There are two basic modes of communication used between neurones in the nervous system: neurotransmission and neuromodulation. When representations of neural circuits are drawn, they tend to include only fast neurotransmission. This can lead us to believe that neurotransmission conveys information through the nervous system and that neuromodulation merely modifies the neurotransmission. Even the words that we use to describe these two forms of communication imply that meaning. However, the very act of modifying neurotransmission conveys information. Therefore, in order to better understand information flow in the nervous system, we need to determine how neuromodulation is integrated into neuronal circuits.

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.

Gender-based differences in cardiovascular mortality may be due to a cardio-protective effect of oestrogens on the myocardium. However, mRNA expression of oestrogen receptors in myocardial tissue of the adult heart has yet to be demonstrated. Furthermore, a calcium antagonistic action of 17[beta]-oestradiol on myocardial tissue has been discussed. Therefore, two subjects were investigated in atrial myocytes of the human, and ventricular myocytes of guinea-pig and rat in this study. (1) Are oestrogen receptors expressed in adult myocardial cells? (2) Is there an influence of oestrogens on the L-type calcium current of cardiac myocytes? Expression of oestrogen receptors was investigated by reverse polymerase chain reaction. L-type calcium current was usually measured by the patch-clamp technique in whole-cell recording mode under selective recording conditions, i.e. overlapping currents were blocked. One series of experiments was performed in perforated patch configuration to avoid internal perfusion. 17[beta]-Oestradiol inhibited L-type calcium current reversibly in all three species. At 10-5 M, the inhibition was 15-20 %. This inhibition was independent of the sex and the species. A full concentration-response curve of 17[beta]-oestradiol on basal L-type current was recorded from female guinea-pig myocytes. The inhibition increased from 2 % at 10-7 M to about 30 % at 10-4 M 17[beta]-oestradiol. The values could be fitted by a sum of two sigmoidal functions with log EC50 values of -6·5 and -4·9 M and Hill slopes of 2·5 for both. The specificity of the 17[beta]-oestradiol action was tested by recording the L-type current in the presence of 17[alpha]-oestradiol and oestrone. 17[alpha]-Oestradiol also inhibited the current, but with a maximal inhibition of only 17 %. The concentration-response curve could be fitted by a single sigmoidal function (log EC50 -6·3 M; Hill slope 0·55). Oestrone did not influence the current at all. The decrease in L-type current after the application of 17[beta]-oestradiol via a rapid perfusion system developed with a time constant of 3·4 s, which was in the same range as that for the influence of isoprenaline. The isoprenaline-stimulated L-type current was much more susceptible to the inhibition by 17[beta]-oestradiol, i.e. in pre-stimulated cells (1) the inhibitory effect is significantly higher (e.g. at 10-5 M, inhibition was 36·3 % compared with 11·2 % in untreated cells) and (2) an inhibitory effect can be seen with oestradiol concentrations as low as 10-9 M. Although the concentrations needed to gain a calcium antagonistic influence on the basal current were much too high to explain a cardio-protective influence of oestrogens, the presence of oestrogen receptors in cardiac myocytes of all three species, together with the shift in concentration dependence following pre-stimulation by isoprenaline, suggest that myocytes are a potential target for oestrogen.

