The role of the paraventricular nucleus (PVN) of the hypothalamus in the cardiovascular response induced by blood volume expansion was examined in anaesthetised rabbits, in which simultaneous recordings were made from a renal sympathetic nerve and one of other sympathetic nerves, the inferior cardiac nerve, a splanchnic nerve and an adrenal nerve. Activation of PVN neurones, by discrete injections (25-100 nl) of d,l-homocysteic acid (DLH, 0.2 M) produced one main pattern of sympathetic nerve activity accompanying a pressor response (57-86 % of PVN sites). This was a decrease in renal sympathetic activity (27 +/- 12 %) and an increase in splanchnic (60 +/- 12 %), adrenal (31 +/- 9 %) and cardiac (42 +/- 8 %) sympathetic activity. Sites in the PVN from which these combinations of nerve activity were obtained were not confined to a specific subnucleus. An increase in renal sympathetic activity which was reversed to a decrease by reducing the volume of DLH injected was obtained at 10 sites in the PVN. These sites were mainly located in the dorsal parvocellular subnucleus. Varying combinations of sympathetic activation were obtained at a minority of sites. It is concluded that the PVN can evoke a differential pattern of sympathetic discharge which may be functionally significant in the control of blood volume regulation, as it mimics that seen on acute volume expansion.

The mode of action of forskolin is of clinical and scientific interest since forskolin has been shown to have potentially therapeutic bone anti-resorptive and anti-hypertensive properties. Forskolin is thought to inhibit the bone resorptive activity of osteoclasts by elevating cytosolic cAMP and to mimic as well as augment the anti-resorptive effect of calcitonin (CT). Other studies have found that forskolin has a dose-dependent dual effect in mouse calavaria, stimulating bone resorption at low doses and having an inhibitory effect at high doses. However, the acute effect of forskolin on osteoclast functional modality has never been studied. The present investigation examined the effect of low (1 mM) and high doses (10 mM) of forskolin on superoxide anion (O2Σ-) generation in isolated bone-resorbing rat osteoclasts. Forskolin was found to have a bimodal cAMP-independent effect on O2Σ- generation, being stimulatory at a low dose and having an inhibitory effect at a higher dose. These findings also suggest that CT-induced inhibition of O2Σ- generation in the osteoclasts is likely to be mediated by cAMP-independent pathways, perhaps involving [Ca2+]i modulation.

In mammals that use the ventilatory system as the principal means of increasing heat loss, raising body temperature causes the adoption of a specialised breathing pattern known as panting and this is mediated by the thermoregulatory system in the preoptic area of the hypothalamus. In these species an additional respiratory drive is also present at raised body temperature, since breathing can reappear at low Pa,CO2 levels, when stimulation of chemoreceptors is minimal. It is not known whether the preoptic area is also the source of this additional drive. Rats do not pant but do possess this additional respiratory drive at raised body temperatures. We have therefore tested whether the preoptic area of the hypothalamus is the source of this additional respiratory drive in rats. Urethane anaesthesia and hyperoxia were used in eleven rats to minimise behavioural and chemical drives to breathe. The presence of the additional respiratory drive was indicated if rhythmic diaphragmatic EMG activity reappeared during hypocapnia (a mean Pa,CO2 level of 21 ± 2 mmHg, n = 11), induced by mechanical ventilation. The additional respiratory drive was absent at normal body temperature (37°C). When the temperature of the whole body was raised using an external source of radiant heat, the additional respiratory drive appeared at 40.6 ± 0.5°C (n = 3). In two further rats this drive was induced at normal body temperature by localised warming in the preoptic area of the intact hypothalamus. The additional respiratory drive appeared at similar temperatures to those in control rats in three rats following isolation of the hypothalamus from more rostral areas of the brain. In contrast, the additional respiratory drive failed to appear at these temperatures in three rats after isolating the hypothalamus from the caudal brainstem, by sectioning pathways medial to the medial forebrain bundle. Since the preoptic area is known to contain thermoreceptors and to receive afferents from peripheral thermoreceptors, the results show that this area is also the source of the additional respiratory drive at raised body temperature in anaesthetised rats. Experimental Physiology (2000) 85.5, 527-537.

