bluuebunny/crossref_metadata_2025 huggingface dataset:
Visualization of nitric oxide formation in cell cultures and living tissue
Authors: Wiklund  , Iversen  , Leone  , Cellek  , Brundin  , Gustafsson  , Moncada
Published: March 2003

We have visualized nitric oxide (NO) released from cell cultures and living tissue. NO was visualized by a reaction with luminol and hydrogen peroxide to yield photons which were counted using a microscope coupled to a photon counting camera. Murine macrophages were activated with interferon‐gamma (IFN‐γ) and endotoxin (LPS). Cultured endothelial cells were stimulated with bradykinin, and neurones in the guinea‐pig myenteric plexus and the rabbit hypogastric nerve trunk were electrically stimulated. There was a marked increase in photons emitted from the cultured cells as well as from the living tissues during stimulation. The stimulation‐induced photon emission was markedly reduced by inhibition of nitric oxide synthase (NOS); removal of L‐arginine from the medium also decreased photon counts. The present method allowed integration times in the order of minutes to improve signal‐to‐noise ratio. However, the high sensitivity of this method also makes it possible to generate an image in seconds, allowing the production of real time films. Photon emission was enhanced under conditions known to increase NO production, and diminished in the presence of NO inhibitors. Thus, this method has demonstrated specificity for the
L‐arginine:NO pathway from a wide range of biological sources such as cultured cells and living tissues, and has the potential for real time imaging of NO formation, with high temporal and spatial resolution.
Phosphorylation and activation of the endothelial nitric oxide synthase by fluid shear stress
Authors: Fisslthaler  , Dimmeler  , Hermann  , Busse  , Fleming
Published: March 2003

Fluid shear stress activates the endothelial nitric oxide (NO) synthase (eNOS) by a mechanism which does not require an increase in the intracellular concentration of free Ca2+ ([Ca2+]i), and is sensitive to several kinase inhibitors. Although phosphorylation of eNOS has been suggested to regulate enzyme activity, the mechanism of eNOS activation is still unclear. Here we demonstrate that fluid shear stress elicits the phosphorylation of eNOS on tyrosine and serine residues. Inhibition of phosphatidylinositol 3‐kinase (PI3K), using wortmannin or a dominant negative mutant of its downstream target, Akt (protein kinase B), prevented the maintained serine phosphorylation and activation of eNOS. Enhancing eNOS phosphorylation by inhibiting serine/threonine phosphatases, increased eNOS activity by approximately twofold, as assessed by the accumulation of intracellular cyclic GMP, without increasing the intracellular concentration of free Ca2+. These data suggest that shear stress activates a pathway involving PI3K and the serine/threonine kinase Akt, which phosphorylates eNOS. This phosphorylation directly increases eNOS activity at resting [Ca2+]i, thus rendering the shear stress‐induced activation of eNOS apparently Ca2+‐independent.
Interactions of the renin–angiotensin system and neuronal nitric oxide synthase in regulation of cyclooxygenase‐2 in the macula densa
Authors: Harris  , Cheng  , Wang  , Zhang  , McKanna
Published: March 2003

Cyclooxygenase‐2 (COX‐2) expression in rat kidney is localized to the macula densa and the immediately proximal cTALH and increases after salt restriction. Either ACE inhibitors or AT1 receptor blockers increase COX‐2 expression in both control and salt‐restricted animals, suggesting that the RAS activation feedback inhibits renal cortical COX‐2 expression. To determine whether increased COX‐2 expression in response to ACE inhibition mediated increases in renin production, rats were treated with Captopril for 1 week with or without the specific COX‐2 inhibitor, SC58236. Plasma renin activity increased significantly in the Captopril group. This increase was partially reversed by simultaneous treatment with SC58236. Kidney renin activity also increased in the Captopril group compared with control, which was also significantly inhibited by SC58236 treatment. Because of the localization of bNOS to MD and surrounding cTALH, the current study investigated the role of NO in the regulation of COX‐2 expression. Rats were fed a normal diet, low salt diet or low salt diet combined with captopril and half of them were treated with the neuronal NOS inhibitor, 7‐NI, and half with vehicle. After 7 days, mRNA was extracted and the microsome proteins purified from renal cortex. COX‐2 mRNA expression was measured by Northern‐blot and normalized with GAPDH. 7‐NI treatment decreased COX‐2 mRNA and immunoreactive COX‐2 expression in each group. In summary, these studies indicate that COX‐2 from macula densa/cTALH is a regulator of renin production and release. Angiotensin II may be a negative regulator of cTALH/macula densa COX‐2 expression, and NO may mediate increased renal cortical COX‐2 expression seen in volume depletion. These studies suggest important interactions between the NO and COX‐2 systems in the regulation of arteriolar tone and the renin–angiotensin system by the macula densa.
Exocytosis and endocytosis in juxtaglomerular cells
Authors: Friis  , Jensen  , Hansen  , Andreasen  , Skøtt
Published: March 2003

