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New Findings What is the central question of this study? The aim was to establish the ability of a newly designed leg exercise technique to produce sustained elevations in shear rate that stimulate flow-mediated dilatation (FMD) in the superficial femoral artery and to determine the repeat trial stability of the FMD response., What is the main finding and its importance? Calf plantar-flexion exercise can be used to increase shear stress and stimulate FMD in the superficial femoral artery. However, the magnitude of FMD varied systematically when multiple trials were repeated in short succession., The superficial femoral artery (SFA) is susceptible to vascular disease, and a technique to assess flow-mediated dilatation (FMD) in this vessel in response to a sustained shear stress stimulus could provide important information about endothelial function. The aim of this study was to establish the ability of a newly designed SFA leg exercise-FMD (LEX-FMD) technique to produce sustained elevations in shear rate, which stimulate FMD, and to determine the repeat trial stability of the FMD response. The SFA FMD stimulated by reactive hyperaemia (RH) and calf plantar-flexion exercise (LEX) was assessed via ultrasound in 19 healthy men ( n = 10) and women ( n = 9). The two experimental visits included either four trials of LEX-FMD or four trials of RH-FMD. The shear stress stimulus was estimated as the shear rate (blood velocity/SFA diameter). Results are expressed as the means ± SD. The LEX steady-state shear rate was consistent between trials ( P = 0.176), whereas the RH shear rate area under the curve was higher in trial 1 versus trials 2-4 ( P < 0.05). The %RH-FMD (four-trial mean 4.9 ± 2.5%) and absolute RH-FMD were not significantly different between trials ( P = 0.465 and P = 0.359, respectively). Both %LEX-FMD and absolute LEX-FMD were higher during trial 3 (4.8 ± 3.4%) than trial 1 (3.6 ± 2.7%; P = 0.026 and P = 0.026, respectively). The magnitude of RH-FMD and LEX-FMD did not differ ( P = 0.241). These results indicate that calf plantar-flexion exercise can be used to increase shear stress and stimulate FMD in the SFA. However, although SFA RH-FMD was stable across four trials, LEX-FMD varied systematically when multiple trials were repeated in rapid succession. [ABSTRACT FROM AUTHOR]

The number of myenteric interstitial cells of Cajal (ICC-MY), responsible for the generation and propagation of the slow wave in the small intestine, has been shown to decrease in diabetes, suggesting impairment of slow-wave (SW) propagation and related motility. To date, however, this expected decrease in SW propagation has neither been recorded nor analysed. Eleven rats were treated with streptozotocin and housed in pairs with 11 age-matched control animals. After 3 or 7 months, segments of duodenum, jejunum and ileum were isolated and divided into two parts. One part was processed for immediate freezing, cryosectioning and immunoprobing using anti-c-Kit antibody to quantify ICC-MY. The second part was superfused in a tissue bath, and SW propagation was recorded with 121 extracellular electrodes. In addition, a cellular automaton was developed to study the effects of increasing the number of inactive cells on overall propagation. The number of ICC-MY was significantly reduced after 3 months of diabetes, but rebounded to control levels after 7 months of diabetes. Slow-wave frequencies, velocities and extracellular amplitudes were unchanged at any stage of diabetes. The cellular automaton showed that SW velocity was not linearly related to the number of inactive cells. The depletion of ICC-MY is not as severe as is often assumed and in fact may rebound after some time. In addition, at least in the streptozotocin model, the initial reduction in ICC-MY is not enough to affect SW propagation. Diabetic intestinal dysfunction may therefore be more affected by impairments of other systems, such as the enteric system or the muscle cells. [ABSTRACT FROM AUTHOR]

The CellML language was developed in response to the need for a high-level language to represent and exchange mathematical models of biological processes. The flexible structure of CellML allows modellers to construct mathematical models of the same biological system in many different ways. However, some modelling styles do not naturally lead to clear abstractions of the biophysical concepts and produce CellML models that are hard to understand and from which it is difficult to isolate parts that may be useful for constructing other models. In this article, we advocate building CellML models which isolate common biophysical concepts and, using these, to build mathematical models of biological processes that provide a close correspondence between the CellML model and the underlying biological process. Subsequently, models of higher complexity can be constructed by reusing these modularized CellML models in part or in whole. Development of CellML models that best describe the underlying biophysical concepts thus avoids the need to code models from scratch and enhances the extensibility, reusability, consistency and interpretation of the models. [ABSTRACT FROM AUTHOR]