Genetic methodologies have provided powerful means for investigating the cellular and molecular mechanisms of biological functions. Cell-cell and cell-extracellular matrix interactions in particular have been studied in different functional systems with genetically modified animals. In the peripheral and central nervous system, many aspects of specific processes based on such interactions, including myelination, synaptic transmission and plasticity, have been elucidated at the cellular and molecular level. Importantly, genetic approaches have greatly advanced the understanding of pathologies resulting from impaired cellular interactions in the brain and the periphery. In this review, some of the most relevant genetic mouse models in cell biology and the methodologies employed for their production will be described. In addition their usefulness for studies of the mechanisms of hereditary neuropathies, learning and memory, and tumorigenesis will be illustrated.

Many recent findings suggest that the nervous system has efferent effects on bone. A putative role of the sensory innervation has been assessed by using a synchronised rat model of bone resorption after treating adult animals with the neurotoxin capsaicin. Fourteen days after capsaicin treatment (50 mg kg-1) the right maxillary molars were extracted to activate a wave of resorption along the mandibular cortex. The rats were killed 4 days later (i.e. at the peak of resorption in this model), and their right mandibles were processed for histometric evaluation of resorption along the cortex and of calcitonin gene-related peptide (CGRP)- and substance P (SP)-immunoreactive (IR) fibres in the dental pulp. CGRP-IR and SP-IR fibres were significantly reduced in numbers by the capsaicin treatment (by 58 and 49 %, respectively), confirming the success of sensory denervation. The resorption surface was significantly reduced (P < 0.005) versus the sham-treated animals. Although the size of the osteoclast population recruited in the site was not modified, the number of actively resorbing osteoclasts was significantly reduced (P < 0.03). However, the activity of the resorbing cells was not modified. Non-specific esterase-positive osteoclast precursors were also significantly few after capsaicin treatment. These data show that the sensory nervous system is involved in the control of bone resorption at two different levels: (1) in the recruitment of osteoclast precursors, and (2) in regulating the access of recruited cells to the bone surface.

The mdx mouse is the most commonly used animal model for Duchenne muscular dystrophy. We tested the null hypothesis that 20 weeks of clenbuterol treatment (∼2 mg kg-1 day-1) of mdx and control mice would have no effect on the absolute and specific force (Po, kN m-2) and absolute and normalised power output (W kg-1) of extensor digitorum longus (EDL) and soleus muscles. For mdx and control mice, clenbuterol treatment produced modest increases in the mass of the two muscles but did not increase absolute or specific force or normalised power output. For absolute power output, only the EDL muscles of mdx mice showed a difference following treatment, with the power output of treated mice being 118 % that of the untreated mice. The modest effects of clenbuterol treatment on the dynamic properties of skeletal muscle provide little support for any improvement in muscle function for the dystrophic condition. Experimental Physiology (2000) 85.3, 295-299.

The aim of this investigation was to determine whether reflex cardiovascular responses were obtained to localised distension of the intrapulmonary arterial and venous circulations in a preparation in which the stimuli to other major reflexogenic areas were controlled and the lung was shown to possess reflex activity. Dogs were anaesthetised with α-chloralose, artificially ventilated, the chests widely opened and a cardiopulmonary bypass established. The intrapulmonary region of the left lung was isolated and perfused through the left pulmonary artery and drained through cannulae in the left pulmonary veins via a Starling resistance. Intrapulmonary arterial and venous pressures were controlled by the rate of inflow of blood and the pressure applied to the Starling resistance. Pressures to the carotid, aortic and coronary baroreceptors and heart chambers were controlled. Responses of vascular resistance were assessed from changes in perfusion pressures to a vascularly isolated hind limb and to the remainder of the subdiaphragmatic circulation (flows constant). The reactivity of the preparation was demonstrated by observing decreases in vascular resistance to large step changes in carotid sinus pressure (systemic vascular resistance decreased by -40 ± 5 %), chemical stimulation of lung receptors by injection into the pulmonary circulation of veratridine or capsaicin (resistance decreased by -32 ± 4 %) and, in the four dogs tested, increasing pulmonary stroke volume to 450 ml (resistance decreased by -24 ± 6 %). However, despite this evidence that the lung was innervated, increases in intrapulmonary arterial pressure from 14 ± 1 to 43 ± 3 mmHg or in intrapulmonary venous pressure from 5 ± 2 to 34 ± 2 mmHg or both did not result in any consistent changes in systemic or limb vascular resistances. In two animals tested, however, there were marked decreases in efferent phrenic nerve activity. These results indicate that increases in pressure confined to the intrapulmonary arterial and venous circulations do not cause consistent reflex vascular responses, even though the preparation was shown to be reflexly active and the lung was shown to be innervated.