The cellular events related to secretion of renin are not well understood. Here we review some of the evidence that has led to the current understanding of renin secretion as a process that involves exocytosis as the predominant mode of secretion. This is based on the observation of occasional fusion events between secretory granules and cell membrane and measurement of intermittent secretion of renin from single afferent arterioles, with a renin content of each secretion episode that corresponds to the renin content of one secretory granule. More recently it has been demonstrated that the afferent arterioles lose a large number of renin granules after acute stimulation without changing the average granular volume. Current electrophysiological techniques have now permitted direct measurements of cell membrane capacitance in juxtaglomerular (JG) cells as a measure of net addition (exocytosis) or removal (endocytosis) of membrane material. With this technique we have shown that cAMP, which is a vasodilator and stimulates renin secretion, enhances net exocytosis at low concentrations, while at higher concentrations membrane retrieval processes are also stimulated. We suggest that both exocytosis and endocytosis are regulated processes in the JG‐cells and both may be important for the long‐term control of renin secretion at the single cell level.
Intracellular localization of dimethylarginine dimethylaminohydrolase overexpressed in an endothelial cell line
Authors: Birdsey  , Leiper  , Vallance
Published: March 2003

Methylarginines are endogenous inhibitors of nitric oxide synthase (NOS) and have been implicated in the regulation of the nitric oxide pathway in health and disease. Cellular concentrations of free methylarginines are determined in part by the activity of dimethylarginine dimethylaminohydrolase (DDAH). There are two isoforms of DDAH which have distinct tissue distributions with some relationship to NOS isoforms. We have determined the intracellular localization of both DDAH isoforms by overexpression of epitope‐tagged DDAH in an immortalized endothelial cell line. Immunofluorescence confocal microscopy and immunoblotting indicate that both isoforms are predominantly cytosolic with no specific association with organelles or the plasma membrane. These data suggest that the key role for DDAH may be to ensure that under normal conditions the levels of methylarginines are kept low throughout the whole cell.
Effect of nervous excitation on acid secretion in horses
Authors: Sandin  , Andrews  , Nadeau  , Nilsson
Published: March 2003

Nervous excitation was induced by various means in horses provided with a gastric cannula. Insulin hypoglycaemia profoundly inhibited the basal acid output and volume secreted from the stomach. No clear effect on acid secretion was noted after administration of bethanechol, as the acid output was covered by the copious secretion of saliva. Atropine almost abolished the basal acid output. Sensoric stimulation by teasing caused a slight but not significant increase in the total acid output. These data suggest that cholinergic excitation might play a role in the stimulation of both volume and acid secretion in the horse. The inhibitory effect seen on these two parameters after insulin hypoglycaemia may hypothetically be ascribed to inhibitory impulses carried in peptide neurones of the vagal nerves or to inhibitory impulses in adrenergic nerves acting directly or indirectly on the parietal cells.
An upper arm model for simulated weightlessness
Authors: Parcell  , Trappe  , Godard  , Williamson  , Fink  , Costill
Published: March 2003

This investigation examined the effects of 4 weeks of non‐dominant arm unloading on the functional and structural characteristics of the triceps brachii muscle of six normo‐active college‐age males (age: 23 ± 1 years, height: 176 ± 4 cm, weight: 76 ± 6 kg). The primary intention of this study was to determine if arm unloading is an effective analogue for simulating the effects of weightlessness on human skeletal muscle. Subjects were tested 2–3 days preceding unloading in a standard arm sling and following removal of the sling. The sling was worn during waking hours to unload the arm. Subjects were allowed to remove the sling during sleep and bathing. Torque production (Nm) during maximal isometric extension at 90° significantly declined (P < 0.05) in response to unloading (53.93 ± 5.07 to 47.90 ± 5.92; 12%). There was no significant change (P > 0.05) in the force–velocity attributes of the triceps over the other measured velocities (1.05, 1.57, 2.09, 3.14, 4.19, 5.24 rad·s–1). Cross‐sectional muscle area (CSA) of the upper arm was smaller (44.3 ± 2.7 to 42.4 ± 2.5 cm2; 4%) following 4 weeks of unloading (P < 0.05). Histochemical analysis of individual muscle fibres demonstrated reductions in fibre CSA of 27 and 18% for type I and type II fibres, respectively. However, these changes were not statistically significant. Electrophoretic analysis of muscle samples revealed a significant increase (40 ± 7 to 58 ± 4%, pre‐ and post‐, respectively) in myosin heavy chain (MHC) type II isoforms following unloading. Reductions in type I MHC isoform composition failed to reach statistical significance (P < 0.08). Amplitude of the integrated electromyographic (IEMG) signal during maximal isometric contraction of the long head of the triceps decreased by 21% in response to the 4‐week unloading period (P < 0.05). The changes in triceps, muscle structure and function found with arm unloading are similar in magnitude and direction to data obtained from humans following exposure to real and simulated weightlessness. These findings demonstrate that arm unloading produces some of the effects seen in response to weightlessness in muscles of the upper arm and provides potential for an additional model to simulate the effects of microgravity on human skeletal muscle.
Human skeletal muscle mitochondrial capacity
Authors: Rasmussen U.F. , Rasmussen H.N.
Published: March 2003

