Skeletal muscle capillarity and fibre cross-sectional area were investigated within and between diaphragm (Diaph), extensor digitorum longus (EDL), soleus (SOL) and tibialis anterior (TA) muscles of control and chronic hypoxic (12 % O2 for 6 weeks) adult male Wistar rats (final body mass ~355 g). Cryostat sections were stained for alkaline phosphatase activity to depict all capillaries, and for succinic dehydrogenase to demonstrate regional differences in oxidative capacity within the muscles. Hypoxia-induced angiogenesis occurred in all muscles (P < 0.01), with capillary-to-fibre ratio (C:F) being higher in the more active and oxidative muscles, Diaph (27 %) and SOL (26 %), than phasically active and glycolytic muscles, TA (21 %) and EDL (15 %). Diaph, SOL and EDL maintained fibre size, and hence showed an increased capillary density (CD) and reduced intramuscular diffusion distance (DD), whereas TA showed fibre hypertrophy and maintained CD and DD compared to control muscles. The extent of angiogenesis among different regions of muscle varied so as to suggest that muscle fibre size has an additional influence on capillary growth during chronic systemic hypoxia, which is progressive over an extended period of systemic hypoxia. Experimental Physiology (2002) 87.3, 287-291.

Diabetes mellitus and alcohol (ethanol) intake are two positively correlated major risk factors for cardiovascular abnormalities. However, the interaction of the two on cardiac function is largely unknown. The purpose of the present study was to examine the impact of genetically predisposed diabetes on acute ethanol exposure-induced cardiac contractile depression at the myocyte level. Ventricular myocytes from spontaneously biobreeding diabetes-prone (BBDP) rats and their diabetes-resistant littermates (BBDR) were stimulated to contract at 0.5 Hz. Contractile properties analysed include: peak shortening amplitude (PS), time-to-PS (TPS), time-to-90 % relengthening (TR90) and maximal velocities of shortening/relengthening (± dL/dt). BBDP rats displayed hyperglycaemia, reduced body weight gain and increased cardiac, hepatic and renal size. Myocytes isolated from BBDP rat hearts exhibited prolonged TPS and TR90 associated with normal PS and ± dL/dt, compared with myocytes from the BBDR group. Acute ethanol exposure (80-640 mg dl-1) caused a concentration-dependent inhibition of PS in both BBDR and BBDP myocytes. However, the degree of inhibition of PS was significantly reduced in BBDP myocytes compared to that of BBDR myocytes. The maximal inhibition was 52.9 % and 28.4 % in BBDR and BBDP groups, respectively. Ethanol significantly depressed ± dL/dt in both BBDR and BBDP myocytes. In addition, ethanol did not affect TPS or TR90 in either the BBDR or BBDP group. Collectively, these results suggest that the ethanol-induced depression in cardiac myocyte contraction may be 'shadowed' by genetically predisposed diabetes. Experimental Physiology (2002) 87.3, 293-298.

We examined whether protein kinase C (PKC) modulates the transport systems involved in bicarbonate movements across the plasma membranes of rat jejunum. Results of enzymatic assays provide evidence that under basal conditions conventional PKC (cPKC) is present in both basolateral membranes (BLMs) and apical (brush border) membranes (BBMs) of the enterocyte. In BLMs the basal expression of the kinase is low compared to expression in BBMs; however, treatment with Ca2+ and phorbol 12-myristate 13-acetate (PMA) causes a significant increase, thus suggesting an asymmetrical kinase translocation. To explore the effect of PKC activation on membrane-bound transport mechanisms, 'in vitro' phosphorylated membrane vesicles were used to perform uptake studies. Results suggest that PKC activation exerts an inhibitory effect on the basolateral Cl--HCO3- antiporter, whereas the basolateral HCO3- conductive pathway seems to be stimulated and Cl- conductance unaffected. The apical, but not basolateral, Na+-H+ exchanger is inhibited by PKC activation. The specificity of the response to PKC was confirmed by using the kinase inhibitor staurosporine or the inactive phorbol ester 4-α-PMA. The inhibition of both apical Na+-H+ and basolateral Cl--HCO3- exchange activities suggests that the overall action of PKC causes a reduction of transepithelial bicarbonate transport. Experimental Physiology (2002) 87.3, 299-309.