We have examined the effect of tenotomy on the expression of myosin heavy chains (MyHC) in regenerating fast and slow skeletal muscles. Degeneration/regeneration of the left soleus and plantaris of Wistar male rats was induced by an injection into the muscle belly of a myotoxin (snake venom: Notechis scutatus scutatus). MyHC isoform content of regenerating plantaris and soleus muscles were studied 21 days after muscle injury using an electrophoretic technique. Tenotomy of the regenerating plantaris (mechanical underload) did not alter its MyHC expression (P > 0.05). In contrast, tenotomy of the regenerating soleus increased its relative levels of MyHC-2b (P < 0.05) and MyHC-2x/d (P < 0.01), and decreased its relative level of MyHC-1 (P < 0.01). Tenotomy of the synergistic gastrocnemius (overload) tended to decrease the relative level of MyHC-2b in regenerating plantaris (P < 0.07). The effect of tenotomy of the synergistic gastronecmius on the regenerating soleus was different: a decrease in the relative levels of MyHC-1 (P < 0.05) and an increase in the relative level of MyHC-neonatal (P < 0.01). In conclusion, and in contrast to a regenerating slow muscle, a change of mechanical loading by tenotomy did not seem to markedly alter the expression of mature MyHC phenotype in a fast regenerating muscle.

Uptakes of oxygen, glucose and lactate by the gravid uterus, fetus and uteroplacental tissues were measured in chronically catheterized pregnant ponies and their fetuses at mid- and late gestation (term 335 days). Rates of O2 uptake by the gravid uterus, fetus and uteroplacental tissues were significant at both gestational ages and were 2- to 3-fold higher in late gestation than the mid-gestation values of 3338 ± 794, 1352 ± 258 and 2035 ± 602 µmol min-1, respectively (n = 4). Similarly, there were significant uptakes of glucose by the gravid uterus, fetus and uteroplacental tissues at both mid- and late gestation. However, unlike O2 uptake, glucose uptake by the uterus and uteroplacental tissues did not increase between mid- and late gestation. No significant uptakes or outputs of lactate were observed by the uterus or uteroplacental tissues at either gestational age, although there was a significant umbilical uptake of lactate in late but not mid-gestation. There was no change in the distribution of uterine O2 uptake between the fetus and uteroplacental tissues with increasing gestational age. The uteroplacental tissues accounted for about 50 % of the uterine O2 uptake at both gestational ages. In contrast, the proportion of the uterine glucose uptake used by the uteroplacental tissues decreased from 73.2 ± 2.1 % (n = 5) at mid-gestation to 61.1 ± 1.9 % (n = 4, P < 0.02) in late gestation. The gestational changes in uteroplacental carbohydrate metabolism in the mare differ from those seen in the ewe and may have important consequences for the duration and outcome of pregnancy in the mare. Experimental Physiology (2000) 85.5, 539-545.

We used the short-circuit current technique to investigate the possible facilitatory role of epithelium-derived prostaglandin E2 (PGE2) release on Cl- secretion in the mouse colon. Carbachol- (CCh)-stimulated Cl- secretion was reduced by pretreatment with either indomethacin (10 µM), or TTX (1 µM), and when added together, these inhibitors revealed net CCh-stimulated K+ secretion. CCh-stimulated Cl- secretion was partially restored to TTX/indomethacin-treated colons by addition of a subsecretory concentration of PGE2 (1 nM). In acutely isolated, unstimulated crypt cells, we measured PGE2 release at a similar level. We conclude that autocrine release of PGs from epithelial cells is sufficient to support the CCh-induced Cl- secretory response and is a likely co-factor in this response.

