It is accepted that whilst hormones such as oxytocin, vasopressin and prostaglandin F2α induce myometrial contractions, essentially via an elevation of intracellular calcium, other ligands, such as β-adrenoceptor agonists, calcitonin gene-related peptide, and prostaglandin E2, promote uterine quiescence via their ability to increase intracellular cyclic AMP levels. At present, the exact factors initiating human parturition remain unknown, and labour may occur due to a loss of uterine quiescence, an increase in uterine contractility, or a combination of both. Whilst many studies have aimed to understand the mechanisms underlying uterine contractility there is a relative paucity of data regarding myometrial relaxation. We have verified the presence of mRNA encoding adenylyl cyclase (AC) isoforms I, II, III, V, VI, VII, VIII and IX in both non-pregnant and pregnant human myometrium, and in isolated myometrial cells maintained in cell culture. Furthermore, by means of immunoblotting and immunocytochemistry, we have demonstrated the expression of these isoforms as membrane-associated AC proteins, and identified changes in individual AC isoform expression during gestation. These findings illustrate the diversity of potential cAMP generating pathways in human myometrium, and the complexity of the signal transduction systems underlying uterine quiescence. Experimental Physiology (2001) 86.2, 265-272.

The factors regulating susceptibility and severity of autoimmune diseases are poorly understood. That neuroendocrine factors are critical modulators in this regard is self-evident. For example, there are major gender differences in susceptibility with women at greater risk than men of, for example, rheumatoid arthritis (RA) and multiple sclerosis (MS). The hypothalamo-pituitary-adrenal (HPA) axis has rightly attracted a considerable amount of attention. Of particular interest has been the hypothesis that susceptibility to autoimmune disease may be related to an impaired responsiveness of the HPA axis; that is, an inability to mount an appropriate cortisol response with which to down-regulate the immune system might allow the immune system to rampage unchecked and attack self. This hypothesis links regulation of the release from the adrenal gland of the potent anti-inflammatory glucocorticoids to the disease process. Endogenous glucocorticoids are crucial for the regulation of the severity of the disease process. The hypothesis proposing a link between a hyporesponsive HPA axis and susceptibility to disease is compelling. However, evidence from a number of sources has suggested that this may not be the entire story and alterations in the activity of the HPA axis have not been consistently observed in patients with RA. This review will concentrate on recent findings concerning the HPA axis in determining susceptibility to, and in regulating the severity of, inflammatory processes in autoimmune disease. These studies have revealed that a single exposure to endotoxin can confer protection to subsequent development of inflammation in an arthritis model in both neonatal and adult rats. Behavioural differences within a single population of rats are associated with differences in the plasma corticosterone responses to stress. However, relative hyporesponsiveness is not reflected by an increase in the severity of inflammation. In humans with RA the dexamethasone-corticotrophin-releasing factor (CRF) test has revealed two distinct sub-populations of patients. Studies in patients with MS have shown that this is not related to depression but rather to the severity of the disease. A better understanding of these complex neuroendocrine interactions may lead to novel clinical interventions. Experimental Physiology (2002) 87.5, 519-525.

The aim of the present study was to determine the influence on renal sympathetic nerve activity of the different chemically coded neuronal phenotypes that project from the paraventricular nucleus (PVN) to the spinal cord. Experiments were carried out on male Wistar rats anaesthetised with chloralose and urethane. Changes in renal sympathetic nerve activity were measured following activation of neurones in the PVN with D,L-homocysteic acid (100 nl, 200 mM), before and following intrathecal application of glutamate, vasopressin, oxytocin, dopamine and their receptor antagonists. Excitatory and inhibitory effects on renal sympathetic nerve activity were elicited by PVN stimulation. PVN excitatory effects were mimicked by intrathecal administration of glutamate and vasopressin and selectively antagonised by intrathecal administration of kynurenic acid and a V1a receptor antagonist, respectively. A low dose of dopamine increased renal sympathetic activity and this was selectively antagonised by haloperidol; however, the latter was without effect on PVN excitatory responses. A high dose of dopamine decreased renal sympathetic nerve activity and this was selectively blocked by a D1 dopamine receptor antagonist (SCH 23390), which also antagonised a minority of inhibitory responses obtained from the caudal extension of the PVN. Oxytocin also had two actions: in 5 rats it inhibited and in 10 rats it increased renal sympathetic nerve activity, both actions being blocked selectively by oxytocin receptor antagonists. Neither of the PVN effects on renal sympathetic nerve activity appeared to be dependent on oxytocin pathways. Tests with intrathecal administration of bicuculline showed that PVN inhibition of renal sympathetic nerve activity was not dependent on spinal GABAA receptor activation. The results show that PVN-induced excitation of sympathetic activity to the kidney is mainly mediated by glutamate or vasopressin neurones whereas dopamine via D1 receptors may mediate some of the PVN inhibitory effects.