The role of extracellular Ca2+ (Ca2+o) in the modulation of cardiac Cl- currents (ICl) such as those activated by cAMP or swelling is uncertain. The effects of Ca2+o and extracellular cadmium (Cd2+o) on Cl- currents in cultured chick cardiac myocytes were investigated in Na+- and K+-free internal and external solutions using the whole-cell patch-clamp technique. In the absence of Na+ and K+ internally and externally, the whole-cell current was predominantly ICl. In the absence of cAMP, removal of Ca2+o (+ 1 mM EGTA) resulted in an increase in the current that was suppressed by reduction of Clo- with a rightward shift of the zero-current potential towards the Cl- reversal potential. We designated this current as a Ca2+-inhibitable ICl. Addition of 0·5 mM Cd2+o with or without removal of Ca2+o also resulted in a 1·5- to 2·0-fold increase in ICl that was attenuated by 1 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). Under similar conditions, ICl activated by Cd2+o (in 1 mM Ca2+o solution) was not further increased by subsequent removal of Ca2+o, suggesting that addition of Cd2+o and removal of Ca2+o activated the same ICl. In contrast, exposure to 1 µM forskolin further enhanced ICl in the presence of Cd2+o. With 10 µM cAMP in the pipette solution, Ca2+-inhibitable ICl could be activated in myocytes that do not possess cAMP-activated Cl- channels, indicating that activation of Ca2+-inhibitable ICl does not require cAMP. In the presence of cAMP, in cells that display the cAMP-activated ICl, removal of Ca2+o resulted in a further increase in ICl, comparable to the Ca2+-inhibitable ICl. The Ca2+-inhibitable ICl was minimized when pipette solutions contained 1·5 µM Ca2+. These results suggest that removal of Ca2+o or application of Cd2+o activates a Ca2+-inhibitable ICl that is distinct from the cAMP-activated ICl.

Hypoxic pulmonary vasoconstriction (HPV) may be mediated, in part, by an oxygen-sensing mechanism intrinsic to pulmonary arterial smooth muscle. It has been proposed that hypoxia inhibits a K+ conductance, which promotes membrane depolarization, subsequent activation of L-type Ca2+ channels and ultimately constriction. We have monitored hypoxia-induced changes in the intracellular Ca2+ concentration ([Ca2+]i) of single myocytes isolated from the rat pulmonary arterial tree using microspectrofluorimetry and ratiometric measurement of indo-1 fluorescence. The basal level of [Ca2+]i was in the range 35-80 nM and cells were quiescent at rest exhibiting no spontaneous oscillations in [Ca2+]i. When the extracellular K+ concentration ([K+]o) was raised to 20 mM, the [Ca2+]i increased from [similar]60 to [similar]100 nM. This increase was abolished by nifedipine, demonstrating the presence, and need for activation, of functional voltage-gated L-type Ca2+ channels. Hypoxia (PO2 [less than or equal to] 30 mmHg; throughout) had little effect on the resting [Ca2+]i in myocytes isolated from either the main intrapulmonary artery, or its primary, secondary or tertiary branches. However, upon raising the [Ca2+]i by increasing [K+]o to 20 mM, hypoxia was found to lower [Ca2+]i, from [similar]110 to [similar]70 nM, in cells isolated from the main conduit and primary branches of the intrapulmonary artery. In marked contrast, when [Ca2+]i was raised, by increasing [K+]o to 20 mM, in myocytes isolated from secondary and tertiary branches of the intrapulmonary artery, hypoxia induced a further reversible increase in the [Ca2+]i from [similar]160 to [similar]240 nM. Neither hypoxia alone nor in combination with 20 mM K+o induced any increase in the [Ca2+]i in the presence of nifedipine. We conclude that hypoxia may modulate [Ca2+]i in rat pulmonary artery myocytes only following its elevation by a depolarizing stimulus.

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).

Feto-maternal vascular (PDF-M) and amniotic-maternal (PDA-M) potential differences were measured simultaneously on seven occasions in six conscious pigs of 100-106 days gestation. Resting values of PDF-M and PDA-M were not significantly different although the range was wide. Fetal intravascular injection of 20 µg adrenaline, but not of saline, was associated with a prompt reversible change, of equal magnitude, in both PDF-M and PDA-M. In some experiments polarity was reversed. Feto-amniotic potential difference did not change. There was no change in fetal plasma K+ and Na+ concentrations. Because of the simultaneous and equal alterations in PDF-M and PDA-M following adrenaline and the anatomical configuration of the pig conceptus, we conclude that the catecholamine modifiable component of PDF-M is generated by electrogenesis in the pig placenta, probably by its chorionic (trophoblastic) cell layer.