It was recently demonstrated that ammonia inhibits sodium absorption in the proximal colon of rats. In order to investigate the effect of luminal ammonia in the distal colon, sodium and chloride transport were measured in Ussing chambers. Under short-circuit conditions, distal colon absorbed sodium and chloride. When luminal ammonia (30 mmol l-1) was present, sodium and chloride absorption was diminished. Inhibition of the two Na+-H+ exchanger isoforms NHE2 and NHE3, which are known to be located in the apical membrane of the distal colon epithelium, failed to influence the effect of ammonia on transepithelial sodium and chloride fluxes. The inhibitory effect of ammonia was eliminated under the following conditions: after block of carbonic anhydrases with acetazolamide, in the presence of an unspecific blocker of Na+-H+ exchangers, and under chloride-free conditions. Ammonia did not alter electrogenic sodium absorption. These results demonstrate that luminal ammonia inhibits sodium and chloride absorption in rat distal colon. We suggest that ammonia inhibits NaCl absorption by interfering with a Na+-H+ exchanger that is not NHE2 or NHE3. Experimental Physiology (2002) 87.3, 311-319.

In this study the effects of changes in extracellular magnesium ([Mg2+]o) and calcium ([Ca2+]o) concentrations on basal and on nerve-mediated and acetylcholine (ACh)-evoked in vitro amylase release and calcium mobilization were investigated in rat parotid gland tissue. In the presence of a normal (2.56 mM) [Ca2+]o, both zero (0 mM) and an elevated (10 mM) [Mg2+]o significantly attenuated basal and ACh-evoked amylase release compared to the response obtained in normal (1.1 mM) [Mg2+]o. During electrical field stimulation (EFS) of parotid tissues, only elevated [Mg2+]o reduced amylase release. In a Ca2+-free medium, both basal and ACh-evoked amylase output were markedly reduced compared to the responses obtained under similar conditions in normal [Ca2+]o. Again, the ACh-induced amylase release in a Ca2+-free solution was larger in normal [Mg2+]o than when the [Mg2+]o was either zero or was elevated to 10 mM. Perturbation of [Mg2+]o had no significant effect on basal intracellular free calcium concentration ([Ca2+]i) in parotid acinar cells loaded with the fluorescent Ca2+ indicator fura-2. Both zero Mg2+ and an elevated [Mg2+]o significantly reduced the ACh-induced rise in the peak and the plateau phase of the Ca2+ transient that was seen in normal [Mg2+]o. In parotid acinar cells loaded with the fluorescent Mg2+ indicator magfura-2, ACh elicited a gradual decrease in intracellular free Mg2+ concentration ([Mg2+]i) to below the basal level. The results indicate that both hypo- and hypermagnesaemia may reduce both basal and ACh-evoked amylase secretion from the salivary gland. As far as the ACh-evoked response is concerned, the effect may be exerted by a decrease in cellular Ca2+ transport. Experimental Physiology (2002) 87.3, 321-326.

The vasoregulatory role of nitric oxide (NO) and angiotensin II type 1 (AT1) receptors in the circulation of the submandibular gland (SMG) of rats was studied. The glandular blood flow was determined by means of laser Doppler flowmetry and rubidium isotope technique. The data obtained by these two methods correlated well (r = 0.77; P < 0.01). The AT1 receptor antagonist candesartan (0.5 mg kg-1, I.V.) reduced the vascular resistance in the SMG by 37 % (P < 0.05). By contrast, the NO synthase blocker L-NAME (15 mg kg-1, I.V.) significantly increased vascular resistance in the SMG both in candesartan-treated (P < 0.001) and non-treated (P < 0.001) animals. The increase in resistance was greater (P < 0.05) after previous blockade of AT1 receptors. These findings suggest that the AT1 receptors have an important role in the vasoregulation of the SMG in the rat. As a result of AT1 blockade, NO-dependent tone of glandular vessels may be enhanced significantly. Experimental Physiology (2002) 87.3, 327-333.

