear accelerator-based cranial stereotactic radiosurgery/therapy (SRS/T), using different beam arrangements. METHODS AND MATERIALS: A standard noncoplanar five-arc beam arrangement and a four-arc technique without a sagittal arc were used to deliver 20 Gy in a single fraction to a midline spherical target volume in the corpus callosum region of an Alderson-Rando anthropomorphic phantom using (i) a 20-mm and (ii) a 40-mm circular collimator. Whole body dose measurements were made using lithium fluoride thermoluminescent dosimetry. Whole body isodose plots in the sagittal and coronal planes and organ doses were compared for the two arcing beam arrangements. An ionization chamber was used to record the exit dose at intervals along the length of the phantom at midline and 4.5 cm off-axis for (i) a single fixed field and (ii) a solitary 90 degrees sagittal arc using a 40-mm circular collimator. RESULTS: The sagittal arc was the major contributor to neck and trunk doses when the five- and four-arc arrangements were compared, with fourfold greater thyroid dose. The gonad dose was increased by the sagittal arc, but was largely due to leakage radiation. The dose from a fixed field exiting down the long axis of the phantom was tenfold greater than that from a solitary 90 degrees sagittal arc. When the fixed field or arc traversed the lung or exited through the pharynx and major upper airways, the dose measurements below the diaphragm were 30-40% higher than those along the exit path of maximum soft tissue density. CONCLUSION: When SRS/T is used in nonmalignant conditions such as cranial arteriovenous malformations or benign tumors the exit paths of arcing beams or fixed fields should be taken into account when deciding upon the final treatment plan. Such consideration should minimize the risk of radiation-induced malignancy, notably in the thyroid gland of younger patients.

A randomized trial of hypofractionated schedules of palliative radiotherapy in the management of bladder carcinoma: results of medical research council trial BA09.

PURPOSE: To compare the efficacy and toxicity of two hypofractionated radiotherapy schedules for the improvement of local symptoms from muscle-invasive bladder cancer. METHODS AND MATERIALS: A multicenter randomized trial was conducted comparing the efficacy and toxicity of two radiotherapy schedules (35 Gy in 10 fractions and 21 Gy in 3 fractions) for symptomatic improvement in patients considered unsuitable for curative treatment through disease stage or comorbidity. The primary outcome measures were overall symptomatic improvement of bladder-related symptoms at 3 months and changes in bladder- and bowel-related symptoms from pretreatment to end-of-treatment and 3-month assessments. Overall symptomatic improvement was defined prospectively as the improvement in one bladder-related symptom of at least one grade at 3 months, with no deterioration in any other bladder-related symptom. RESULTS: Five hundred patients were recruited, but data on symptomatic improvement at 3 months was only available on 272 patients. Of these, 68% achieved symptomatic improvement (71% for 35 Gy, 64% for 21 Gy), with no evidence of a difference in efficacy or toxicity between the two arms. There was no evidence of a difference in survival between the two schedules (hazard ratio [HR] = 0.99, 95% CI 0.82-1.21, p = 0. 933). CONCLUSION: This is the largest prospective trial to date in the palliative treatment of bladder cancer, and provides baseline data against which other results may be compared. The use of 21 Gy in 3 fractions appears as effective as 35 Gy in 10 fractions, although modest differences in survival, symptomatic improvement rates, and toxicity can not be reliably excluded.

Binding of N-(2-bromo ethyl)-N-ethyl-N 1 -naphthylmethylamine HBr (SY28) to the proteins of guinea-pig vas deferens.

1. Vasa deferentia from guinea-pigs were exposed to (14)C-SY28 HBr (5.32 x 10(-6)M) washed, lipid extracted, and broken up.2. The tissue was digested with papain, hydrolyzed with 6 N HCl, and gel filtration performed.3. Aliquots at each stage were used for paper chromatography and autoradiography.4. Three labelled spots (in addition to unbound 2-halogenoalkylamine) were located from acid hydrolysate and a fourth from gel filtrate.5. Selected amino-acids were complexed with (14)C-SY28 and the procedure repeated. The R(F) values were compared with those of the same amino-acids with and without 6 N HCl.6. Histidine, aspartic acid, arginine and serine gave a significant degree of binding of (14)C-SY28.7. The theoretical implications are discussed.

Cardiovascular and respiratory effects of cannabis in cat and rat.