Under aerobic work, the oxygen consumption and major ATP production occur in the mitochondria and it is therefore a relevant question whether the in vivo rates can be accounted for by mitochondrial capacities measured in vitro. Mitochondria were isolated from human quadriceps muscle biopsies in yields of ≈45%. The tissue content of total creatine, mitochondrial protein and different cytochromes was estimated. A number of activities were measured in functional assays of the mitochondria: pyruvate, ketoglutarate, glutamate and succinate dehydrogenases, palmitoyl‐carnitine respiration, cytochrome oxidase, the respiratory chain and the ATP synthesis. The activities involved in carbohydrate oxidation could account for in vivo oxygen uptakes of 15–16 mmol O2 min–1 kg–1 or slightly above the value measured at maximal work rates in the knee‐extensor model of Saltin and co‐workers, i.e. without limitation from the cardiac output. This probably indicates that the maximal oxygen consumption of the muscle is limited by the mitochondrial capacities. The in vitro activities of fatty acid oxidation corresponded to only 39% of those of carbohydrate oxidation. The maximal rate of free energy production from aerobic metabolism of glycogen was calculated from the mitochondrial activities and estimates of the ΔG or ATP hydrolysis and the efficiency of the actin–myosin reaction. The resultant value was 20 W kg–1 or ≈70% of the maximal in vivo work rates of which 10–20% probably are sustained by the anaerobic ATP production. The lack of aerobic in vitro ATP synthesis might reflect termination of some critical interplay between cytoplasm and mitochondria.
Nutritive and non‐nutritive blood flow: rest and exercise
Authors: Clark M.G. , Rattigan S. , Clerk L.H. , Vincent M.A. , Clark A.D.H. , Youd J.M. , Newman J.M.B.
Published: March 2003

There is growing evidence to support the notion of two vascular routes within, or closely associated with skeletal muscle. One route is in intimate contact with muscle cells (hence is known as ‘nutritive’) and the other functions as a vascular shunt (and has had the interesting misnomer of ‘non‐nutritive’). Recent findings suggest that the ‘non‐nutritive’ route may, in part, be those vessels in closely associated (interlacing?) connective tissue that nourishes attached fat cells, and may form the basis of ‘marbling’ of muscle in obesity. In addition, embolism studies using various size microspheres indicate that the ‘non‐nutritive’ vessels are likely to be capillaries fed by terminal arterioles that branch from the same transverse arterioles as those supplying terminal arterioles of the muscle capillaries (i.e. two vascular systems operating in parallel). The proportion of flow distributed between the two routes is tightly regulated and controls muscle metabolism and contraction by regulating hormone and substrate delivery as well as product removal. Because a high proportion of nutritive flow may elevate the set point for basal metabolism, a low proportion of nutritive flow in muscle at rest confers an evolutionary advantage, particularly when food is scarce. In addition, the proportion of flow that is carried by the non‐nutritive routes at rest affords a flow reserve that can be switched to the nutritive route to amplify nutrient supply during exercise. Alternatively the non‐nutritive route may allow flow to escape when active muscle contraction compresses its nutritive capillaries. Thus rhythmic oscillation of blood flow between the non‐nutritive and nutritive networks may aid the muscle pump.
Adenosine and muscle vasodilatation in acute systemic hypoxia
Authors: Marshall J.M.
Published: March 2003

Adenosine is released by skeletal and cardiac muscles when their metabolism increases: it serves to couple O2 supply with O2 demand by causing vasodilatation. This review argues that adenosine plays a similar role in skeletal muscle in systemic hypoxia. It accounts for ≈50% of the increase in muscle vascular conductance and, within muscle, it causes dilatation of individual arterioles, thus maximizing the distribution of O2 and allowing O2 consumption to remain constant when O2 delivery is reduced. In vivo and in vitro studies have indicated that adenosine can induce dilatation in several different ways. This review argues that during systemic hypoxia, adenosine is predominantly released from the endothelium and acts on endothelial A1 receptors to produce dilatation in a nitric oxide (NO)‐dependent manner. A1 receptor stimulation increases the synthesis of NO by a process initiated by opening of ATP‐sensitive K+ (KATP) channels. Moreover, recent findings suggest that prostaglandins also make a major contribution to the hypoxia‐induced dilatation, but that the dilator pathways for adenosine, NO and prostaglandins are interdependent. In addition, adenosine released from the skeletal muscle fibres contributes indirectly to the dilatation by stimulating A1 and A2 receptors on the muscle fibres, opening KATP channels and allowing efflux of K+, which is a vasodilator. Finally, by acting on endothelial A1 receptors, adenosine attenuates the vasoconstrictor effects of constant or bursting patterns of sympathetic activity. This limits the extent to which the sympathetic nervous system can reduce O2 delivery to muscle when it is already compromized by systemic hypoxia.
Perfusion, diffusion and their heterogeneities limiting blood–tissue O2 transfer in muscle
Authors: Piiper J.
Published: March 2003

The relative roles of blood flow (perfusion) and diffusion in O2 supply to exercising muscle can be estimated using a simple model consisting of an O2‐consuming tissue block in contact with blood (perfusion Q˙, slope of O2 equilibirum curve β) through a resistance to O2 diffusion (O2‐diffusing capacity D). The decisive variable is the ‘equilibration index’Y=D/(Q˙β). With decreasing Y, diffusion limitation increases and perfusion limitation decreases (Y > 3 indicates predominant perfusion limitation; 3 > Y > 0.1, combined perfusion and diffusion limitation, Y < 0.1, prevailing diffusion limitation). On the basis of literature data on humans at maximum O2 uptake, O2 supply to muscle is shown to be always limited by both perfusion and diffusion. In nomoxia, perfusion limitation is prevalent, but in hypoxia diffusion limitation becomes predominant. The underlying model assumes perfect homogeneity of muscles with respect to O2 requirement, diffusion conditions and blood flow. In numerous studies on isolated and in situ muscles a pronounced heterogeneity of blood flow has been found, also during exercise and at maximal O2 uptake. It is shown that with unequal distribution of blood flow and/or O2‐diffusing capacity the efficiency of O2 transfer is reduced with reference to the homogeneous model. Therefore, the diffusing capacity value calculated on the basis of the homogeneous model is an underestimate of the true diffusing capacity and diffusion limitation is overestimated.
Importance of post‐exercise hypotension in plasma volume restoration
Authors: Hayes  , Lucas  , Shi
Published: March 2003