Adult rat submandibular glands have a rich autonomic innervation, with parasympathetic and sympathetic nerves working in synergy rather than antagonistically. Ligation of the secretory duct rapidly causes atrophy and the loss of most acini, which are the main target cell for parasympathetic nerves. Following deligation, there is a recovery of gland structure and function, as assessed by autonomimetic stimulation. This study examines whether the parasympathetic nerves reattach to new target cells to form functional neuro-effector junctions. Under recovery anaesthesia, the submandibular duct of adult male rats was ligated via an intra-oral approach to avoid damaging the chorda-lingual nerve. Four weeks later, rats were either killed or anaesthetized and the ligation clip removed. Following a further 8 weeks, both submandibular ducts were cannulated under terminal anaesthesia. Salivary flows were then stimulated electrically (chorda-lingual nerve at 2, 5 and 10 Hz) and subsequently by methacholine (whole-body infusion at two doses). Glands were excised, weighed and divided for further in vitro studies or fixed for histological examination. Ligation of ducts caused 75% loss of gland weight, with the loss of most acinar cells. Of the remaining acini, only 50% were innervated despite unchanged choline acetyltransferase activity, suggesting few parasympathetic nerves had died. Following deligation, submandibular glands recovered half their weight and had normal morphology. Salivary flows from both glands (per unit of gland tissue) were similar when evoked by methacholine but greater from the deligated glands when evoked by nerve stimulation. This suggests that parasympathetic nerves had reattached to new target cells in the recovered glands at a greater ratio than normal, confirming reinnervation of the regenerating gland. [ABSTRACT FROM AUTHOR]

The effect of resistance training on arterial stiffening is controversial. We tested the hypothesis that resistance training would not alter central arterial compliance. Young healthy men (age, 23 ± 3.9 (mean ± s.e.m.) years; n= 28,) were whole-body resistance trained five times a week for 12 weeks, using a rotating 3-day split-body routine. Resting brachial blood pressure (BP), carotid pulse pressure, carotid cross-sectional compliance (CSC), carotid initima–media thickness (IMT) and left ventricular dimensions were evaluated before beginning exercise (PRE), after 6 weeks of exercise (MID) and at the end of 12 weeks of exercise (POST). CSC was measured using the pressure-sonography method. Results indicate reductions in brachial (61.1 ± 1.4 versus 57.6 ± 1.2 mmHg; P < 0.01) and carotid pulse pressure (52.2 ± 1.9 versus 46.8 ± 2.0 mmHg; P < 0.01) PRE to POST. In contrast, carotid CSC, β-stiffness index, IMT and cardiac dimensions were unchanged. In young men, central arterial compliance is unaltered with 12 weeks of resistance training and the mechanisms responsible for cardiac hypertrophy and reduced arterial compliance are either not inherent to all resistance-training programmes or may require a prolonged stimulus. [ABSTRACT FROM AUTHOR]

Procedures that reduce contraction are used to facilitate optical measurements of membrane potential, but it is unclear to what extent they affect the excitability of the heart. This study has examined the electrophysiological consequences of a range of extracellular [Ca2+] (0.7-2.5 mmol 1-1 ), 2,3-butane-dione monoxime (BDM; 1-20 mmol 1-1) and cytochalasin-D (CytoD; 1-5 µmol 1-1 ). Methods. Monophasic action potentials (MAPs) were recorded from the basal epicardial surface of the left ventricle of isolated rabbit hearts. Conduction delay (CD) and time to 90% repolarisation of the monophasic action potential (MAPD90) were measured. The effects of BDM and Cyto-D on restitution were studied at a [Ca2+] of 1.9 mmol 1-1. Restitution curves for MAPD90 were generated using a standard S1-S2 protocol. Results. All manoeuvres decreased left ventricular developed pressure (LVDP): 0.7 mmol 1-1 Ca2+ to 74.0 ± 6.1%, 20 mmol 1-1 BDM to 4.5 ± 1.0%, and 5 µmol 1-1 Cyto-D to 12.8 ± 3.5% of control value. CD decreased from a control value (33.3 ± 1.0 ms, n = 16) to 93.0 ± 2.2% in 0.7 mmol 1-1 Ca2+, but increased to 133.7 ± 10.5% in 20 mmol 1-1 BDM and 127.4 ± 10.6% in 5 µmol 1-1 Cyto-D. At 350 ms pacing cycle length, MAPD90 (control = 119.6 ± 1.7 ms n = 16) was prolonged by reduced extracellular [Ca2+]. BDM had no effects on MAPD90 at control pacing rates. Cyto-D caused a significant prolongation (to 115.0 ± 3.0% of control, n = 6) at the highest concentration studied (5 µmol 1-1). Both BDM (20 mmol 1-1) and Cyto-D (3 µmol 1-1) flattened the restitution curves but neither agent altered maximum MAPD90. Conclusions. Extracellular... [ABSTRACT FROM AUTHOR]