This investigation tested the hypothesis that the effects of nitric oxide synthase on myocardial capillary perfusion were reduced during myocardial stunning. Anaesthetized open-chest rabbits were assigned to either a control group or a group treated with a nitric oxide synthase inhibitor. To induce myocardial stunning, a coronary artery was occluded (for 15 min) and then reperfused (for 15 min) twice. During reperfusion, rabbits were given either saline or NG-nitro-L-arginine methyl ester (L-NAME, 30 mg kg-1) followed by I.V. injection of 150 mg kg-1 fluorescein isothiocyanate (FITC)-labelled dextran (molecular weight, 150 000) for 14 s. Fluorescence microscopy was used to identify the perfused vessels and an alkaline phosphatase stain was used to locate the total microvasculature. The 'closest-individual' method was used to estimate the geometric distribution of capillaries. No significant differences were observed in the total volume fraction (mm3 of capillaries per mm3 of tissue) between the control and stunned regions in either the saline- (0.045 ± 0.008 and 0.042 ± 0.009, respectively) or the L-NAME-treated hearts (0.060 ± 0.010 and 0.049 ± 0.005, respectively). There were no significant differences in the percentage volume fraction of perfused capillaries between the control and the stunned regions (49 ± 4 % and 54 ± 4 %, respectively) in saline-treated hearts. In hearts treated with L-NAME, the percentage of perfused capillaries was significantly reduced. The reduction was significantly greater in the control region (~27 %) than the stunned region (~17 %). Closest-individual analysis of the perfused capillary distribution in both groups demonstrated a similar unchanged distribution. Thus, nitric oxide synthase is an important regulator of basal coronary capillary perfusion, and its effects are significantly reduced by myocardial stunning. Experimental Physiology (2002) 87.3, 335-342.

The acute and long-term effects of blockade of nitric oxide (NO) production were studied in six chronically catheterised fetal sheep aged from 116 and 118 days; six untreated fetal sheep received injections of saline. Injection of 10 mg (kg maternal body wt)-1 of the nitric oxide synthase (NOS) inhibitor Nω-nitro-L-arginine (NOLA) to the fetus, caused an immediate rise in fetal mean arterial pressure (MAP, P < 0.005) and a reflex fall in fetal heart rate (FHR, P < 0.001). Plasma renin concentration (PRC) fell from 8.4 ± 3.3 to 1.5 ± 0.3 ng ml-1 h-1 (P < 0.001) and was dependent on MAP (P = 0.001). Glomerular filtration rate (GFR) tended to increase, but renal blood flow (RBF) velocity decreased (P < 0.001). Thus filtration fraction (FF) increased (P < 0.025). Urine flow and sodium excretion increased (P < 0.001 for both). Fractional sodium reabsorption decreased (P < 0.05). In fetuses treated with NOLA, arterial pressure was found to affect glomerular haemodynamics and renal tubular handling of sodium. No such relationships were observed in untreated fetuses. The vascular responses to acetylcholine tended to be less (P = 0.07) and the responses to noradrenaline were enhanced in NOLA-treated fetuses. There were no changes in untreated fetuses. Fetuses were then injected twice daily with either 5 mg kg-1 NOLA or saline for the next 2 days. On the 4th day, injection of 10 mg kg-1 NOLA did not have any effects on MAP, FHR or renal function. However, the pressor responses to angiotensin II (Ang II) were enhanced (P < 0.005), as was the response to noradrenaline but to a lesser extent. It is concluded that endothelial production of NO maintains normal fetal blood pressure, renal vascular resistance and fetal renal function. When NO production was blocked by repeated injections of NOLA, other vasodilator pathways took over the maintenance of cardiovascular and renal vascular tone. However, alterations in both cardiovascular and renal function were still present. That is, there was increased pressor sensitivity to exogenous Ang II and unmasking of effects of arterial pressure on glomerular and tubular function. Experimental Physiology (2002) 87.3, 343-351.

Physiological changes occurring in the mother during pregnancy can determine the outcome of pregnancy in terms of birthweight and neonatal viability. Maternal adaptations include plasma volume expansion linked to enhanced activity of the renin-angiotensin system (RAS). The present study was designed to determine whether these changes occur very early in gestation, and the extent to which maternal nutrient restriction may compromise the maternal RAS. Using sheep, we have investigated the effects of pregnancy per se, maternal nutrient restriction and later restoration of maternal diet on maternal body weight, plasma volume and plasma renin concentration (PRC), and angiotensinogen (Aogen) and arginine vasopressin (AVP) concentration. During the period of placental growth (i.e. 28-80 days gestation) ewes were fed either a nutrient-restricted (NR) diet or were well fed (WF). NR ewes consumed between 3.2 and 3.8 MJ day-1 of metabolisable energy (ME) which is close to 60 % of requirements taking into account the ME required for both ewe maintenance and growth of the conceptus in order to produce a 4.5 kg lamb at term. WF ewes consumed 150 % of ME requirements. Restoration of maternal diet between 80 and 140 days gestation (i.e. fed to satiety and consuming between 8 and 10.9 MJ day-1, which is close to 150 % of ME requirements) followed previous nutrient restriction. Between pre-conception and 28 days gestation, plasma volume increased in conjunction with a decline in PRC and Aogen concentration. During the period of nutrient restriction ewe body weight did not increase and plasma volume was lower in NR than WF ewes. During this time there was no effect of maternal nutrition on PRC; however, Aogen concentration was lower in the NR group. From 80 days gestation following the rise in food intake for previously NR ewes, greater increases in ewe body weight, plasma volume and PRC occurred up to term compared with ewes that were well fed throughout gestation. Plasma AVP concentration was not significantly affected by either maternal nutrition or gestational age. In conclusion, the stimulus of moderately severe maternal nutrient restriction evoked smaller rises in maternal weight, plasma volume and Aogen concentration than occurred in ewes that were well fed throughout gestation. Following the restoration of maternal diet after 80 days gestation, PRC gradually rose to peak at term. These adaptations in the maternal RAS during the critical period of placental growth may have long-term effects on fetal development. Experimental Physiology (2002) 87.3, 353-359.