The aim of this study was to test the hypothesis that post‐exercise hypotension was the mechanism for the plasma volume and albumin gain during recovery. Seven healthy young men completed two experiments (≥1 week apart) to exercise continuously at 65% of peak aerobic capacity for 60 min followed by the recovery without (experiment 1) or with phenylephrine infusion (experiment 2) to counteract post‐exercise hypotension. Heart rate, arterial pressure (Finapres), plasma volume (PV, Evans blue dye dilution), haematocrit, haemoglobin, plasma total solutes (refractometer), albumin, total proteins (colorimetric method), [Na+] and [K+] were not different prior to the experiments. Exercise decreased PV –13.7% (–521 mL) and –14.2% (–566 mL) at the end of 60 min in experiments 1 and 2, respectively, associated with increases in the concentrations of plasma albumin, total protein and solutes. These changes were similar between the two experiments. Following 30 min recovery in experiment 1 the decreased PV was not significantly different from the baseline. Although the volume restoration was complete at the end of 90 min recovery, the change in the albumin concentration was still above zero, indicating a gain of 11 g albumin (P < 0.05). When phenylephrine was infused during recovery, there was no gain in intravascular albumin associated with a sustained decrease in PV (–7% or –280 mL,P < 0.05) observed at the end of experiment 2. These data suggest that post‐exercise hypotension may be the mechanism for a gain of intravascular albumin via the lymph return, which enhances plasma water retention and PV restoration during recovery from exercise induced hypovolaemia, even without rehydration.
Hyperglycaemia alters the endothelium‐dependent relaxation of canine coronary arteries
Authors: Kocsis  , Pacher  , Pósa  , Nieszner  , Pogátsa  , Koltai
Published: March 2003

The aim of the present study was to investigate the effects of experimental diabetes and hyperglycaemia per se on the endothelium‐dependent relaxation of isolated canine coronary arteries and to analyse the possible involvement of the cyclooxygenase pathway in the alterations induced by hyperglycaemia. Rings from the left anterior descending coronary arteries of 18 metabolically healthy, six alloxan‐diabetic and six insulin‐treated alloxan diabetic dogs were set up for isometric tension recording. Diabetic coronaries as well as healthy vessels subjected to in vitro hyperglycaemia (25.5 mmol L–1 glucose) showed impaired (P < 0.05) relaxation to acetylcholine (3 nmol L–1–10 μmol L–1) compared with normoglycaemic, i.e. metabolically healthy and insulin‐treated diabetic controls, either before or after indomethacin (3 μmol L–1) administration. The maximal dilation elicited by acetylcholine was further decreased (P < 0.05) by the cyclooxygenase inhibitor in the diabetic coronaries only. Relaxation to sodium nitroprusside did not differ among groups. These results suggest that hyperglycaemia may result in impaired endothelium‐dependent dilation of coronary arteries. Diminished relaxation of diabetic coronaries is worsened by the inhibition of the synthesis of vasodilator cyclooxygenase products.
Changes in capillary luminal diameter in rat soleus muscle after hind‐limb suspension
Authors: Kano  , Shimegi  , Takahashi  , Masuda  , Katsuta
Published: March 2003

This study examined the time course change of the capillary luminal diameter and the number of capillaries in the rat soleus muscle during hind‐limb suspension. Male Wistar rats were divided into 1 and 3 weeks of hind‐limb suspension (HS) groups (HS‐1 and HS‐3). The HS groups were compared with age‐matched control groups. All morphometric parameters with respect to capillary and muscle fibre cross‐sectional area were determined in perfusion‐fixed soleus muscles. After 1 and 3 weeks of hind‐limb suspension, the mean muscle fibre cross‐sectional area was significantly decreased in HS‐1 (–32.0%) and HS‐3 (–59.3%) compared with age‐matched control groups. Despite a lower capillary‐to‐fibre ratio (HS‐1, –19.3%; HS‐3, –21.2%), the capillary density was unchanged in HS‐1 and significantly increased in HS‐3 compared with age‐matched control groups. The mean capillary luminal diameter was significantly smaller in HS‐1 (–19.9%) and HS‐3 (–21.9%) than in the age‐matched control groups. The capillary‐to‐fibre perimeter ratio which indicates the capillary surface area available for gas exchange between blood and tissue did not significantly differ between control groups and HS groups. In conclusion, the morphometrical adaptations in rat soleus with the suspension involved changes in both the capillary luminal diameter and number of capillaries, and the change in capillary surface area was proportional to the degree of muscle atrophy in HS groups.
PKCα translocation is microtubule‐dependent in passaged smooth muscle cells
Authors: Battistella‐Patterson A.S. , Fultz M.E. , Li C. , Geng W. , Norton M. , Wright G.L.
Published: March 2003