Early life and preconception environmental stimuli can affect adult health-related phenotypes. Exercise training is an environmental stimulus affecting many systems throughout the body and appears to alter offspring phenotypes. The aim of this study was to examine the influence of parental exercise training, or 'exercise ancestry', on morphological and metabolic phenotypes in two generations of mouse offspring. The F0 C57BL/6 mice were exposed to voluntary exercise (EX) or sedentary lifestyle (SED) and bred with like-exposed mates to produce an F1 generation. The F1 mice of both ancestries were sedentary and killed at 8 weeks or bred with littermates to produce an F2 generation, which was also sedentary and killed at 8 weeks. Small but broad generation- and sex-specific effects of exercise ancestry were observed for body mass, fat and muscle mass, serum insulin, glucose tolerance and muscle gene expression. The F1 EX females were lighter than F1 SED females and had lower absolute tibialis anterior and omental fat masses. Serum insulin was higher in F1 EX females compared with F1 SED females. The F2 EX females had impaired glucose tolerance compared with F2 SED females. Analysis of skeletal muscle mRNA levels revealed several generation- and sex-specific differences in mRNA levels for multiple genes, especially those related to metabolic genes (e.g. F1 EX males had lower mRNA levels of Hk2, Ppard, Ppargc1a, Adipoq and Scd1 than F1 SED males). These results provide preliminary evidence that parental exercise training can influence health-related phenotypes in mouse offspring.

Altering [K+]o might have different effects on action potential duration (APD) in myocytes from different regions. Therefore, the effects of [K+]o on regional differences in action potential characteristics were investigated in sub-endocardial, mid-myocardial and sub-epicardial myocytes isolated from the base of guinea-pig left ventricular free wall using three different [K+]o (2.7, 5.4 and 8.1 mM KCl). Action potentials were recorded using the switch-clamp technique at 0.5 Hz. Increasing [K+]o from 2.7 to 8.1 mM shortened the action potential duration to 90 % repolarization (APD90; mean APD90 values in sub-endocardial, mid-myocardial and sub-epicardial myocytes were, respectively, 295 +/- 9, 286 +/- 9 and 266 +/- 8 ms in 2.7 mM [K+]o, 270 +/- 7, 255 +/- 7 and 215 +/- 7 ms in 5.4 mM [K+]o, 234 + 7, 212 +/- 10 and 155 +/- 8 ms in 8.1 mM [K+]o), depolarized the resting potential, and reduced the amplitude of the action potential. The effect of increasing [K+]o on action potential characteristics was more pronounced in sub-epicardial myocytes than in sub-endocardial and mid-myocardial myocytes. The regional differences in APD90 in 5.4 mM [K+]o were increased in 8.1 mM [K+]o and abolished in 2.7 mM [K+]o. In conclusion, changing [K+]o produces more pronounced effects on action potentials in sub-epicardial myocytes than in sub-endocardial myocytes, modifying the normal heterogeneity of action potentials. The differences in the response of sub-epicardium and sub-endocardium to [K+]o may contribute to the flattening or inversion of the T wave commonly seen in patients presenting with hypokalaemia and the upright and tall T waves observed in electrocardiograms recorded during hyperkalaemia, although the underlying ionic currents remain to be determined.

The effects of osmotic stress on the cytosolic Ca2+ concentration ([Ca2+]i) in the endothelium of excised intact rat aorta were investigated using the indicator fura-2 and the patch clamp technique. Hyperosmotic stress evoked a reversible rise in endothelial [Ca2+]i in the presence but not absence of extracellular Ca2+, indicating that it evoked Ca2+ entry without release from intracellular stores. Hyposmotic stress was without significant effect. Cytochalasins B and D reduced the effect of hyperosmotic stress but not acetylcholine on endothelial [Ca2+]i. In endothelium isolated from underlying smooth muscle, hyperosmotic stress produced a rise in the [Ca2+]i and depolarisation of the endothelial membrane potential whereas hyposmotic stress was without effect. Mechanosensitive cationic channels recorded in cell-attached patches were activated by hyperosmotic solutions applied to the endothelium and inhibited by hyposmotic solutions. These data suggest that shrinkage of endothelial cells evokes an increase in [Ca2+]i by opening a pathway for Ca2+ entry from the extracellular space. The mechanosensitive ionic channels which we have previously described may be responsible for this response.

Shear stress is an important modulator of endothelial function which can affect vessel tone, endothelial morphology and gene transcription. However, the mechanism by which the endothelium detects changes in shear stress is the subject of conflicting reports. The possible contributions of changes in the cytosolic Ca2+ concentration ([Ca2+]i) and of membrane hyperpolarisation are disputed. Here we have investigated the effects of shear stress on these variables in native endothelium in situ in rat aorta and hepatic portal vein. Endothelial [Ca2+]i was recorded using the fluorescent indicator fura-2. The [Ca2+]i in unstimulated rat aortic endothelium was 86 +/- 12 nM (n = 9). Acetylcholine (ACh) evoked relaxation of the aorta and a biphasic increase in endothelial [Ca2+]i. Shear stress that evoked relaxation comparable to that evoked by ACh did not affect the endothelial [Ca2+]i. Endothelial membrane potential was recorded using the patch clamp technique. The resting membrane potentials of rat aortic and portal vein endothelium were -47 +/- 3.8 mV (n = 14) and -68 +/- 4.5 mV (n = 9), respectively. Shear stress did not affect the endothelial membrane potential of rat aorta, but evoked a small (-1.6 +/- 0.3 mV, n = 9) hyperpolarisation of the endothelium of rat portal vein in all preparations studied. These results suggest that neither changes in [Ca2+]i nor hyperpolarisation are involved in the reception of shear stress by native rat aortic endothelium in situ, but that shear stress induces small hyperpolarisations of endothelium of portal vein, a mechanism that may be present and of greater physiological significance in other parts of vascular system.

