Atherosclerosis is the leading cause of mortality in the developed world. In the United States alone there are approximately 700 000 deaths per year attributable to coronary artery disease, stroke and peripheral vascular diseases (National Center for Health Statistics, 1994). Although national data demonstrate an increase in coronary artery disease prevalence since 1970 (Morano, 1996), there has been a significant decline in mortality from atherosclerosis over the past three decades. Much of this decline may be attributable to risk factor modification. Intriguingly, many of the risk factors for atherosclerosis (hypercholesterolaemia, hypertension, homocysteinaemia, diabetes mellitus and exposure to tobacco) are associated with a reduced elaboration of nitric oxide by the endothelium (endothelium-derived nitric oxide (EDNO)) (Lüscher, Raij & Vanhoutte, 1987; Celermajer et al. 1993; Wu & Meininger, 1995). The result is a perturbation in the normal functions of the endothelium to maintain appropriate vessel calibre and resist the development of atherosclerosis. This endothelial impairment occurs well before any structural changes of atherogenesis are detected. Indeed when otherwise normal vessels are exposed in vitro to oxidized lipoproteins, an impairment in EDNO-dependent vasodilatation can be detected within minutes.It is well documented that risk factor modification has reduced the incidence of morbidity and mortality due to cardiovascular disease (Pasternak, Grundy, Levy & Thompson, 1996). More recently, modification of risk factors has also been shown to restore endothelial function and it may be by this mechanism that morbidity and mortality are curtailed. For example, cigarette smoking, which has been significantly linked to the occurrence of coronary artery disease, is associated with endothelial vasodilator dysfunction (Celermajer et al. 1993). Cessation of smoking appears to improve endothelial vasodilator dysfunction and reverses this increased risk within a few years (Rosenberg, Kaufman, Helmrich & Shapiro, 1985; Ockene, Kuller, Svendsen & Meilahn, 1990). Likewise, lowering cholesterol has been shown to improve endothelial function (Stroes, Koomans, de Bruin & Rabelink, 1995; Treasure et al. 1995) and reduce the incidence of initial and recurrent coronary artery disease events (Sacks et al. 1996). The effect of these therapies on endothelial function may explain their beneficial impact upon clinical events. Indeed, the effect of lipid-lowering therapy on mortality greatly exceeds the rather modest effect upon angiographic measures of lesion progression (Fuster, Gotto, Libby, Loscalzo & McGill, 1996). The success of risk factor modification in reversing endothelial dysfunction and clinical events serves to illustrate the importance of restoring endothelial function by other means. A greater understanding of the involvement of the nitric oxide system in the pathophysiology of vascular disease may provide the basis for new therapeutic strategies.

Knowledge of pleural liquid pressure (Pliq) and composition is crucial for studies concerning intrapleural fluid dynamics, and pleural fluid turnover. We measured Pliq at intercostal and costal levels in anaesthetized spontaneously breathing rabbits using a minimally invasive method that assures a long-lasting hydraulic continuity between the pleural liquid and the recording system. Polyethylene tubes were glued either to the exposed endothoracic fascia or inserted into a rib to provide a sealed connection to the recording system. After inducing a pneumothorax with nitrous oxide (N2O) via an intrapleural cannula, a hole ([similar]0·7 mm2) was pierced in the parietal pleura through the tube lumen. The tubes were then connected to pressure transducers and the whole system was filled with heparinized saline to the level of the parietal pleura; finally the pneumothorax was removed after N2O washout and Pliq recordings were performed. A different kind of tube was used to obtain microsamples of pleural fluid (2·5-3 µl) during spontaneous breathing; colloid osmotic pressure of the microsamples ([Pi]liq) was measured with an osmometer, and averaged 9·3 ± 1·5 cmH2O (n = 70 samples). When pooled and plotted against lung height end-expiratory intercostal and costal Pliq data scattered along a single regression line with a slope of -0·83 and -0·90 cmH2O cm-1 in supine and prone animals, respectively. End-inspiratory costal Pliq was significantly more subatmospheric than intercostal in the ventral region of the chest (P < 0·05), and less subatmospheric in the dorsal region, regardless of posture. The techniques presented here could be helpful in gaining a greater insight into the physiology and pathophysiology of the pleural space in terms of pleural fluid dynamics and turnover.

Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism for matching the ventilation/perfusion ratio in the lung, but the signal transduction pathway through which hypoxia induces vasoconstriction remains unclear. We hypothesized that the decrease in K+ current induced by hypoxia is a key mechanism for HPV, and examined the effects of the substances which are expected to accumulate during hypoxia on the activity of large conductance Ca2+-activated K+ (BKCa) channels. Pulmonary and ear arterial smooth muscle cells were isolated from the rabbit using enzymatic digestion, and large conductance Ca2+-activated K+ current (IBK,Ca) was recorded in symmetrical K+ concentrations using the inside-out mode of the patch-clamp technique. Increasing the Na+ concentration on the intracellular side suppressed IBK,Cadose dependently: 4·6, 20·9, 35·5 and 44·6 % reduction with 4, 8, 12 and 16 mM Na+, respectively. Mg2+ also reduced IBK,Ca, and the maximum reduction was obtained at 0·5 mM. Lactate, adenosine, ADP and ATP did not significantly affect IBK,Ca. There was no difference between pulmonary and ear arterial smooth muscle cells in their response to the above substances; this finding rules out modulation of BKCa channels by the various factors thought to accumulate during hypoxia as a major mechanism involved in the decrease in the K+ conductance of pulmonary arteries in hypoxia.

The neuroendocrine response to surgical trauma is a complex set of hormonal and metabolic changes evoked by anxiety, blood loss, tissue damage, visceral handling, and also by the anaesthetic drugs and procedures (Traynor & Hall, 1981). A useful approach to understanding the surgery-induced stress syndrome is to individualize these stressful stimuli in order to explore the specific effects of anxiety, anaesthesia or surgery itself on the endocrine and metabolic responses that are observed altogether. The relative importance of each element contributing to surgical stress should be better characterized in order to provide the rationale for improving the management of surgical patients. For instance, it remains unclear whether the physiological alterations induced by surgical stress are a single syndrome arising from the addition of several stressors or comprise parallel responses to each stressor, with some convergent end-points.

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.

It has recently been shown that a sodium background current, ib,Na, exists in cardiac muscle cells whose effect is to depolarize the membrane so that the resting potential, Vm, is positive to the potassium equilibrium potential, EK. In ventricular cells, where ib,Na is smallest, Vm is about 10 mV positive to EK (EK = -87 mV at 37¡C). Yet, replacement of Na+ ions by large impermeant cations does not cause the expected hyperpolarization. We have studied this problem in guinea-pig myocytes using a single microelectrode recording technique in combination with a rapid external solution switch. Cells depolarized [less than or equal to] 0·5 mV from potentials between -80 and -73 mV and hyperpolarized up to 5 mV from potentials between -73 and -64 mV when 70 mM choline chloride or N-methyl-D-glucamine chloride were used to replace 70 mM Na+ in the bathing solution. Replacement by 70 mM lithium chloride, however, only caused hyperpolarization in very depolarized cells when the voltage change was much smaller. The changes were complete almost as soon as the solution change, i.e. within 250 ms, indicating that the actions are attributable to the external solution change rather than to secondary changes in intracellular concentrations. Patch clamp recording was used to investigate the mechanism involved. These experiments showed that the presence or absence of the inward rectifier current iK1 determines in which direction Na+ removal acts. In the absence of iK1 the changes are attributable to removal of ib,Na, whereas in the presence of iK1 the changes resemble the i(V) relation for iK1, implying that Na+ regulates iK1 in a way that can mask the changes in ib,Na. These results explain why removal of Na+ does not lead to hyperpolarization in ventricular cells as would be expected if changes in ib,Na were solely responsible. Computer reconstruction shows that the effects may be attributed to actions of sodium removal on the conductance and gating of iK1.

Under physiological conditions, calcium-dependent ([Ca2+]i-dependent) Cl- currents (ICl(Ca)) have been suggested to participate in the repolarizing processes. In this paper, the possible contribution of ICl(Ca) to transient inward currents and, hence to arrhythmias, has been studied in myocytes from the working myocardium and from the conductive system. Single atrial, ventricular and Purkinje cells, isolated enzymatically from rabbit heart, have been studied under whole-cell voltage-clamp and were internally perfused with the fluorescent Ca2+ indicator, fura-2 (100 µM). Ca2+ release from the sarcoplasmic reticulum was either induced by external application of caffeine or occurred spontaneously in Ca2+-overloaded cells. Membrane currents accompanying Ca2+ transients showed linear current-voltage characteristics between -60 and +80 mV as evidenced from fast voltage ramps. When intra- and extracellular Cl- concentrations were kept symmetrical in the absence of the Na+-Ca2+ exchange mechanism, transient currents had a reversal potential close to 0 mV. Reduction of external Cl- concentration shifted this reversal potential towards the new Cl- equilibrium potential. Neither the time course of the transient currents nor the shift in their reversal potentials was significantly affected by the presence of Na+. Approximately 90 % of this current was blocked by the application of the Cl- channel blocker, anthracene-9-carboxylic acid (0·5 mM) at +80 mV. It is concluded, that [Ca2+]i-activated transient membrane currents in atrial, ventricular and Purkinje cells of rabbit heart are mainly due to the activation of a [Ca2+]i-dependent Cl- current.