The translocation of protein kinase C (PKC) isozymes from their inactive cell locus to a variety of cytoskeletal, organelle, and plasmalemmal sites is thought to play an important role in their activation and substrate specificity. We have utilized confocal microscopy to compare phorbol 12, 13 dibutyrate (PDB) – stimulated translocation of PKCα in cultured cells derived from rat vascular smooth muscle. In enzymatically dispersed, passaged smooth muscle cells, PKCα was uniformly distributed throughout the unstimulated cell. PDB stimulation resulted in extensive association of the PKCα into filamentous strands with subsequent accumulation of the isoform in the peri‐nuclear region of the cell. Dual immunostaining indicated that PKCα was extensively colocalized with microtubules in the interval immediately following PDB stimulation but was largely disassociated from microtubules at 10 min, at which time the translocation of PKCα to the peri‐nucleus/nucleus was nearly complete. It was further found that the use of colchicine to disrupt the microtubules caused the loss of PKCα translocation to the peri‐nuclear region. By comparison, cytochalasin B disruption of actin microfilaments had no significant effect on this parameter. The data suggest that PDB stimulation results in a transient association of PKCα with cell microtubules and that the microtubules play an important role in the translocation of PKCα from the cytosol in passaged cells derived from rat aortic smooth muscle.
Exercise‐induced sympathetic activation is correlated with cerebral hemisphere laterality, but not handedness
Authors: Saito M.
Published: March 2003

To investigate whether sympathetic responses are correlated with central laterality or handedness, muscle sympathetic nerve activity (MSNA), heart rate (HR) and blood pressure (BP) were compared between right (RA) and left arm (LA) grip exercise with volitional maximum effort (MVHG) for 2 min and post‐exercise arterial occlusion (PEAO) in right‐ and left‐handed volunteers. MVHG and PEAO led to a greater increase in MSNA in RA than in LA exercise (180 vs. 150%,P=0.004; 140 vs. 85%,P=0.005). MVHG elevated HR to a significantly lesser extent in RA than in LA (35 vs. 46%,P=0.030), and the difference was maintained during PEAO. The BP rise during MVHG and PEAO was the same in RA and in LA. Muscle sympathetic nerve activity, HR and BP responses during MVHG and PEAO showed no difference between the dominant and non‐dominant arm. These results suggested that the effects of central motor command and metaboreflex on sympathetic outflow to the vasculature and the heart may be selectively modulated partly by hemispherical laterality.
Desmin‐related myopathies in mice and man
Authors: Carlsson L. , Thornell L.‐E.
Published: March 2003

Desmin, the main intermediate filament (IF) protein in skeletal and heart muscle cells, is of great importance as a part of the cytoskeleton. The IFs surround and interlink myofibrils, and connect the peripheral myofibrils with the sarcolemma. In myotendinous junctions and neuromuscular junctions of skeletal muscle fibres, desmin is enriched. In the heart, desmin is increased at intercalated discs, the attachment between cardiomyocytes, and it is the main component in Purkinje fibres of the conduction system. Desmin is the first muscle‐specific protein to appear during myogenesis. Nevertheless, lack of desmin, as shown from experiments with desmin knockout (K/O) mice, does not influence myogenesis or myofibrillogenesis. However, the desmin knock‐out mice postnatally develop a cardiomyopathy and a muscle dystrophy in highly used skeletal muscles. In other skeletal muscles the organization of myofibrils is remarkably unaffected. Thus, the main consequence of the lack of desmin is that the muscle fibres become more susceptible to damage. The loss of membrane integrity leads to a dystrophic process, with degeneration and fibrosis. In the heart cardiac failure develops, whereas in affected skeletal muscles regenerative attempts are seen. In humans, accumulations of desmin have been a hallmark for presumptive desmin myopathies. Recent investigations have shown that some families with such a myopathy have a defect in the gene coding for αB‐crystallin, whereas others have mutations in the desmin gene. Typical features of these patients are cardiac affections and muscle weakness. Thus, mutations in the desmin gene is pathogenic for a distinct type of muscle disorder.
5‐Hydroxytryptamine‐induced microvascular pressure transients in lungs of anaesthetized rabbits
Authors: Sen N. , Glucksberg M. R. , Bhattacharya S.
Published: March 2003

We determined lung microvascular pressure transients induced by 5‐hydroxytryptamine (5HT), by the micropuncture technique. We mechanically ventilated anaesthetized (halothane 0.8%), open‐chested rabbits, in which we recorded pulmonary artery (PA), left atrial (LA) and carotid artery pressures and cardiac output. For 4‐min periods of stopped ventilation, we constantly inflated the lung with airway pressure of 7 cmH2O, then micropunctured the lung to determine pressures in arterioles and venules of 20–25 μm diameter. An intravenous bolus infusion of 5HT (100 μg), increased total pulmonary vascular resistance by 59%. Prior to 5HT infusion, the arterial, microvascular and venous segments comprised 30, 50 and 19% of the total pulmonary vascular pressure drop, respectively. However 14 s after 5HT infusion, the PA‐arteriole pressure difference (arterial pressure drop) increased 46%, while the venule‐LA pressure difference (venous pressure drop) increased >100%. The arteriole–venule pressure difference (microvascular pressure drop) was abolished. The increase in the arterial pressure drop was maintained for 4.8 min, whereas the increased venous pressure drop reverted to baseline in <1 min. We conclude that in the rabbit lung in situ, a 5HT bolus causes sustained arterial constriction and a strong but transient venous constriction.
Expression of heat shock protein 72 in atrophied rat skeletal muscles
Authors: Oishi Y. , Ishihara A. , Talmadge R. J. , Ohira Y. , Taniguchi K. , Matsumoto H. , Roy R. R. , Edgerton V. R.
Published: March 2003