Uridine triphosphate (UTP) evoked inhibition of adrenaline-evoked cAMP accumulation in cultured equine epithelial cells (EC50, 1.8 +/- 0.2 microM) and this effect was mimicked by 5-Br-UTP (EC50, 6.6 +/- 1.8 microM) and uridine diphosphate (UDP; EC50, 96 +/- 26 microM). This inhibitory action of UTP was abolished by pre-treating cells with pertussis toxin (10 ng ml-1, 24 h). UTP (EC50, 2.3 +/- 0.3 microM) and 5-Br-UTP (EC50, 29.4 +/- 9.4 microM) also increased intracellular free calcium ([Ca2+]i) whilst UDP did not; the two effects are thus differentially sensitive to these pyrimidine nucleotides. ATP evoked cAMP accumulation in control cells and this response was unaffected by pertussis toxin. There is, therefore, no indication that ATP activates the pertussis toxin-sensitive inhibitory pathway. The UTP-evoked inhibition of cAMP accumulation was abolished by isobutylmethylxanthine (IBMX, 5 mM) and so the negative control over cAMP levels appears to be mediated by receptors that are selectively activated by pyrimidine nucleotides and permit control over phosphodiesterase activity.

A method is described that enables the cell membrane of isolated rat ventricular myocytes to be permeabilized and resealed while maintaining cell viability. Streptolysin O, a cholesterol-binding cytolysin, was used to form pores in the surface membrane; subsequent incubation with 5% fetal bovine serum was used to reverse this permeabilization. The efficacy of membrane permeabilization and resealing was ascertained using a simultaneous double-staining technique using propidium iodide, a marker for cells with permeabilized membranes, and fluorescein diacetate, a marker for viable cells. This procedure allowed a distinction to be made between dead cells, unpermeabilized cells and viable cells that had been successfully permeabilized and resealed. The accessibility of the cell interior during permeabilization was investigated by including fluorescein isothiocyanate (FITC)-labelled dextrans (11, 38 and 148 kDa) and bovine serum albumin (67 kDa) in the permeabilization buffer, and localizing the FITC label using confocal microscopy following resealing. The confocal images showed that these molecules entered the cells and were retained after resealing. Following the permeabilization-resealing protocol, cells appeared to have both normal morphology and response to electrical stimulation. Thus this appears to be a cheap, simple and effective method to introduce relatively large molecules into cardiac myocytes.

Exposure of cardiac muscle to metabolic poisons reduces the availability of cellular ATP and cardiac dysfunction ensues. In this study rat ventricular myocytes were exposed to 2-deoxyglucose, iodoacetate and cyanide to induce complete metabolic blockade. Changes in contraction, cytosolic Ca2+ and pH were determined during metabolic blockade and following restoration of mitochondrial ATP production. Metabolic blockade resulted in a rapid failure of contractions and Ca2+ transients, a rise of diastolic Ca2+, a cytosolic acidosis and ultimately a rigor contracture. Washing out cyanide during the development of the rigor contracture led to a rapid relaxation of the contracture, a fall in cytosolic Ca2+ and a rapid, partial reversal of the cytosolic acidosis. The partial reversal of the cytosolic acidosis and fall of cytosolic Ca2+ were abolished in the presence of oligomycin. This suggests that the rapid partial recovery of cytosolic acidosis could result from the rephosphorylation of ADP to ATP by the mitochondrial F1,F0-ATPase (a reaction that consumes protons).

The inward transport of two purines, adenosine and hypoxanthine, at 37 degrees C by horse erythrocytes was compared. No mediated transport of adenosine was detected in horse erythrocytes, nor was saturable, high-affinity binding of the potent facilitated-diffusion inhibitor nitrobenzylthioinosine demonstrable in horse erythrocyte membranes. In contrast, erythrocytes from most horses possessed a saturable sodium-dependent hypoxanthine transporter (apparent K(m), 100 +/- 28 microM; Vmax, 0.20 +/- 0.08 mmol (l cells)-1 h-1; means +/- S.E.M., n = 5). Guanine inhibited hypoxanthine influx (apparent Ki, 24 +/- 6 microM), but adenine and xanthine had no effect. Unlike human erythrocytes, no sodium-independent hypoxanthine transporter was detected in horse erythrocytes. There are, however, a small number of animals (approximately 15%) whose erythrocytes fail to transport hypoxanthine. This variation appears to be under genetic control, but the precise nature of the control is unknown.