1. In anaesthetized rats, intravenous administration of cannabis extract (10 mg/kg), Delta(1)-tetrahydrocannabinol (THC) (0.5 mg/kg) and Delta(6)-THC (0.5 mg/kg) caused a reduction in systemic blood pressure, pulse rate and respiratory rate.2. Neither cannabinol (1 mg/kg, i.v.) nor cannabidiol (1 mg/kg, i.v.) had any observed effects on the cardiovascular and respiratory systems of the rat.3. Pretreatment of rats with atropine (1 mg/kg, i.v.) reduced the hypotension and bradycardia caused by Delta(1)-THC or the extract.4. In anaesthetized cats with autoperfused hindquarters, cannabis extract (10 mg/kg, i.v.) and Delta(1)-THC (0.2 mg/kg, i.v.) caused hypotension, bradycardia, depression of respiratory rate and reduction of hindlimb perfusion pressure.5. Both cannabis extract and Delta(1)-THC potentiated reflex vasodilation and direct vasoconstriction in the hindlimb induced by intravenous noradrenaline in the cat; they reduced reflex hindlimb vasoconstriction elicited by histamine, acetylcholine or bilateral carotid occlusion.6. Tolerance to these cardiovascular and respiratory effects of cannabis extract developed in rats which had been treated i.p. with the extract at (50 mg/kg) per day for 14 days.

Response to acetylcholine and nicotine of the perfused vessels of the rabbit ear.

1. The vessels of the isolated rabbit ear were perfused at 23 mbar with Krebs solution with (tonic) and without (atonic) noradrenaline (5.9 x 10(-7)M) at selected temperatures of 20 degrees -38 degrees C. Peripheral resistance units (PRU) were calculated from the observed peak flow rates and alterations caused by drugs expressed as Delta% PRU.2. ACh is constrictor in the atonic vessel.3. ACh is a vasodilator of the tonic vessel perfused with NA. This effect is potentiated by anticholinesterase and by denervation, unaffected by botulinum toxin and antagonized by atropine. ACh also dilates the vessel perfused with vasopressin.4. Increasing the temperature reduces the responses to ACh but increases the effect of anticholinesterase.5. Nicotine causes a dose dependent dilatation of the tonic vessels, reduced but not abolished by C(6), by atropine, by botulinum toxin and by denervation.6. Nicotine causes a dose dependent constriction of the atonic vessels, abolished by C(6) and by phentolamine, reduced by denervation, but unaffected by botulinum toxin.

The actions of prostaglandins E 1 and F 2 on the on the perfused vessels of the isolated rabbit ear.

1. In the isolated rabbit ear vascular bed, perfused with Krebs solution, prostaglandins E(1) and F(2alpha) produce dose-dependent, phentolamine-sensitive constrictions.2. These are absent if the animal is pre-treated with reserpine or if the ear is denervated in advance.3. If noradrenaline or vasopressin is added to the Krebs solution, vascular resistance is high and PGE(1) and PGF(2alpha) produce vasodilatation which is unaffected by hyoscine or propranolol.4. Perfusion with theophylline, with added ATP, ADP or 3'5'-AMP, or pre-treatment of the animal with stilboestrol antagonizes the dilator response to PGE(1) in the presence of noradrenaline, which may be reversed. Most of the responses to PGF(2alpha) are reversed. These treatments elevate the level of 3'5'-AMP in tissues.5. It is postulated that prostaglandins exert a regulatory action on 3'5'-AMP levels through inhibition of adenyl cyclase and/or phosphodiesterase and that the resulting rising or falling level of 3'5'-AMP determines the nature of the response by the smooth muscle to the released noradrenaline.

The effect of morpine dependence on the vesicular content of adrenergic nerves in relation to arteriolar smooth muscle in the pancreas of the rat.

1. Sibling male Wistar rats were kept in groups of three under identical conditions. Two groups were made morphine dependent in 28 days and the third served as control. The state of dependence was established by recording the development of tolerance to the analgesic action of the drug and the effect of acute withdrawal of morphine and injection of nalorphine on one test group.2. Animals in the other test group and the control group were killed and specimens of pancreas collected and prepared for examination by electron microscopy. The adrenergic innervation of pancreatic arterioles was located and photographed and the effect of morphinization on the percentage of granular and agranular small vesicles in the axons determined.3. Morphinization produces a significant reduction in the granular or dense core vesicle population of these adrenergic axons.

The effect of delta1-tetrahydrocannabinol on the release of (3H-(-)-noradrenaline from the isolated vas deferens of the rat.

1 The amount of tritium released upon transmural stimulation of the isolated vas of the rat incubated with [(3)H]-Delta(1)-tetrahydrocannabinol (THC) (2.8 muM) was significantly greater than the non-stimulated overflow.2 There was no difference between non-stimulated overflow and the effect of transmural stimulation of vasa equilibrated with [(14)C]-sorbitol (6.5 mg/ml).3 The difference between non-stimulated and stimulated efflux from vasa equilibrated with [(3)H]-Delta(1)-THC was abolished in rats pretreated for 24 h with 6-hydroxydopamine (250 mg/kg).4 The amount of tritium released upon transmural stimulation of the vas incubated with [(3)H]-noradrenaline (6 muM) was significantly greater than the non-stimulated overflow.5 Delta(1)-THC (560 nM and 14 muM) caused a significant dose-dependent reduction of both non-stimulated and stimulated tritium efflux, especially the latter.