Changes in the expression of heat shock protein 72 (HSP72) in response to atrophic‐inducing perturbations of muscle involving chronic mechanical unloading and denervation were determined. Adult male Wistar rats were assigned randomly to a sedentary cage control (CON), hind limb unloading (HU, via tail suspension), HU plus tenotomy (HU + TEN), HU plus denervation (HU + DEN), or HU + TEN + DEN group. Tenotomy and DEN involved cutting the Achilles tendon and removing a segment of the sciatic nerve, respectively. After 5 days, HSP72 levels in the soleus of the HU + DEN and HU + TEN + DEN groups were 42 (P < 0.05) and 53% (P < 0.01) less than CON, respectively. Soleus weight decreased in both groups. Heat shock protein 72 levels in the plantaris of the HU + TEN, HU + DEN, and HU + TEN + DEN groups were 31, 25, and 30% lower than CON, respectively (P < 0.05). Plantaris weight decreased in the HU + DEN and HU + TEN + DEN, but not in the HU + TEN group. Hind limb unloading alone had little effect on the HSP72 level in either muscle. Reduced levels of HSP72 were associated with a decreased soleus (r=0.62, P < 0.01) and plantaris (r=0.78, P < 0.001) weight. These results indicate that the levels of HSP72 in both a slow and a fast rat plantarflexor are responsive to a chronic decrease in the levels of loading and/or activation and suggest that the neuromuscular activity level and the presence of innervation of a muscle are important factors that induce HSP72 expression.
Effects of C‐peptide on forearm blood flow and brachial artery dilatation in patients with type 1 diabetes mellitus
Authors: Fernqvist‐Forbes E. , Johansson B.‐L. , Eriksson M. J.
Published: March 2003

Recent studies suggest that C‐peptide increases blood flow in both exercising and resting forearm in patients with type 1 diabetes. Now we have studied the effect of C‐peptide administration on endothelial‐mediated and non‐endothelial‐mediated arterial responses as well as central haemodynamics in 10 patients with type 1 diabetes in a placebo‐controlled double‐blind study. Euglycaemia was maintained with an i.v. insulin infusion before and during the study. A high‐resolution ultrasound technique and Doppler echocardiography were used to assess haemodynamic functions. Brachial artery blood flow and brachial artery diameter were measured in the basal state, 1 and 10 min after reactive hyperaemia and 4 min after sublingual glyceryl trinitrate administration (GTN; endothelial‐independent vasodilatation), both before and after the end of 60‐min C‐peptide (6 pmol kg–1 min–1) or saline infusion periods. Echocardiographic measurements were also performed before and at the end of the infusion periods. Seven healthy age‐matched males served as controls for vascular studies. The patients showed a blunted brachial dilatation after reactive hyperaemia in comparison with the healthy controls (2.1 ± 0.5% vs. 9.3 ± 0.3%, P < 0.001), indicating a disturbed endothelial function. C‐peptide infusion compared with saline resulted in increased basal blood flow (33 ± 6%, P < 0.001) and brachial arterial dilatation (4 ± 1%, P < 0.05). Left ventricular ejection fraction seemed to be improved (5 ± 2%, P < 0.05) at the end of C‐peptide infusion compared with placebo. The vascular response to reactive hyperaemia and GTN was not affected by C‐peptide infusion. Our results demonstrate that physiological concentrations of C‐peptide increase resting forearm blood flow, brachial artery diameter and left ventricular systolic function in patients with type 1 diabetes.
Effects of sprint and endurance training on passive stress–strain relation of fast‐ and slow‐twitch skeletal muscle in Wistar rat
Authors: Muñiz J. , Del Rio J. , Huerta M. , Marin J. L.
Published: March 2003

We investigated the effects of endurance and sprint training on the passive mechanical properties of fast‐twitch (FT) and slow‐twitch (ST) skeletal muscles. Eight‐week‐old male Wistar rats (n=18) were divided into three groups: control (C), sprint‐trained (S) and endurance‐trained (E). The trained animals exercised for 10 weeks on a treadmill. Under anaesthesia, Plantaris and Soleus muscles were deformed cyclically in vivo at 0.33 mm s–1 with length increments of 1 mm in successive cycles until rupture. The rupture of muscle occurs at belly. Stress–strain relation were constructed using the maximum stress and maximum strain in each cycle. The data were fitted to an S‐shaped curve. The curve‐fitting parameters for trained and untrained muscles showed significant statistical differences. Stress and strain at rupture and maximum deformation energy were statistically greater for trained ST muscles (both groups) than for the controls. The changes induced by the present training protocols were not significant in Plantaris. The above results suggest the plasticity of passive structure caused by activity‐demands.
Targeted proteomics in the kidney using ensembles of antibodies
Authors: Knepper M. A. , Masilamani S.
Published: March 2003

Building on extensive physiological characterization of sodium transport mechanisms along the renal tubule over the past 30 years, complementary DNAs for almost all of the major transporters and channels responsible for renal tubular sodium reabsorption have been cloned over the past 10 years. The consequence is the generation of a broad range of cDNA and antibody probes which can be used to investigate physiological mechanisms on a molecular level. An ensemble of such probes can be exploited for comprehensive analysis of integrative physiological processes, approaches which are referred to as ‘physiological genomics’ or ‘physiological proteomics’. In this review, we describe a targeted proteomic approach to comprehensive analysis of sodium transporter and water channel protein abundance along the renal tubule using an ensemble of rabbit polyclonal antibodies directed to the major sodium transporters and water channels expressed in each renal tubule segment. We discuss preparation and characterization of the antibodies, strategies for quantification of transporter protein abundance, and provide examples of the application of antibody‐based targeted proteomics analysis of kidney tissue, revealing the effects of elevations of circulating aldosterone levels and circulating vasopressin levels on sodium transporter, sodium channel, and water channel abundance in kidney.
Genetic manipulation of b‐adrenergic signalling in heart failure
Authors: Davidson M. J. , Koch W. J.
Published: March 2003