A systematic examination has been made of the active and passive electrophysiological properties and synaptic inputs of forty-four randomly impaled submucosal neurones in the proximal colon of the guinea-pig to compare these characteristics directly with those of submucosal neurones in the caecum (n = 70) and descending colon (n = 45). Within each of the three electrophysiological classes of submucosal neurones identified (S, S/AH and AH), no statistically significant regional differences were found with respect to the resting membrane potential, membrane time constant or input resistance between neurones of the proximal colon, descending colon and caecum. Of submucosal neurones from the proximal colon, forty-three of forty-four (98%) received fast excitatory synaptic potentials (fast EPSPs); thirty-nine (91%) were S neurones and the others were S/AH neurones; only one of the forty-four cells (2%) was an AH neurone. An idazoxan-sensitive slow inhibitory postsynaptic potential (slow IPSP) was induced in thirty of forty-three S and S/AH neurones (70%) of the proximal colon, compared with sixty-one of sixty-six caecal neurones (92%) and twelve of forty-one neurones (29%) in the descending colon. The mean (+/- S.E.M.) amplitude of the slow IPSP in proximal colonic neurones was 17 +/- 1 mV (range, 6-30 mV; n = 30), compared with the significantly larger synaptic response (25 +/- 1 mV; range, 7-38 mV; n = 66; P < 0.05) recorded in the caecum; the mean slow IPSP amplitude in the descending colon was significantly smaller (12 +/- 2 mV; range, 5-27 mV; n = 12; P < 0.05) than that in the caecum. In the proximal colon and caecum, only those neurones with a slow IPSP had a hyperpolarizing response to noradrenaline, whereas about 50% of those neurons of the descending colon that lacked a slow IPSP were hyperpolarized by noradrenaline, acting via alpha 2-adrenoceptors. Thus, the electrophysiological characteristics of the submucosal neurones of the proximal colon more closely resemble those of the caecum than those of the descending colon, of which many do not have a functional noradrenergic synaptic input. Furthermore, the results confirm that there are fundamental regional differences in the guinea-pig large intestine with respect to the synaptic organization of submucosal neurones of particular electrophysiological classes.

This study investigates whether an increase in L-type calcium current (ICa) could explain the prolongation of the action potential associated with the cardiac hypertrophy produced by repeated administration of isoprenaline. Hypertrophy was induced by daily injection of isoprenaline (5 mg/kg i.p.) for 7 days in male Wistar rats. Under whole-cell voltage-clamp conditions, ICa was evoked in Na(+)- and K(+)-free solution, by step depolarizations from a holding potential of -45 mV in single left ventricular myocytes isolated from control and hypertrophied rat hearts. In the test group, heart weight to body weight ratio and cell membrane capacitance were increased by 30 and 34%, respectively. Peak ICa was increased by 26% (control, -1.46 +/- 0.06 nA, n = 17; hypertrophy, -1.85 +/- 0.13 nA, n = 19; P < 0.02). However, when normalized for cell capacitance, there was no significant difference in peak current density (control, -12.1 +/- 0.5 pA/pF; hypertrophy, -11.5 +/- 0.6 pA/pF). The voltage dependence of ICa was similar in both cell types. No change was observed either in the steady-state activation or inactivation kinetics, or in the time course of inactivation. The recovery from inactivation of ICa, when fitted with monoexponential function with time constant tau rec, was not changed significantly by hypertrophy (control, tau rec = 115 +/- 23 ms, n = 9; hypertrophy, tau rec = 120 +/- 12 ms, n = 15). The increased calcium current occurs in parallel with the increase in cell size. The prolonged action potential duration seen in this model must be explained by changes in currents other than L-type calcium current.

The aim of the present experiment was to determine whether the potassium channel opener 2-(2,2-bis(difluoromethyl)-6-nitro-3,4-dihydro-2H-1, 4-benzoxazine-4-yl)pyridine-N-oxide (ZM260384) was capable of accelerating the decline in skeletal muscle function during restricted blood flow in vivo. Cats (3.0-4.5 kg body weight) were anaesthetized with alphaxalone-alphadalone and breathed spontaneously following tracheotomy. Isometric tension was measured in the extensor digitorum longus-anterior tibialis (EDL-TA) muscle group. Ischaemia was induced by perfusing the hindlimb with the animal's own blood at a rate of 12.5 ml min-1 using a roller pump and stimulating the common peroneal nerve to induce repetitive submaximal tetanic contractions in the EDL-TA. The number of stimulation voltage increments required each minute to maintain a constant level of submaximal mechanical output and the time to exhaustion were used as indices of the rate of tension decline. The rate of tension decline in the ischaemic EDL-TA in the presence of ZM260384 at 3 mg kg-1, a maximally hypotensive dose predicted to be within the dose range required to exert direct effects on skeletal muscle, was measured and compared with the rate of tension decline in the presence of ZM260384 at 0.03 mg kg-1, also maximally hypotensive dose but below the predicted dose range for skeletal muscle effects. The number of voltage increments per minute was 1.93 +/- 0.07 and 1.48 +/- 0.14 (P < 0.05) in the presence of 3 and 0.03 mg kg-1 ZM260384, respectively. Time to exhaustion was 17.5 +/- 4.2 and 7.2 +/- 0.8 min (P < 0.05) in the presence of 3 and 0.03 mg kg-1 ZM260384, respectively. Given that there was no difference between these two groups in any haemodynamic variable measured, the results of the present study suggest that ZM260384 (3 mg kg-1) increases the rate of isometric force loss in ischemic skeletal muscle in vivo.