We have investigated the mediators and mechanisms underlying the vasodilator effects of the potent vasoactive peptide, endothelin-1 (ET-1) and its isomers ET-2 and ET-3 in human skin, in vivo, using cutaneous microdialysis to quantify the release of mediators within the dermal response and scanning laser Doppler imaging to measure changes in blood flux. The effects of local anaesthesia, inhibition of nitric oxide synthase (NOS) by L-NAME and ET receptor blockade on the ET-induced vascular response were also investigated. ET-1, -2 and -3 all caused a dose-dependent area of pallor surrounded by a long-lasting flare which was accompanied by a short-lived burning pruritus. The concentration of nitric oxide (NO) in dialysate collected within the pallor response to 5 µM ET-1 (1.43 ± 0.64 µM, n = 5) was not significantly different from baseline levels collected prior to injection (0.86 ± 0.38 µM) whilst that in the flare increased to reach a peak value of 2.28 ± 0.61 µM at between 4 and 10 min after intradermal injection (P < 0.004). Pretreatment with local anaesthetic slowed the development of the flare and significantly reduced its size by up to 52 % at 20 min after injection (P < 0.05) but had no significant effect on the central pallor. L-NAME, delivered by dialysis also caused a significant reduction in the ET-1-induced flare (P < 0.005). Bosentan, the non-selective ETA/ETB antagonist, when given by dialysis at the site of injection, reduced the area of both the ET-1-induced pallor and surrounding flare by 41 and 26 %, respectively. No significant increase in tissue histamine was measured within either the pallor or flare response to ET-1, -2 or -3. Together these data confirm that the vasodilator response to endothelin-1 in human skin is neurogenic in origin and that it is in part mediated by the local release of nitric oxide. There appears to be little evidence for the involvement of mast cell-derived histamine in the initiation or modulation of ET-induced vasodilatation, in vivo.

The paraventricular nucleus of the hypothalamus plays a pivotol role in the regulation of plasma volume. Part of the response to an increase in volume load is an inhibition of renal sympathetic nerve activity. The present experiments were designed to determine which subnuclei of the paraventricular nucleus are involved in this sympatho-inhibitory response. Experiments were performed on anaesthetised rats. Activated neurones were recognised by the expression of the early gene c-fos, identified by immunohistochemical labelling of its protein product Fos. Plasma volume loading with 4 % Ficoll 70, using an infusion-withdrawal procedure (2 ml over 1 min) repeated 15 times over 1 h revealed a total of 775 ± 101 (n = 6) Fos-positive neurones scattered throughout both the magnocellular and parvocellular subnuclei. In comparison, sustained hypertension resulted in 452 ± 56 (n = 3) Fos-positive neurones similarly distributed, whereas a normotensive control group (n = 3) displayed 115 ± 18 Fos-positive neurones. Because of this lack of a specific effect we used a more selective stimulation of right atrial receptors via a balloon placed at the junction of the superior vena cava and the right atrium so it did not impede venous return. Inflation of the balloon inhibited renal sympathetic nerve activity (36 ± 5 %, n = 7) and repetitive inflation over 1 h resulted in c-fos activation of a small number of neurones (54 ± 14) located only in the parvocellular subnuclei. Whether these are inhibitory interneurones acting within the paraventricular nucleus, or spinally projecting neurones which inhibit or excite renal sympathetic activity by an action in the spinal cord remains to be determined. Experimental Physiology (2002) 87.1, 25-32.