The aim of the present study was to investigate the mechanism of the retrograde transfer of ovarian steroid hormones from the ovarian lymphatic and venous effluent to the arterial blood supplying the ovary. In the first experiment, reproductive organs were collected from gilts in the luteal (n = 10) and follicular (n = 10) phase of the oestrous cycle. The ovary with the mesovarium was isolated and perfused through the ovarian artery with warmed, oxygenated autologous blood. The concentrations of progesterone and oestradiol in ovarian arterial blood increased on passing through the ovarian artery to the ovary, in the luteal phase, from 20.3 ± 2.1 to 31.4 ± 3.9 ng ml-1 (P < 0.001) and from 6.2 ± 0.8 to 11.4 ± 1.4 pg ml-1 (P < 0.001), respectively, and in the follicular phase, from 1.2 ± 0.2 to 2.2 ± 0.4 ng ml-1 (P < 0.001) and from 8.2 ± 1.8 to 13.2 ± 2.3 pg ml-1 (P < 0.001), respectively. Approximately 17.5 ± 3.9 % of the progesterone and 12.6 ± 1.7 % of the oestradiol found in the ovarian venous effluent was retrogradely transferred from the ovarian venous blood to the ovary in the luteal phase. In the follicular phase, these values were 10.1 ± 2.0 % and 8.6 ± 1.4 %, respectively. The efficiency of retrograde transfer of oestradiol and the rate of retrograde transfer of progesterone differed between phases of the oestrous cycle (P < 0.05 and P < 0.0001, respectively). A direct relationship between the concentration of the steroids in the venous effluent and the efficiency and rate of the retrograde transfer to the ovary was not found. In the second experiment (luteal phase, n = 10; follicular phase, n = 5), the concentration of progesterone and oestradiol increased in both ovarian arterial blood (P < 0.0001) and in the venous effluent (P < 0.0001) after administration of the steroids into the lymphatic vessels of the isolated mesovarium with separated ovary. In the third experiment (follicular phase, n = 5), with the mesovarium isolated after the ovary was removed and ovarian venous blood flowing out under the force of gravity (without the blood pressure in the ovarian vein), it was demonstrated that the veno-venous network covering the branches of the ovarian artery was supplied with the blood flowing out from the mesovarian tissue and that the filling of the veno-venous network was dependent on the blood pressure in the ovarian artery. We conclude that the effective retrograde transfer of steroid hormones from ovarian venous and lymphatic effluent to the ovary is accomplished not only by the classical counter-current exchange mechanism, but also as a result of complex processes that may be dependent on a specific part of the circulation of the blood and lymph in the periovarian vascular complex of the mesovarium. Experimental Physiology (2002) 87.3, 361-371.