The whole-cell patch clamp technique was used to investigate the effect of different charge carriers upon ultra-slow voltage-dependent inactivation of L-type Ca2+ channel current in ferret ventricular myocytes at 37 degrees C. Intracellular Ca2+ was buffered with 10 mM EGTA and the membrane potential held at -40 mV. With Ba2+ as the charge carrier, the L-type current decayed throughout 20 s pulses to 0 mV as a result of ultra-slow voltage-dependent inactivation. In contrast, with Ca2+ as the charge carrier, there was no such slow decay of the current as the current decayed almost completely in the first approximately 100 ms as a result of Ca(2+)-dependent inactivation. However, with Ca2+ as the charge carrier it is still possible that ultra-slow voltage-dependent inactivation occurs. A conditioning-test pulse protocol and a second protocol were used to test for the development of ultra-slow inactivation during 20 or 30 s pulses to 0 mV with Ca2+ as the charge carrier. Ultra-slow inactivation did occur and it was qualitatively similar to that with Ba2+ as the charge carrier. The onset of ultra-slow inactivation with Ca2+ as the charge carrier could be described by the sum of two exponentials with time constants of 0.3 and 6.7 s. Recovery from ultra-slow inactivation with Ca2+ as the charge carrier was also measured with a conditioning-test pulse protocol and was best described by the sum of two exponentials with time constants of 0.5 and 6.2 s. We conclude that ultra-slow inactivation of the L-type current does occur with the physiological charge carrier, Ca2+, but it is normally masked by Ca(2+)-dependent inactivation.

Ventricular myocytes, isolated from the guinea-pig, were stimulated to contract by 100 ms long voltage clamp pulses from -80 to 0 mV at 0.5 and 3 Hz. An increase in frequency from 0.5 to 3 Hz led to a positive inotropic effect. Contraction-voltage relationships (CVR) were determined at each frequency. The CVR at 0.5 Hz was bell shaped and peaked between 0 and +20 mV, displaying a voltage dependence similar to the L-type Ca2+ current (ICa). At 3 Hz, contractions continued to increase at positive voltages, giving a more sigmoidal CVR. At 0.5 Hz, TTX reduced the size of steady-state contractions to 91 +/- 2% of control values, but had no effect on the shape of the CVR. At 3 Hz, TTX significantly reduced (P < 0.05) the magnitude of contractions at positive voltages (> or = +20 mV) but had no significant effect on contractions at voltages negative to 0 mV. These data illustrate that intracellular sodium activity (aNa(i)) and, in particular, Na+ entry due to the sodium current (INa) are important in determining the voltage dependence of contraction at positive voltages. Thapsigargin (2.5 microM), a blocker of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, reduced the size of steady-state contractions at 0 mV to 65 +/- 7% at 0.5 Hz. Increasing frequency to 3 Hz abolished the positive inotropy seen under control conditions. With thapsigargin present, contractions at 0.5 Hz were reduced at all potentials and the CVR was bell shaped. At 3 Hz the CVR was sigmoidal in shape. Contractions were significantly inhibited by thapsigargin at all potentials, but most significantly at more positive potentials (> or = +20 mV). These data show that, at normal body temperature, the shape of the CVR of guinea-pig ventricular myocytes changes with stimulation rate. Due to the voltage dependence of ICa, contractions evoked at positive voltages at 3 Hz must be supported by other mechanisms. The sensitivity of such contractions to TTX and thapsigargin suggests the involvement of both a Na(+)-dependent process and the SR. One possibility is that when aiNa and the Ca2+ content of the SR are raised at higher stimulation rates, enhanced Ca2+ entry via reverse Na(+)-Ca2+ exchange leads to a direct activation of the myofilaments and, to a lesser extent, the release of Ca2+ from the SR.