Our recent data have confirmed that maximum oxygen delivery in racing Thoroughbreds is positively correlated to left ventricular mass measured by echocardiography. A similar, but weaker relationship also exists between left ventricular mass and Timeform performance rating in commercial racehorses. The relationship of the Thoroughbred heart to racing success and the special problems that selective breeding for aerobic capacity have had in this species are reviewed in this article. Experimental Physiology (2003) 88.5, 659-663.

L-Arginine transport is mediated by the cationic/neutral amino acid transport system y+L and cationic amino acid transporters y+/CATs in human umbilical vein endothelial cells (HUVECs). System y+/CATs activity may be rate-limiting for nitric oxide (NO) synthesis, but no reports have demonstrated system y+L involvement in NO synthesis in endothelium. We investigated the role of system y+L in NO synthesis in HUVECs. Transport of 1.5 µM L-arginine was inhibited (P < 0.05) by L-lysine (Ki, 1.4 µM), L-leucine (Ki, 1.8 µM) and L-phenylalanine (Ki, 4.1 µM), but was unaltered (P > 0.05) by L-alanine or L-cysteine. The system y+/CATs inhibitor, N-ethylmaleimide (NEM), did not alter 1.5 µM L-arginine transport, but inhibited (92 ± 3 %) 100 µM L-arginine transport. L-Arginine transport in the presence of NEM was saturable (Vmax, 0.37 ± 0.02 pmol (µg protein)-1 min-1; Km, 1.5 ± 0.3 µM) and competitively inhibited by L-leucine in the presence of Na+ (Vmax, 0.49 ± 0.06 pmol (µg protein)-1 min-1; Km, 6.5 ± 0.9 µM). HUVECs express SLC3A2/4F2hc, SLC7A7/4F2-lc2 and SLC7A6/4F2-lc3 genes encoding for the high-affinity transport system y+L. NG-Nitro-L-arginine methyl ester and L-leucine, but not NEM, inhibited NO synthesis in medium containing 1.5 µM L-arginine. Cells exposed to 25 mM D-glucose (24 h) exhibited reduced system y+L activity (Vmax, 0.15 ± 0.008 pmol (µg protein)-1 min-1; Km, 1.4 ± 0.3 µM) and NO synthesis. However, 25 mM D-glucose increased NO synthesis and L-arginine transport via system y+. Thus, L-arginine transport through system y+L plays a role in NO synthesis, which could be a determining factor in pathological conditions where the endothelial L-arginine-NO pathway is altered, such as in diabetes mellitus. Experimental Physiology (2003) 88.6, 699-710.

Epidemiological and experimental evidence suggests that maternal undernutrition during pregnancy may alter development of fetal organ systems. We have demonstrated previously that fetal hypothalamic-pituitary-adrenal (HPA) axis responses to exogenous corticotropin-releasing hormone (CRH) + arginine vasopressin (AVP), or adrenocorticotrophin hormone (ACTH), are reduced in fetuses of mildly undernourished ewes. To examine these effects further we tested HPA axis responses to acute isocapnic hypoxaemia in fetal sheep at 114-129 days gestation (dGA), following 15 % reduction in maternal nutritional intake between 0 and 70 dGA. Fetuses from control (C) and nutrient-restricted (R) ewes were chronically catheterised and plasma ACTH and cortisol responses were determined at 114-115, 120-123 and 126-129 dGA during hypoxaemia (1 h) induced by lowering the maternal inspired O2 fraction (FI,O2). Basal plasma cortisol concentrations and HPA axis responses at 114-115 and 120-123 dGA did not differ between C and R fetuses. At 126-129 dGA, both plasma ACTH (P < 0.01) and cortisol (P < 0.05) responses were smaller in R fetuses compared to C fetuses. Fetal blood gas status, fetal body weight, body proportions and organ weights did not differ between the groups. We conclude that mild maternal undernutrition alters development of the fetal HPA axis producing a reduction in pituitary and adrenal responsiveness to endogenous stimuli.