Most of the transcranial Doppler (TCD) experimental studies on cerebral haemodynamics have been performed in the rabbit because of the similarity between its Willis circle and that of the human, but these studies have mainly been limited to the basilar artery. The present study was aimed at extending the use of TCD sonography to all other large cerebral arteries. In anaesthetised rabbits, these arteries were insonated from three different recording sites, i.e. top-cranial, suboccipital and orbital, using a two-channel pulsed Doppler device equipped with 4 and 8 MHz probes. First, discrimination between intra- and extracranial arteries was achieved through a standard 'rebreathing' test (hypercapnic-hypoxic stimulation). The distinctive blood velocity response patterns, reflecting the different extents of metabolic reactivity in intra- and extracranial territories, are described and discussed. Intracranial arteries were then identified on the basis of their response to ipsi- and contralateral common carotid artery occlusion. This procedure allowed recording from the following arteries: anterior common trunk, anterior cerebral, internal carotid, middle cerebral and basilar; the latter could be simultaneously monitored with any of the others. This study provides an experimental model allowing investigation of regional differences in the haemodynamic response to neurogenic and pharmacological stimuli.

The goal of the present study was twofold: (1) to detect the possible storage of dietary polyamines (PAs) in various tissues and (2) to investigate the role of dietary PAs in the differentiation of the pig intestinal epithelium. A first experimental series was designed to assess the accumulation of either milk PAs (mostly spermidine) or orally administered spermine (SPM) in piglet red blood cells (RBCs) and plasma, a preliminary stage in their distribution to growing and storage organs. Though PA concentrations of piglet RBCs and plasma were generally significantly higher than their sow counterparts, our experimental conditions failed to demonstrate that this increase could stem from ingested PAs. A second experimental series dealt with the determination of disaccharidase specific activities in proximal and distal parts of piglet gut on the 26th and 29th days after birth (preweaning time). In agreement with observations made previously on rat pups, we observed an increase in maltase specific activity (SA) at the end of the suckling period (the observed increase in sucrase SA was not significant). However, orally administered SPM did not affect this activity. Compared to the constant protein concentrations observed in both parts of the gut, the pancreatic protein content decreased sharply between the 26th and 29th postnatal days. At the same time pancreatic concentrations of spermidine (SPD) also decreased, suggesting that some pancreatic PAs were released as the organ secreted its proteins. In accordance with this hypothesis, we recorded SPM and SPD in pancreatic juice. The increases in PA concentrations seemed to follow the protein secretion pattern (i.e. PA concentrations reached a maximal value when the protein concentration was highest). The presence of PAs in pancreatic juice could be indicative of a control mechanism exerted by the pancreas on PA-induced growth and differentiation of porcine intestinal epithelium. Experimental Physiology (2000) 85.3, 301-308.

Incrementally applied static stretch over 3 weeks resulted in a 72 % increase in the weight of the in situ latissimus dorsi muscle in rabbits. True growth rather than tissue oedema was confirmed by increases in the protein content (130 %), the cross-sectional area of the type I fibres (30 %) and the muscle length (i.e. number of sarcomeres in series increased 25 %). Despite an increase in the proportion of fibres staining positive for the enzyme succinate dehydrogenase (SDH), the myosin ATPase stain showed no appreciable fibre type transformation. While total power output in the stretched muscle was unchanged, its maximum mass specific power output, as determined by oscillatory work loops, was decreased by 50 %. The cross-sectional area that was occupied by connective tissue increased from 15 to 19 % in the stretched muscles, with a concomitant increase in passive energy dissipation. Some incrementally stretched muscles were then allowed an additional 3 weeks of maintained stretch to determine whether the adaptive changes would be preserved or reversed. Previous gains in muscle weight, length and area of type I fibres all remained. In contrast, the connective tissue content and the passive properties returned to control values during this period.