To investigate the role of hypoxia as a stimulus to the early upregulation of vascular endothelial growth factor (VEGF) in fast skeletal muscles during chronic low frequency stimulation, blood flow, oxygen consumption, VEGF expression and capillary:fibre ratio were measured in chronically stimulated tibialis anterior and extensor digitorum longus (EDL) muscles in rabbits and rats. No differences were found in blood flow, oxygen consumption and extraction between rabbit muscles stimulated for 2 or 4 days (8 h on-16 h off) and controls. Muscle PO2 polarographically measured immediately at the end of stimulation on day 2 was also no different from control under resting conditions (10.7 ± 1.6 vs. 9.5 ± 1.2 Torr, n.s.). Unlike control muscles, however, PO2 in 2 day stimulated muscles did not increase immediately after a further acute bout of contractions. This difference was not apparent after similar acute contractions in 4 day stimulated muscles. The involvement of VEGF in early angiogenesis in stimulated muscles was studied in serial cryosections of rat EDL. The proportion of capillaries positively immunostained for VEGF increased from 25 ± 1 % to 40 ± 1 % (P < 0.05) in muscles removed on day 2 immediately at the end of chronic stimulation; it decreased slightly after 16 h rest, and increased again after 4 days of stimulation. Capillary:fibre ratio was unchanged throughout the experiment. Capillary cell proliferation increased only after the rest period on day 2 (20-fold increase) and day 4 (12-fold increase), indicating angiogenesis in progress. Thus the timing of transient hypoxia and increase in capillary-linked VEGF in stimulated muscles, albeit in different species, was similar, and increased VEGF staining and capillary cell proliferation occurred even after the hypoxia had resolved. This suggests (1) a connection between hypoxia and VEGF during the early stages of stimulation, although ensuing capillary proliferation may thereafter rapidly correct for local hypoxia, and (2) that the subsequent angiogenesis and VEGF expression are dependent on factors other than hypoxia. Experimental Physiology (2002) 87.3, 373-381.

The rapid increase in body mass that often occurs following creatine (Cr) supplementation is believed to be due to intracellular water retention. The purpose of this study was to determine whether Cr consumption alters the magnetic resonance (MR) transverse relaxation (T2) distribution of skeletal muscle. Transverse relaxation can be used to model water compartments within a cell or tissue. In this double-blind study, subjects were asked to supplement their normal diet with creatine monohydrate (20 g day-1 for 5 days) mixed with a grape drink (Creatine group, n = 7), or the grape drink alone (Placebo group, n = 8). Phosphorous MR spectroscopy was used to determine the effectiveness of the supplementation protocol. Subjects that responded to the Cr supplementation (i.e. showed a > 5 % increase in the ratio of the levels of phosphocreatine (PCr) and ATP) were placed in the Creatine group. Both proton MR imaging and spectroscopy were used to acquire T2 data, at 1.89 T, from the flexor digitorum profundus muscle of each subject before and after supplementation. Following the supplementation period, the Creatine group showed a gain in body mass (1.2 ± 0.8 kg, P < 0.05, mean ± S.D.), and an increase in PCr/ATP ratio (23.8 ± 16.4 %, P < 0.001). Neither group showed any changes in intracellular pH or T2 calculated from MR images. However, the spectroscopy data revealed at least three components (> 5 ms) at approximately 20, 40 and 125 ms in both groups. Only in the Creatine group was there an increase in the apparent proton concentration of the two shorter components combined (+5.0 ± 4.7 %, P < 0.05). According to the cellular water compartment model, the changes observed in the shorter T2 components are consistent with an increase in intracellular water. Experimental Physiology (2002) 87.3, 383-389.

We have investigated the febrile responses of New Zealand White rabbits to a Gram-negative pyrogen (bacterial lipopolysaccharide (LPS) from Salmonella typhosa), commonly associated with systemic infection, and a Gram-positive pyrogen (Staphylococcus aureus), more frequently associated with superficial soft tissue infection, each administered via one of four different routes (intravenous, intramuscular, subcutaneous or intraperitoneal) at each of three different doses (LPS: 0.1, 1 and 10 µg kg-1; S. aureus: 1.5 × 107, 1.5 × 108 and 1.5 × 109 cell walls kg-1). Intravenous administration of LPS evoked rapid, dose-dependent biphasic fever. Injection of LPS by the other routes also evoked dose-dependent fever. However, these fevers were monophasic, had increased latency of onset, and were of lower amplitude. It is important to note that a dose of approximately 10 and 100 times that of the standard intravenous dose was required to produce a similar peak rise in temperature when administered subcutaneously and intraperitoneally, respectively. Intravenous injection of the highest dose of S. aureus evoked dose-dependent biphasic fever, with short latency of onset, which was very similar to that induced by intravenous LPS. At lower doses, intravenous S. aureus induced monophasic fever. No fever occurred when the same doses of S. aureus were administered by any other route. We conclude that any of the four routes may be used for the study of LPS-induced fever, provided that the doses are adjusted. However, studies of S. aureus-induced fever, and detection of contamination with either pyrogen, requires intravenous injection. Experimental Physiology (2002) 87.3, 391-399.