The effect of physical training on an individual's ability to withstand an orthostatic stress is unclear. This study was undertaken to determine the effects on orthostatic tolerance in healthy volunteers of training at a level appropriate for unfit subjects and cardiorespiratory patients. In 11 asymptomatic, untrained subjects the following assessments were made: plasma volume by Evans Blue dye dilution and blood volume derived from haematocrit; carotid baroreceptor sensitivity from the slope of the regression of change in cardiac interval against pressure applied to a neck chamber; orthostatic tolerance as time to presyncope in a test of head-up tilting combined with lower body suction; exercise test relating heart rate to oxygen consumption. Subjects were then given a training schedule (5BX/XBX, Royal Canadian Air Force) involving 11-12 min of mixed exercises per day until an age/sex related 'target' was reached. Following training all subjects showed evidence of improved fitness, seen as decreases in heart rate at an oxygen uptake (VO2) of 1Σ5 l min-1 and in the elevation of the regression line between heart rate and VO2. All also had increases in plasma and blood volumes and decreases in baroreceptor sensitivity. Seven of the subjects showed increases in orthostatic tolerance. Improvement in orthostatic tolerance was related to a low initial tolerance, and was associated with increases in plasma volume and decreases in baroreceptor sensitivity. These results show that moderate exercise training increases orthostatic tolerance in subjects who do not already have a high initial tolerance and suggest that training may be of value in the management of untrained patients with attacks of syncope due to orthostatic intolerance.

Chromogranin A (CgA) is a member of a family of highly acidic proteins, chromogranins, which are co-stored in the adrenergic neurons and paraneurons and co-released with adrenaline and noradrenaline (NAd) in response to adequate stimulation. The present study provides novel evidence that CgA-like immunoreactivity (IR) is stored in the exocrine cells in the granular convoluted tubule, and is secreted into saliva by stimulation with NAd and acetylcholine (ACh) in the isolated and perfused rat submandibular gland. NAd at 1 µM produced maximum secretion of CgA-like IR (<< 0·9 mM) and a marked increase in salivary flow. Further increases in NAd concentration (10 or 100 μM) yielded concentration-dependent decreases in both responses. ACh at 1 μM produced maximum salivary flow and a slight elevation of CgA-like IR secretion (6 μM); 100 μM ACh decreased the salivary flow but increased the CgA-like IR secretion (0·6 mM). Electron microscopic examination showed vigorous compound exocytosis of secretory granules in the cells of the granular convoluted tubule when the submandibular gland was stimulated with 1 μM NAd. These results provide an experimental basis for the view that the salivary CgA-like IR secretion may be a sensitive and quantitative index of the activity of the sympathetic nervous system innervating the gland.

Neurological testing tools for measuring and monitoring somatosensory function lack resolution and are often dependent on the clinician testing. In this study we have measured perceptual threshold (PT) to electrical stimulation of the skin and compared it with two-point discriminative ability (TPDA) in 12 control subjects. Tests were made on both sides of the body at American Spinal Injury Association (ASIA) key points on seven spinal dermatomes (C3 (neck), C4 (shoulder), C5 (upper arm), C6 (thumb), T8 (abdomen), L3 (knee), L5 (foot)) and in the mandibular (chin) and maxillary (cheek) fields of the trigeminal (V) nerve. Electrical stimulation (0·5 ms pulse width; 3 Hz) was applied via a self-adhesive cathode and an anode strapped to the wrist or ankle. The stimulus intensity was adjusted and PT was recorded as the lowest current at which the subject reported sensation. Sites were tested in random order. Indices for both TPDA and PT differed according to the dermatome tested but there was no correlation between TPDA and PT for any dermatome. There was good correlation between results from equivalent dermatomes on left and right sides for both PT and TPDA. Women frequently had lower mean (± s.e.) PTs and better TPDA than men; differences were significant (P < 0·05) for PT on the knee (women, 1·31 ± 0·15 mA; men, 2·05 ± 0·26 mA) and the foot (women, 2·90 ± 0·19 mA; men, 4·13 ± 0·28 mA) and for TPDA on the thumb (women, 3·8 ± 0·2 mm; men, 7·8 ± 1·3 mm) and the knee (women, 17·8 ± 1·6 mm; men, 27·1 ± 4·0 mm). Four subjects repeated the experiment on another day and the results correlated well with the first test for PT (r2, 0·62) and TPDA (r2, 0·48). PT differs between dermatomes in a predictable way but does not relate to TPDA. PT is easy to measure and may be a useful assessment tool with which to monitor recovery or deterioration in neuropathies, neurotrauma or after surgery. Experimental Physiology (2001) 86.1, 127-130.