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are able to lyse suitable target cells. CTLs recognize a specific peptide epitope presented by major histocompatibility complex (MHC) class I molecules on the target cells, whereas NK cells lyse targets that express no or low MHC class I molecules. Using perforin-deficient mice, we provide evidence that both NK cells and CTLs exclusively use perforin-dependent cytolysis as an effector mechanism in vitro, as well as in vivo. This review summarizes the most important role of perforin-dependent cytolysis in a wide variety of bacterial and viral infections, in tumour surveillance, in immunopathology and in autoimmunity.

Intravenous injection of growth hormone in anaesthetized pigs has been shown to cause coronary vasoconstriction by antagonizing the vasodilatory effects of β2-adrenergic receptors. Because nitric oxide is believed to modulate or mediate β2-adrenergic effects, the present study was undertaken in the same experimental model to determine the role of nitric oxide in the above response to growth hormone. In fourteen pigs anaesthetized with sodium pentobarbitone, changes in left circumflex or anterior descending coronary blood flow caused by intravenous injection of 0.05 i.u. kg-1 of growth hormone at constant heart rate and arterial blood pressure were assessed using electromagnetic flowmeters. In a first control group of six pigs, growth hormone caused a decrease in coronary blood flow which averaged 13.1 % of the baseline values. In a second group of eight pigs, intravenous administration of Nω-nitro-L-arginine methyl ester (L-NAME) was used to block the endothelial release of nitric oxide. In these pigs, the subsequent injection of growth hormone did not cause any significant changes in coronary blood flow, even when performed after reversing the increase in arterial blood pressure and coronary vascular resistance caused by L-NAME with continuous intravenous infusion of papaverine. These results indicated that the coronary vasoconstricting effect of growth hormone, known to involve antagonism of β2-adrenergic vasodilatory effect, was mediated by inhibition of nitric oxide release.

We investigated the influence of exogenous thyroxine (T4) administration in conjunction with level of feeding on adipose tissue and liver growth in postnatal lambs. Pairs of lambs were fed either 100 g (i.e. low fed) or 200 g (i.e. high fed) of milk powder per litre of reconstituted milk replacer over the first month of life. Half of the pairs of lambs were fed a bolus dose of T4 (15 mg (kg body weight)-1) daily until 8 days of age. Perirenal adipose tissue and hepatic tissue were sampled at either 8 or 35 days of age. High fed lambs grew faster, possessed more adipose tissue and had larger livers than low fed lambs at 8 and 35 days of age. T4 administration resulted in a lower thermogenic activity (i.e. GDP binding) in adipose tissue at 8 days of age in low, but not high fed lambs. There was no difference between groups in colonic temperature or oxygen consumption. Between 17 and 35 days of age high fed lambs previously treated with T4 had lower daily milk consumption than untreated siblings, but still attained the same growth rate. Plasma insulin-like growth factor-I concentrations were greater in high than low fed lambs, a relationship that was not influenced by T4 treatment. In adipose tissue, iodothyronine 5′ deiodinase activity was not influenced by T4 administration and was greater in high than low fed lambs. Hepatic iodothyronine 5′ deiodinase activity was not influenced by T4 administration in low fed lambs, but was reduced by T4 in high fed lambs. In conclusion, T4 administration over the first 8 days of life can accelerate the rate of decline in thermogenic activity of uncoupling protein-1. This effect is not observed when the level of feeding is increased. Following withdrawal of T4 treatment, high fed T4-treated lambs were able to maintain the same growth rate as untreated lambs despite having a lower food intake.

The magnitude of diuresis and natriuretis produced by oxytocin in the female rat has been shown to be dependent on the stage of the oestrous cycle. A study has been performed to determine the role of ovarian steroids in modulating the renal response to oxytocin infused at a rate of 100 fmol min-1 in hypotonic saline. Observations were performed on ovariectomised rats with and without steroid treatment and rats with suppressed oestrous cycles following treatment with the long-acting gonadotrophin-releasing hormone analogue Zoladex, given as a 100 mg S.C. depot, or the antioestrogen tamoxifen, given as three daily injections of 1 mg. Steroid treatment comprised a single dose of 10 µg oestradiol benzoate or 2.0 mg progesterone, or a combination of the two hormones given 30 h apart. Ovariectomy had no significant effect on plasma oxytocin concentrations, although progesterone treatment produced an increase. The natriuresis in ovariectomised animals of 27 % was smaller (P < 0.05) than that seen on the day of pro-oestrus and closer to that seen in the intact rat at oestrus. The responses in animals with suppressed oestrous cycles following treatment with tamoxifen were not significantly different from those observed following ovariectomy, neither was the diuresis following Zoladex. The renal responses were greater following treatment with oestradiol than in the ovariectomised group. Thus ovarian steroids do influence the renal responsiveness to oxytocin with oestradiol augmenting the response.