Both neurogenic influences and the regulatory neuropeptide substance P (SP) have been implicated in the development of joint inflammation. Using the laser Doppler perfusion imaging technique to quantify relative changes in joint blood flow, the effects of nerve stimulation and topical SP application were examined in normal and chronically inflamed rat knee joints. Synovial inflammation was induced by unilateral intra-articular injection of Freund's complete adjuvant and experiments were carried out 1 week and 3 weeks later. Normal knees showed a frequency-dependent vasoconstriction in response to saphenous nerve stimulation over the range of 5-30 Hz and a dose-dependent vasodilation in response to SP administration. These vasoactive responses were completely abolished in the chronically inflamed knee joint, the abolition persisting throughout the investigation. Since articular cartilage is critically dependent on synovial fluid formation for its nutrition, loss of neurovascular control of the synovial microcirculation could contribute to the degenerative changes that commonly accompany chronic inflammatory joint diseases.

An imaging technique (laser Doppler perfusion imaging, LDI), based on measurement of backscattered Doppler-broadened laser radiation, was used to provide two-dimensional images of perfusion in the exposed rat knee joint capsule. In control animals, frequency-dependent constriction of knee joint blood vessels occurred during electrical stimulation of sympathetic nerve fibres whilst dose-dependent vasodilatation of these vessels was elicited by direct application of the sensory neuropeptide substance P (SP) to the capsule. Intra-articular injection of the neurotoxic agent capsaicin did not affect vasoconstrictor responses when tested 5-7 days later, but substantially reduced dilator responses to SP. These findings indicate that capsaicin is selectively neurotoxic for sensory unmyelinated fibres but not sympathetic postganglionic fibres and is also capable of modifying receptor-mediated effects of SP. LDI is a useful method for mapping tissue perfusion, particularly in structures such as joints where the spatial distribution of blood flow is heterogeneous.

The aim of the present experiment was to investigate the relative contribution of nitric oxide produced in endothelial cells to functional and reactive hyperaemia in the hindlimb of anaesthetized cats. Cats (2.5-3.4 kg) were anaesthetized with alphadalone-alphalaxone, and breathed spontaneously following tracheotomy. Left hindlimb blood flow was measured with a flow probe and hyperaemia responses were monitored following 10 s occlusion of the left external iliac artery and during 20 min stimulation of the sciatic and femoral nerves at 3 Hz. This was repeated following nitric oxide synthase inhibition with NG-nitro-L-arginine methyl ester (L-NAME, 100 mg kg-1, I.V.). Following L-NAME administration, baseline hindlimb blood flow and arterial blood pressure were restored by infusion of sodium nitroprusside (range, 0.3-2.25 micrograms kg-1 min-1, I.V.). Following arterial occlusion, L-NAME reduced the peak reactive hyperaemia (6.5 +/- 0.8 vs. 4.5 +/- 1.0 ml min-1 kg-1, P < 0.05) and blood flow repayment (9.9 +/- 2.3 vs. 6.1 +/- 2.6 ml, P < 0.05) responses. In contrast, the total functional hyperaemia response during hindlimb contraction was not altered (264.7 +/- 68.2 vs. 264.4 +/- 62.8 ml kg-1, n.s.). The results of the study suggest that the production of nitric oxide from endothelial cells does not contribute to functional hyperaemia in contracting skeletal muscle, but plays a large role in reactive hyperaemia. The results imply that flow-dependent dilatation of feed arteries is mediated by NO in reactive hyperaemia.

Since the retinal vessels have no sympathetic innervation, blood flow in response to raised blood pressure is dependent on autoregulation. To determine the effect of hypertension on retinal haemodynamics and the autoregulatory capacity of the retinal circulation under conditions of normoglycaemia and hyperglycaemia, retinal blood flow was measured before and after raising the systemic blood pressure in ten normal control subjects, ten diabetic subjects with blood glucose < 10 mmol l-1 and ten diabetic subjects with blood glucose > 15 mmol l-1. A controlled rise in systemic blood pressure was achieved using an intravenous infusion of tyramine. Retinal volume flow was determined from red cell velocity using laser Doppler velocimetry and from retinal vessel diameter measurements using digital image analysis of fundus photographs. With a 40% increase in mean arterial blood pressure (MAP), there was a significant increase in retinal blood flow of 32.9 +/- 7.1% in non-diabetic controls. In diabetics at the low blood glucose level, the increase in retinal blood flow was significant at 30% increase in MAP (23.6 +/- 8.7%, P = 0.032) and at 40% increase (49.9 +/- 12.03%, P = 0.004). Diabetics with high blood glucose failed to autoregulate at any of the increased levels of MAP (15% increase, 27.0 +/- 11.1%; 30% increase, 66.9 +/- 19.8%; and 40% increase, 101.9 +/- 21.4%; P < 0.022). The coefficients of autoregulation showed that in non-diabetic controls, retinal vascular autoregulation broke down with increases in MAP of between 30 and 40%. In diabetic subjects, it broke down between 15 and 30% in normoglycaemia and at less than 15% in hyperglycaemia. This study demonstrates an impairment in retinal vascular autoregulation in response to raised systemic blood pressure in diabetic subjects, more so at an elevated blood glucose level, thus providing a mechanism for the detrimental effect of hypertension on diabetic retinopathy.