Current applications of gene transfer in farm animals include the improvement of product quality and quantity, disease resistance, the production of valuable proteins in the mammary gland or other organs, the genetic modification of pigs for xenotransplantation and the generation of new animal models in cases where rodent models are not sufficient for studying the problem under evaluation. Although DNA microinjection into pronuclei of zygotes from various farm animal species has happened since 1985, the efficiency of this method is low. Further drawbacks are related to the random integration process which may cause mosaicism, insertional mutations and varying expression due to position effects. Sperm-mediated gene transfer is not routinely established yet, although the mechanisms of binding and internalisation of DNA by sperm cells is becoming increasingly clearer. New protocols for the use of retroviral vectors to infect metaphase II oocytes which are subsequently fertilised resulted in efficient production of transgenic cattle. In spite of extensive efforts to establish pluripotent stem cells from farm animal species, no germ-line competent cells have been reported in mammalian species other than mouse so far. However, recent success in cloning sheep, cattle, goats and pigs from cultured cells provides an alternative route for efficient and targeted genetic modifications of farm animals.

Maximal exercise reduces the cerebral metabolic ratio (O2/(glucose + 1/2lactate)) to < 4 from a resting value close to 6, and only part of this decrease is explained by the 'intent' to exercise. This study evaluated whether sensory stimulation of brain by muscle ischaemia would reduce the cerebral metabolic ratio. In 10 healthy human subjects the cerebral arterial-venous differences (a-v differences) for O2, glucose and lactate were assessed before, during and after three bouts of 10 min cycling with equal workload: (1) control exercise at light intensity, (2) exercise that elicited a high rating of perceived exertion due to a 100 mmHg thigh cuff, and (3) exercise followed by 5 min of post-exercise muscle ischaemia that increased blood pressure by ~ 20 %. Control exercise did not significantly affect the a-v differences. However, during the recovery from exercise with thigh cuffs the cerebral metabolic ratio decreased from a resting value of 5.4 ± 0.2 to 4.0 ± 0.4 (mean ± S.E.M.; P < 0.05) as a discrete lactate efflux from the brain at rest shifted to a slight uptake. Also, following post-exercise muscle ischaemia, the cerebral metabolic ratio decreased to 4.5 ± 0.3 (P < 0.05). The results support the hypothesis that during exercise, cerebral metabolism is influenced both by the mental effort to exercise and by sensory input from skeletal muscles. Experimental Physiology (2003) 88.2, 297-302.

It has been known for a long time that isolated mitochondria are able to accumulate large amounts of calcium ions. Before the discovery that endoplasmic reticulum (ER) was the main Ca2+-storing cellular organelle, mitochondria were thought to play a major role in cytoplasmic Ca2+ homeostasis (Carafoli, 2002). After IP3 was discovered and it was shown that IP3 receptors were localized in ER membrane and that Ca2+-binding proteins such as calsequestrin could store large amounts of Ca2+ in the ER, the role of mitochondria in the regulation of cytoplasmic Ca2+ was questioned. However, in recent years, mostly due to the development of new methods, there has been increasing evidence that mitochondria could be an important cytoplasmic Ca2+ sink, especially under conditions of a high cellular Ca2+ load. Experimental Physiology (2003) 88.1, 91-97.

This study compared the concentration of ethanol, both absolute and relative to water content, in sweat and blood. Ten male volunteers consumed approximately 13 mmol (kg body weight)-1 of ethanol. Blood and sweat samples were collected approximately 1, 2 and 3 h following ingestion. Sweat was collected following pilocarpine iontophoresis using an anaerobic technique that prevented ethanol evaporation. In addition, the water content of sweat and blood samples was determined. The correlation between sweat and blood ethanol, expressed in mmol l-1, was r = 0.98. The slope of the relationship was 0.81. When corrected for the water content in each sample, and expressed as mmoles per litre of water, the correlation remained very high (r = 0.97) while the slope increased to 1.01. These results suggest that rapid and complete equilibrium of ethanol occurs across the sweat gland epithelium.