Heart failure (HF) represents one of the leading causes for hospitalization in developed nations. Despite advances in the management of coronary artery disease, no significant improvements in prognosis have been achieved for HF over the last several decades. Heart failure itself represents a final common endpoint for several disease entities, including hypertension, coronary artery disease, and cardiomyopathy. However, certain biochemical features remain common to the failing myocardium. Foremost amongst these are alterations in the β‐adrenergic receptor signalling cascade. Recent advances in transgenic and gene therapy techniques have presented novel therapeutic strategies for the management of HF via enhancement of β‐adrenergic signalling. In this review, we will discuss the biochemical changes that accompany HF as well as corresponding therapeutic strategies. We will then review the evidence from transgenic mouse work supporting the use of adrenergic receptor augmentation in the failing heart and more recent in vivo applications of gene therapy directed at reversing or preventing HF.
Extramuscular myofascial force transmission within the rat anterior tibial compartment: proximo‐distal differences in muscle force
Authors: Huijing P. A. , Baan G. C.
Published: March 2003

Intramuscular connective tissues are continuous to extramuscular connective tissues. If force is transmitted there, differences should be present between force at proximal and distal attachments of muscles. Extensor digitorum longus (EDL), tibialis anterior (TA), and extensor hallucis longus muscles (EHL) were excited simultaneously and maximally. Only EDL length was changed, exclusively by moving the position of its proximal tendon. Distal force exerted by TA + EHL complex was not affected significantly. Proximal and distal EDL isometric force were not equal for most EDL lengths: Fprox – Fdist ranged from 0 to ≈+22.7% of Fprox at higher lengths and from 0 to ≈–24.5% at the lowest lengths. It is concluded that extramuscular connections transmit force from muscle. Significant proximo‐distal differences of EDL force remained after repeated measurements, regardless of length order, although their length dependence was altered. Measurements of both proximal and distal EDL force were highly reproducible, if EDL did not attain higher lengths than target length. After being active at high lengths, proximal and distal length–force curves were altered at low lengths but not for the highest length range. Extensor digitorum longus length–active force hysteresis was present for proximal as well as distal EDL measurements with increasing and decreasing isometric length order. Further isolating EDL removed the proximo‐distal difference for active EDL force. However a decreased difference for passive EDL force remained, which was ascribed to remaining extramuscular connective tissue linkages. It is concluded that extramuscular myofascial force transmission is an important feature of muscle that is not isolated from its surrounding tissues.
Modulation of vascular tone and reactivity by nitric oxide in porcine pulmonary arteries and veins
Authors: Bäck M. , Walch L. , Norel X. , Gascard J.‐P. , Mazmanian G. , Brink C.
Published: March 2003

Isolated porcine pulmonary vessels were studied in order to evaluate the role of nitric oxide in arteries and veins. Leukotriene C4 and noradrenaline contracted porcine pulmonary arteries but induced only negligible contractions of porcine pulmonary veins. After treatment with the nitric oxide synthase inhibitor Nω‐nitro‐L‐arginine (L‐NOARG), significant contractions to leukotriene C4 and noradrenaline were uncovered in pulmonary veins. In arterial preparations, L‐NOARG caused a less marked potentiation of noradrenaline‐induced contractions and did not alter leukotriene C4‐induced contractions. Endothelium‐dependent relaxations to acetylcholine were greater in veins compared with arteries whereas the endothelium‐independent relaxations to the nitric oxide donor sodium nitroprusside (SNP) and the cyclic nucleotide analogue 8‐bromo‐cGMP were similar in the two preparations. Taken together these data suggest that the apparent insensitivity of porcine pulmonary veins to leukotriene C4 and noradrenaline was because of release of nitric oxide. The effect of nitric oxide synthase inhibition was less pronounced in porcine pulmonary arteries, suggesting a preferential functional role of nitric oxide in porcine pulmonary veins, originating in a greater production of nitric oxide by veins as opposed to arteries.
Effect of endurance training on oestrogen receptor alpha transcripts in rat skeletal muscle
Authors: LEMOINE S. , GRANIER P. , TIFFOCHE C. , BERTHON P. M. , RANNOU‐BEKONO F. , THIEULANT M.‐L. , CARRÉ F. , DELAMARCHE P.
Published: March 2003

Endurance training induces, in female rats, alterations of oestrous cycle with decrease in plasma oestradiol levels. Moreover, it is well known that oestradiol concentrations modify oestrogen receptor levels. In order to further explain the effects of oestrogens on skeletal muscles, we hypothesized that endurance training modifies the levels of oestrogen receptor alpha messenger ribonucleic acid (ERα mRNA) in rat gastrocnemius muscle. Wistar rats were separated into four groups: male controls (Cm) (n=7), female controls (Cf) (n=6), male trained (Em) (n=7) and female trained (Ef) (n=6). The endurance training programme was performed for 7 weeks, 5 days week–1 and consisted of 1 h of continuous running on an adapted motor‐driven treadmill. At the end of the training session, the gastrocnemius muscle was isolated, weighed and semiquantification of ERα mRNA was performed using the reverse transcriptase‐polymerase chain reaction (RT‐PCR) technique. The citrate synthase (CS) activity of the gastrocnemius muscle was measured by a fluorimetric method. The CS activity of the male and female gastrocnemius muscle, respectively, 100 ± 7% in Cm (n=7) vs. 120 ± 14% in Em (n=6, P < 0.01) and 100 ± 13% in Cf (n=6) vs. 138 ± 23% in Ef (n=6, P < 0.01) was significantly increased after 7 weeks of training. The ERα mRNA levels were significantly increased in Ef compared with Cf (0.49 ± 0.15 vs. 0.31 ± 0.11, P < 0.01) but not in Em compared with Cm (0.37 ± 0.15 vs. 0.37 ± 0.13). In conclusion, these results demonstrate that 7 weeks of endurance training increased the level of transcripts encoding ERα in rats with the increase restricted to the females.
PKC‐α shows variable patterns of translocation in response to different stimulatory agents
Authors: LI C. , FULTZ M. E. , WRIGHT G. L.
Published: March 2003