Force-velocity data at different temperatures (range, 10-35¡C) from intact fibre bundles are analysed to determine the temperature dependence of the maximal mechanical power output of fast and slow rat muscles. At 35¡C, the maximal mechanical power was [similar]250 kW m-3 (= 250 µW mm-3) in fast (probably an underestimate) and [similar]100 kW m-3 in slow muscle. Within the more physiological temperature range (25-35¡C), the temperature coefficient (Q10) of maximum power was 2-2·5. In both muscles, the maximal power at 10¡C was only about 3-5 % of that at 35¡C, the decrease being particularly pronounced at temperatures below 20¡C (Q10 of 5-7).

The effects of cyclical, passive manipulation (PM, 30 min day-1, 5 days week-1 for 6 months) mimicking the length excursions observed during stepping on the mechanical and associated biochemical properties of the inactive cat soleus muscle were determined in five cats. Inactivity was produced via spinal cord isolation (SI), i.e. complete spinal cord transections at low thoracic and high sacral levels and bilateral dorsal rhizotomy between the transection sites. Passive manipulation was administered to one leg of each SI cat. Compared with normal controls, SI resulted in [similar]70 % decrease in weight, an 80 % decrease in maximum tetanic tension (Po) and an [similar]100 % increase in maximum rate of shortening (Vmax) and myosin adenosine triphosphatase (mATPase) activity of the soleus. The passive manipulation regime partially ameliorated these effects. When compared with the control SI soleus, the SI-PM soleus weight and maximum tetanic tension were 12 and 21 % higher, respectively, and the Vmax and mATPase activity 21 and 12 % (p > 0·05) lower, respectively. Thus, inactivity resulted in a smaller and faster muscle, whereas passive manipulation for only 30 min a day tended to maintain these properties closer to normal control values. The results suggest a potential therapeutic effect of short bouts of cyclical, passive manipulation on otherwise inactive skeletal muscles.

To characterize an 'electrocholecystogram' (EChG) for the normal gall bladder (GB) that might act as a standard for the evaluation of pathological electrocholecystograms of the diseased GB, ten mongrel dogs (14·4 ± 3·2 kg) were studied. Under anaesthesia, the abdomen was opened and three silver-silver chloride electrodes were sutured to the GB serosa. The GB pressure was measured by a perfused catheter introduced through the fundus. The response of the myoelectrical activity of the GB to balloon distension was tested and the direction of electrical waves was defined by GB myotomies. Pacesetter potentials (PPs) were recorded exhibiting the same frequency, amplitude and velocity of conduction from the three electrodes of each dog on all test days. Action potentials (APs) followed the PPs randomly and were associated with a rise of the GB pressure. Balloon distension of the GB caused increased PP and AP frequency. The electrical waves showed proximally but not distally to GB myotomy, indicating that they propagate proximo-distally towards the cystic duct. To conclude, the GB possesses electrical waves which are probably initiated from a 'pacemaker' at the GB fundus. A normal EChG could be characterized in the canine model. It is suggested that this EChG might show changes in gall bladder diseases and might thus act as an investigative tool, provided it can be developed to be performed percutaneously.

Electromanometric recordings of caecal pressures were made in chronically cannulated ponies. Endoscopy allowed direct observation of the caecal base and cupola while recording intracaecal pressures and enabled a functional interpretation of caecal pressure profiles. Using gas-tight seals, the caecal gas cap baseline pressure (tone) was usually below atmosphere, becoming less negative postprandially. Diurnal variations were observed in the amplitude and frequency of caecal pressure profiles and the direction of caecal movements, during which intra-caecal gas cap pressures briefly became positive. A paraprandial (psychic) phase of enhanced caecal motility was observed. Manipulation of the caecal gas cap volume, by insufflation of nitrogen gas, demonstrated the capacity of the caecal base to regulate gas pressures and to eliminate fermenting gases.