The aim of this study was to determine the site within the brain at which inhibition of renal sympathetic nerve activity (RSNA) occurs following right atrial receptor stimulation. The atrial receptors were stimulated by inflating a balloon at the right vena cava-atrium junction and the reflex effect was observed before and during application of neurotransmitter agonists and antagonists into the paraventricular nucleus (PVN), or intrathecally to the spinal cord. Balloon inflation reduced RSNA by 29.1 ± 3 % without changing blood pressure in anaesthetised Wistar rats. Microinjection of the GABAA receptor antagonist bicuculline (0.025 mM, 100 nl) into the PVN increased RSNA by 42.3 ± 5 % and this was changed little by balloon inflation when PVN increased RSNA by 50.6 ± 6.3 %. Microinjection of the nitric oxide synthase (NOS) inhibitors L-NAME (0.1 mM, 100 nl) or L-NMMA (0.2 mM, 100 nl) into PVN elicited increases in RSNA of 36 ± 8 % or 54 ± 10 %, respectively. Balloon inflation during PVN stimulation plus NOS inhibition resulted in RSNA activity of 8 ± 4 % or -1 ± 1 %, respectively, compared to baseline control. Baseline RSNA was similar throughout this series of tests ranging from 9.1 ± 1.3 to 11.5 ± 1.1 spike counts s-1. To rule out the possibility that the atrial reflex inhibition was in part dependent on a dopamine-mediated PVN-spinal projection pathway inhibiting RSNA at a spinal locus, a dopamine D1 receptor antagonist SCH 23390 was intrathecally applied to the spinal cord. The effect of subsequent balloon inflation on RSNA was not significantly reduced. It was concluded that atrial receptor activation causes an inhibition of RSNA at the PVN and that this effect is mediated by GABA. Experimental Physiology (2003) 88.3, 335-342.

Heat shock proteins (HSPs) are expressed when cells are exposed to various types of stress and they may provide protection against cellular insult. Previous data have shown increases in HSP expression following acute exhaustive exercise in rats (Locke et al. 1990, 1995; Salo et al. 1991) and humans (Liu et al. 1999); however, it is not known if chronic exercise will increase resting levels of HSPs. The purpose of this study was to determine if basal protein levels of HSP 72/73 and HSP 60 are increased in cardiac and skeletal muscle of endurance trained Fischer 344 rats. Heart, soleus (SOL) and lateral gastrocnemius (LG) muscles were removed and hearts were sectioned into left ventricle (LV), right ventricle (RV) and atria (AT). Endurance training improved myocardial citrate synthase activity by 88, 90 and 77 % and cytochrome c oxidase activity by 58, 51 and 89 % in LV, RV and AT, respectively. LV and RV oxidative enzyme activities were greater when compared to AT for both trained and untrained rats (P < 0.05). HSP 72/73 expression was significantly greater (P < 0.05) in LV, RV and SOL from endurance trained versus from control rats (26, 45 and 67 %, respectively). HSP 60 was also increased (P < 0.05) in LV, RV and SOL in trained relative to untrained rats. HSP 72/73 and HSP 60 were unchanged in AT and LG after training. These results indicate that endurance training increases the basal expression of stress proteins and this observation is consistent with the hypothesis that endurance training may activate a protective mechanism to stress.

The regulation of vascular tone includes modulation of contractile element calcium sensitivity. We tested the involvement of the Rho-associated protein kinase p160ROCK in tone and calcium sensitivity of cannulated rat mesenteric small arteries. These vessels developed basal tone and showed myogenic responses upon pressure steps, resulting from an increase in calcium in combination with a high contractile element calcium sensitivity. Y-27632, believed to be a specific p160ROCK inhibitor, caused concentration-dependent inhibition of basal tone, with near full inhibition at 3 µM. At this concentration, myogenic responses were absent and stepwise pressure elevation resulted in severe vascular distension. Y-27632 did not affect pressure-induced changes in intracellular calcium but rather reduced pressure-induced as well as phenylephrine-induced calcium sensitisation. Thus in the presence of the blocker, for a given calcium concentration, tone was greatly reduced, and the divergence in sensitivity between pressure and phenylephrine as stimuli on the one hand and potassium on the other disappeared. K+ (125 mM) and ionomycin still caused contraction in the presence of the p160ROCK blocker. These data show that in pressurised small arteries the Rho-p160ROCK pathway is active in the absence of vasoconstrictors, keeping the vessels in a state of high calcium sensitivity and basal tone. Experimental Physiology (2001) 86.5, 585-592.