Reports from numerous laboratories suggest that protein kinase C (PKC) translocation to substrate target sites may vary depending on cell type and experimental conditions. We have proposed that acutely variable targeting of PKC to different substrate sites could greatly expand the functional properties of individual isoforms in individual cell types (Li et al., 2001). Confocal microscopy and PKCα‐enhanced green fluorescent protein (PKCα–EGFP) fusion protein expression were utilized to investigate the spatial and temporal pattern of PKCα translocation to different stimulating agents in A7r5 smooth muscle cells. Phorbol 12, 13 dibutyrate (PDBu 10–8 M) caused a slow but irreversible relocation of the fusion protein from the cytosol to the plasmalemma. By comparison, thapsigargin (10–5 M) and A23 187 (2 × 10–5 M) induced a rapidly transient translocation to the cell membrane which was completed within 4 min. In contrast to these agents, angiotensin II (Ang II, 10–6 M) caused only partial relocalization of cytosolic PKCα–EGFP to brightly fluorescing patches at the cell periphery. Localization at peripheral patches was completed within seconds and the fusion protein returned to the cytosol within 2 min. The PKC inhibitor staurosporine blocked cellular contraction to PDBu but not A23 187 and had no effect on PKCα–EGFP translocation. By comparison, the calcium chelators EDTA and BAPTA‐AM blocked the contraction to A23 187, attenuated the contraction to PDBu, and abolished the translocation of PKCα–EGFP by both agents. The results show that in a single cell type the spatial and temporal characteristics of individual PKC isoform translocation may differ markedly. This further suggests the existence of potentially complex mechanisms which regulate the rate and location of target site availability.
Bradykinin is not involved in angiotensin converting enzyme modulation of ovarian steroidogenesis and prostaglandin production in frog Rana esculenta
Authors: BRAMUCCI M. , MIANO A. , GOBBETTI A. , ZERANI M. , QUASSINTI L. , MACCARI E. , MURRI O. , AMICI D.
Published: March 2003

Angiotensin converting enzyme (ACE) was demonstrated to modulate the production of 17β‐estradiol, progesterone and prostaglandin E2 (PGE2) in frog ovary of Rana esculenta. However, the activity was not mediated by angiotensin II (Ang II). In an attempt to identify the peptide involved in the pathway modulated by ACE, bradykinin, another physiological substrate of ACE, was chosen and incubated in the presence of the membrane suspension purified from the frog ovary homogenate. The hydrolytic products were analysed by reverse‐phase high‐pressure liquid chromatography (HPLC) analysis and the results showed that bradykinin was metabolized by membrane suspension. The presence of the protease inhibitors in the incubation mixture indicated ACE and neutral endopeptidase as being responsible for the bradykinin hydrolysis. Frog ovary was incubated in vitro in the presence of bradykinin (10 μM), bradykinin receptor antagonist NPC 567 (1 mg mL–1), bradykinin fragment (1–7) (10 μM), ACE (2.5 mU mL–1), captopril (0.1 mM) and lisinopril (0.1 mM). The results showed no modulating activity by bradykinin on ovarian 17β‐estradiol and PGE2 production, thus demonstrating that it was not involved in the ACE‐modulated pathway.
Nandrolone decanoate treatment induces changes in contractile responses of rat untrained fast‐twitch skeletal muscle
Authors: JOUMAA W. H. , SERRURIER B. , BIGARD X. , LÉOTY C.
Published: March 2003

This investigation was designed to examine whether short‐term administration of anabolic–androgenic steroids (AAS) (nandrolone decanoate) could produce changes in contractile responses of untrained rat fast‐ (edl) and slow‐ (soleus) twitch skeletal muscle. Twenty male rats were divided into two groups, one group received weekly (for 6 weeks) an intramuscular injection of AAS, nandrolone decanoate (15 mg kg–1) and the second group received weekly the similar doses of vehicle (sterile peanut oil). In edl intact isolated small bundles (two to four cells), it was found that nandrolone decanoate treatment increases the K+ contracture tension (146 mM) relative to maximum tension by 56%, whereas no change was observed in the time to peak tension and in the time constant of relaxation. By contrast, in treated soleus muscle, compared with control, no significant modification was found in the K+ contracture characteristics. The change in edl contractile responses was associated with a shift to more negative potential of the voltage‐dependence activation and the steady‐state inactivation curves which also shifted leftward in treated soleus fibres. Furthermore, in edl skinned Triton X‐100 fibres, the Ca2+ sensitivity of contractile proteins (pCa50) was increased, while electrophoresis analysis indicates no significant effect of nandrolone decanoate treatment on myosin heavy chain (MHC) isoforms. The present results show that nandrolone decanoate treatment produces more pronounced changes in untrained fast muscle function rather than soleus by acting at different levels of the excitation–contraction coupling mechanism without changes in the MHC isoforms and that contractile responses became similar to those found in soleus muscle.