We investigated anaerobic threshold (thetaL) gas exchange kinetics and maximal oxygen uptake (VO2,max) among older men with reduced left ventricular end-diastolic filling (LVDF). Ten men (mean age, 73 years) with LVDF impairment and low fitness, but without other cardiovascular dysfunction were studied. Treatments compared to control included: 5 days, high intensity exercise training protocol; 5 days, calcium channel blockade (240 mg verapamil); 21 days, detraining/washout; and 5 days, combined treatments. Results indicated no changes in resting left ventricular systolic function with any treatment. Significant resting diastolic function changes included increased early:late flow velocity (control, 0.87; training, 1.28; verapamil, 1.32), and a decreased isovolumic relaxation time (control, 0.10 s; training, 0.08 s; verapamil, 0.08 s). The combined treatments were not additive. Sub-threshold oxygen uptake kinetics (tauVO2, s) were significantly faster following either training or verapamil (tauVO2,control, 62 ± 12; tauVO2,training, 44 ± 9; tauVO2,verapamil, 48 ± 10) and combined treatments (tauVO2, 41 ± 8). V O2,max (ml kg-1 min-1) was significantly increased (control, 21.8 ± 2.2; training, 27.3 ± 2.2; verapamil, 25.2 ± 3.4; combined treatments, 26.9 ± 2.3). Increasing ventricular preload with either exercise training or calcium channel blockade was coincident with faster tauVO2 and increased VO2,max. Experimental Physiology (2000) 85.5, 547-555.

Genetically modified mice have become an invaluable tool in modern biomedical and basic research. This review provides an overview of knockout and transgenic mice studied with regard to their cutaneous wound healing properties. In addition, several gene transfer studies are briefly introduced, which have further highlighted our knowledge on individual gene function in wound healing

Mammals that use the ventilatory system as the principal means of increasing heat loss, i.e. that pant, show two fundamental changes in the control of breathing at raised temperatures. First, alveolar ventilation increases by more than, rather than in proportion to, the increase in CO2 production. Second, hypocapnia no longer causes apnoea. Rats do not use the ventilatory system as the principal means of increasing heat loss, so we have investigated whether rats also show these two changes at raised temperatures. Breathing was detected from diaphragmatic electromyogram (EMG) activity. Anaesthesia and hyperoxia were used to minimise behavioural and hypoxic drives to ventilation and arterial PCO2 (Pa,CO2) was controlled using mechanical ventilation. At 36.6 ± 0.1 °C, breathing was absent as long as Pa,CO2 was held below a threshold level of 32.9 ± 0.7 mm Hg (n = 14) under steady-state conditions. When body temperature in rats was raised above 37 °C, both fundamental changes in the control of breathing became apparent. First, at 39 °C the mean Pa,CO2 level during spontaneous breathing (39.6 ± 5.4 mm Hg, n = 4) fell by 3.9 ± 1.4 mm Hg (P < 0.05, Student's paired t test). Second, at 39.9 ± 0.1 °C breathing was present when mean Pa,CO2 levels were only 18.2 ± 1.5 mm Hg (n = 14), the lowest mean Pa,CO2 level we could achieve with mechanical ventilation. We calculate, however, that at 39.9 °C, the drive to breathe from the increased CO2 production alone would not sustain breathing below a Pa,CO2 level of 27.8 ± 1.4 mm Hg (n = 13). In rats at raised body temperatures therefore a respiratory drive exists that is in addition to that related to the increase in CO2 production. Experimental Physiology (2000) 85.3, 309-319.