1.Tissues weighing from 0·006 to 3·0 g were dissected and put directly into 15 ml screw cap polypropylene tubes with a conical base (maximum of 3 g per tube). It proved unnecessary to mince tissue, even though smaller pieces may aid quicker digestion. Five millilitres of 2 M KOH in 99 % (IMS) ethanol with 0·5 % Tween-80 was then added. Tissue digestion was usually completed in 2-4 h using a dry heating block held at 60°C, with intermittent shaking. Samples were routinely processed using fresh tissues, although storage of frozen tissue (in the dark at -20°C) introduced no detectable error in BF estimation and tended to aid tissue maceration.2.After digestion was completed the tubes were centrifuged at 3000 r.p.m. (1500 g) for 15 min.3.The supernatant was carefully aspirated until < 500 µl was left, thereby reducing the possibility of accidental loss of microspheres.4.After 1 ml dH2O was added the tubes were quickly vortexed to prevent microsphere flocculation and aid resuspension of remaining pellets, while the subsequent addition of ethanoic Tween (100 % ethanol + 0·5 % Tween-80) allowed complete sedimentation by centrifugation.5.Nine millilitres of ethanoic Tween-80 was added, and the tubes were vortexed and spun at 1500 g for 15 min.6.The supernatant was aspirated as above (Step 3).7.Five millilitres of 100 mM phosphate buffer (pH 7·00) was added to neutralize the pellet as alkaline solutions quench fluorescence. Using aqueous solutions increased the possibility of microsphere loss by adhesion to the surface of tubes or aggregation, which could readily be seen by eye when tubes were examined under bright light, and hence the buffer was followed by addition of 4 ml absolute ethanol and further vortexing, before spinning (1500 g) for 20 min.8.The supernatant was aspirated, leaving up to 300 µl depending on the amount of tissue residue, and the remaining microspheres and pellet were quickly vortexed to ensure complete resuspension.9.The tubes were left to evaporate in an oven at 60°C and then briefly vortexed during this period to disperse more of the flocculent, until around 100 µl fluid remained. This improves solvent extraction of microspheres, which may be less efficient in a completely dry pellet.10.Two or three millilitres of solvent (di(ethylene glycol) ethyl ether acetate, 98 %; Aldrich Chemical Co., Poole, Dorset, UK) was added according to the expected fluorescence intensity, and vortexed several times over 3-5 min. After 30 min the tubes were sonicated in a water bath for 5 min to complete dye extraction by the solvent. The tubes were kept as far as possible in the dark after the solvent was added to avoid photo-bleaching of the fluorescent dyes.11.After sonication, the tubes were occasionally spun (1500 g) once more to sediment any undigested/undissolved material, though this was rarely necessary in our experiments. Readings should be completed within 1 h to avoid loss of signal intensity.

The oxytocin receptor (OTR) is part of an ancient hormone system expressed in diverse phyla in relation to acute reproductive smooth muscle responses, such as egg-laying, birth, or milk letdown. The regulation of the OTR gene, while correlating with steroid levels in vivo, remains elusive. There appear to be both inhibitory and stimulatory influences acting upon a constitutive pattern of basal expression. We have found no evidence, however, for an effect of the sex steroids either directly on gene transcription, or on the receptor itself at the protein level. In the prostatic carcinoma cell line Du145, we have shown that up-regulation of the OTR gene transcription can be effected by cAMP. In an attempt to characterize the expression of the OTR protein in vivo, we have shown, using ligand-blotting, that the OTR can be expressed at different sizes in transfected cells and in myometrium. Also, in the myometrium at term, immunohistochemistry suggests that there is both an increase in OTR protein per cell, as well as in the number of smooth muscle cells expressing OTR, emphasizing that perinatal changes are the results of both individual gene activation events and gross cellular differentiation. The OTR is a valuable model system reflecting molecular changes in the perinatal period. When we understand how this important molecule is regulated, we will also be a long way towards understanding the mechanisms controlling myometrial contractility at birth. Experimental Physiology (2001) 86.2, 289-296.