The neurohypophysial hormone vasopressin contributes to control of urine output and, while urine flow shows a clear daily rhythm, there has been debate as to whether this is true of neurohypophysial hormones. A study was performed on fifteen adult males, with a mean age of 25 years, over a 24 h period, nine blood samples being taken at regular intervals for the determination of neurohypophysial hormones and indices of fluid balance. Samples were taken via an indwelling cannula so that sleep was undisturbed. A daily variation in the plasma concentrations of oxytocin and vasopressin was demonstrated with concentrations reaching a nadir in the late afternoon. Concentrations of both hormones peaked at 02.00 h. Vasopressin concentrations were inversely correlated with packed cell volume, indicating that the altered hormone release was affecting fluid retention. Consistent with this was the observation that the relationship of plasma osmolality to vasopressin depended on the time of day. To determine the effect of ageing, a similar study was performed on nine healthy elderly subjects with a mean age of 70 years. The nocturnal peak of vasopressin was markedly attenuated, while oxytocin release was similar to that in the younger group. These observations confirm the existence of a daily rhythm in the plasma concentrations of neurohypophysial hormones and indicate that the amplitude of the vasopressin change decreases with age.

During prolonged exercise in a cold environment, fatigue is generally associated with a depletion of endogenous glycogen stores. This has lead many authors to hypothesize that the carbohydrate (CHO) content of fluids ingested in cool environments should be high, yet this hypothesis has not been specifically examined. In the present study, six healthy males cycled to exhaustion at approximately 80 % of their maximum oxygen consumption ([Vdot]O2,max) with either no drink (ND), a 15 % CHO-electrolyte drink (15 % CHO) or a 2 % CHO-electrolyte drink (2 % CHO). Dietary intake and exercise were replicated 2 days prior to each trial. Mean (S.D.) ambient temperature was 10·0 (0·3)°C with a relative humidity of 72 (2) % and an air velocity of approximately 0·7 m s-1 on all trials. Weighted mean skin temperature was calculated, and rectal temperature and heart rate were recorded at rest, during exercise and at exhaustion. Venous samples were drawn before and during exercise and at exhaustion for determination of haemoglobin, haematocrit, blood metabolites and serum electrolytes and osmolality. Expired air was collected for calculation of [Vdot]O2 and respiratory exchange ratio which were used to estimate rates of fuel oxidation. Ratings of perceived exertion (RPE) were also obtained. Exercise capacity was not different (P = 0·49) between trials, with median (range) times to exhaustion of 90·6 (66·8-106·0), 97·7 (60·9-112·0) and 102·0 (77·1-170·7) min for the ND, 15 % CHO and 2 % CHO trials, respectively. The 15 % CHO drink significantly (P < 0·05) elevated blood glucose concentration and total CHO oxidation compared with the ND trial. The 2 % CHO drink significantly (P < 0·05) reduced the estimated change in plasma volume and resulted in a lower serum osmolality than the other two trials. No differences were observed in any thermoregulatory or cardiorespiratory responses between trials. These data suggest that maintenance of blood-borne substrate and/or plasma volume does not influence exercise capacity in the cold, as is commonly reported during this type and intensity of exercise in warmer environments.

Using microdialysis, we measured nitric oxide (NO) levels in healthy human skin, in vivo, before and during the local inflammatory response to histamine. Basal dialysate NO concentration, assayed using an amperometric technique, was 0·49 ± 0·06 µM (mean ± S.E.M., 21 probes, 14 subjects). Histamine injection produced transient increases in NO concentration within both the weal and flare which was blocked by the NO synthase inhibitor, NG-nitro-L-arginine-methyl ester (L-NAME). Dialysate NO concentration also increased following transdermal delivery of the nitrosovasodilator, glyceryl trinitrate. Thus, using microdialysis, it is possible to quantify NO production in human skin in vivo and study its modulation during the acute inflammatory response.