We have explored the factors that may regulate membrane permeability in a cell line (NCL-SG3) derived from the human sweat gland epithelium. Ionomycin increased the rate of 125I-efflux from preloaded cells and this action appeared to be due to an increase in intracellular free calcium ([Ca2+]i). The ionomycin-evoked increase in 125I- efflux was reduced in cells that were exposed either to barium or to valinomycin in the presence of a high concentration of external potassium. It thus appears that a fraction of the ionomycin-evoked increase in 125I- efflux is due to the activation of potassium channels and experiments using 86Rb+ also suggested that ionomycin increased the rate of potassium efflux, an effect which was totally abolished by barium. Blockade of Na(+)-K(+)-2Cl- cotransport and of Cl- -HCO3- exchange reduced the basal rate of 125I- efflux and the ionomycin-evoked increase in 125I-efflux from control cells and from cells depolarized by valinomycin. These transport systems thus contribute to anion efflux, although [Ca2+]i-dependent chloride channels also appear to be present. Acetylcholine increases [Ca2+]i in the secretory cells of human sweat glands, but this neurotransmitter did not increase [Ca2+]i in NCL-SG3 cells and so membrane permeability was not under cholinergic control. Adrenaline did not increase [Ca2+]i, but this hormone did evoke cyclic-3',5'-adenosine monophosphate (cyclic AMP) production. However, membrane permeability was not under adrenergic control, as the cells did not appear to express functional, cyclic AMP-dependent anion channels. This may be because they were not fully differentiated under the culture conditions. ATP consistently evoked a dose-dependent increase in anion efflux that appeared to be mediated by [Ca2+]i. The increase in [Ca2+]i was initiated by the release of calcium from a limited internal store and was subsequently sustained by calcium influx. UTP and ADP also increased [Ca2+]i, whereas adenosine, AMP and alpha,beta-methylene ATP were without effect. These data thus suggest that a subclass of type 2 purine receptor, which is functionally coupled to phosphoinositidase C, is present in these cells.

The occurrence and disposition of immunoreactivity for neuropeptide Y (NPY) was investigated in the submucosa of guinea-pig caecum. There were few NPY-immunoreactive neurones but intraganglionic varicose fibres were so numerous and strongly immunoreactive that some somata may have been obscured. In each neurone tested, exogenous application of NPY (1-100 nM) depressed the duration and amplitude of the slow inhibitory postsynaptic potential. In contrast, NPY (1-300 nM) had no effects on the resting membrane potential, input resistance and fast and slow excitatory synaptic transmission.

The degree to which stretch-activated channels operate during physiological length changes in multicellular heart preparations, or how much the channels could contribute to length-dependent activation, is not known. We studied the relationship between muscle length and contractile force in guinea-pig papillary muscles superfused with gadolinium chloride (10 microM), a stretch-activated channel blocker, and compared the effects to those with nifedipine (0.25 microM), a calcium channel blocker. Gadolinium reduced contractile force statistically significantly more at the longer muscle lengths than at the short muscle lengths. This did not apply with nifedipine, although a marginally greater effect at longer lengths was perceptible. The results can only partly be explained by gadolinium having a non-specific action via the calcium channel, or Na(+)-Ca2+ exchange, and are consistent with the possibility that stretch-activated channels contribute to length-dependent activation in cardiac muscle, and thus to 'Starling's Law of the Heart'.

Sweat glands isolated from skin obtained from normal subjects and patients with cystic fibrosis (CF) were pre-loaded with 86Rb+ and superfused with a physiological salt solution and the rate of 86Rb+ efflux was measured as an indicator of cellular potassium permeability. Acetylcholine always evoked a permeability increase in the glands from control subjects and this response could be resolved into calcium-dependent and calcium-independent components. Sweat glands from CF patients did not show such consistent responses. In three individuals the glands were abnormally insensitive to acetylcholine but normal responsiveness was seen in a fourth case. It is proposed that CF can induce dysfunction of calcium-dependent control processes in sweat glands.

The changes in cytoplasmic free calcium ([Ca2+]i) which occur in isolated human sweat glands during cholinergic stimulation have been studied indirectly by monitoring potassium permeability. The acetylcholine-evoked permeability increase normally consists of transient and sustained phases which are attributed to the mobilization of intracellular calcium stores and to calcium influx respectively. Such consistent responses to acetylcholine could not be obtained during superfusion with bicarbonate-free, HEPES-buffered solutions. The human sweat gland in vitro therefore appears to have a strict requirement for bicarbonate. The sustained component of the response was not affected by total removal of external sodium, suggesting that calcium influx does not occur via a sodium-dependent system. The transient component, however, was abolished when external sodium was replaced by N-methyl-D-glucammonium (NMDG+). It therefore appears that secretagogue-evoked mobilization of cytoplasmic calcium is dependent, in some way, upon external sodium. This dependence is not, however, absolute as the response was essentially normal when sodium was replaced by lithium.