The extrusion mechanism for intracellular Mg2+ was investigated in voltage-clamped snail neurones using Mg2+-sensitive microelectrodes and mag-fura-2. The intracellular free magnesium ion concentration ([Mg2+]i) of snail neurones voltage clamped to -60 mV was estimated to be 0·57 ± 0·06 mM (mean ± S.E.M.; n = 12) using Mg2+-sensitive microelectrodes and 0·62 ± 0·05 mM (n = 15) using mag-fura-2 . Raising extracellular MgCl2 from 5 to 20 mM caused an average increase in [Mg2+]i of 0·25 ± 0·04 mM (n = 7). In three experiments, removing extracellular MgCl2 caused an average decrease in [Mg2+]i of 0·1 mM. Replacing extracellular Na+ with N-methyl-D-glucamine (NMDG) caused a rise in [Mg2+]i of 1·8 ± 0·5 mM (n = 7); [Mg2+]i recovered to resting levels when extracellular Na+ was restored. Iontophoretic injections of MgCl2 were used to raise [Mg2+]i. The rate of recovery from such increases in [Mg2+]i (calculated from the slope of the recovery) was inhibited by 85-100 % (n = 5) in the absence of extracellular Na2+ compared with control conditions. Raising extracellular Ca2+ from 7 to 35 mM caused a reversible rise in [Mg2+]i of 0·4 ± 0·05 mM (mean ± S.E.M., n = 7). It was concluded that in snail neurones the main mechanism for [Mg2+]i extrusion is a Na+-Mg2+ exchanger which may be partially inhibited be high extracellular Ca2+ concentrations.

The intracellular mechanisms responsible for inhibition of osteoclast activity are of significant interest in the search for more effective ways of managing bone diseases associated with enhanced bone resorption. Previous studies have suggested that the protein kinase C (PKC) pathway is an important inhibitory second messenger in osteoclasts. We, therefore, investigated the effects of the synthetic peptide fragments, PKC(530-558) and (19-36), which correspond to parts of the catalytic and regulatory domains of PKC, on the activity of isolated osteoclasts. These fragments have been shown to activate and inhibit PKC, respectively, in biochemical studies employing isolated rat brain PKC, but have rarely been employed in studies of cellular activity. PKC(19-36), an enzyme inhibitor (PKC-I), had no effect by itself on osteoclastic bone resorption. However, PKC(530-558), a PKC activator (PKC-A), caused a dose-responsive inhibition of bone resorption, which was accompanied by a rapid and distinctive change in osteoclast morphology. This effect was reversible: (a) upon removal of PKC-A, (b) upon continuous exposure to this fragment for more than 36 h, or (c) in the presence of PKC-I. In conclusion, a short synthetic peptide fragment of PKC (PKC-A) significantly inhibits osteoclastic bone resorption; this, together with the fact that the inhibitory effect is abolished in the presence of PKC-I, provides further evidence for an important physiological role for the PKC pathway in the regulation of osteoclast activity. Selective activation of this pathway may have important therapeutic implications for the management of bone diseases associated with enhanced resorption.

The analysis of picomolar quantities of magnesium by electrothermal atomic absorption spectrophotometry (EAAS) was studied using a Perkin-Elmer-Zeeman 3030 spectrophotometer. The absorbance signal was not heavily dependent on the atomization temperature, but was greatly reduced when ashing temperatures in excess of 1200¡C were applied. The magnesium signal was significantly depressed in the presence of excess chloride in the sample matrix. However, use of NH4NO3 as a matrix modifier was sufficient to overcome this artefact. The analytical sensitivity was 0·15 absorbance units pmol-1 and the detection limit was 0·04 pmol. Using nanolitre constriction pipettes to dispense standards, the mean coefficient of variation was 5 %. Measurement of magnesium handling in the rat proximal convoluted tubule revealed a significant correlation between the tubular fluid-to-plasma ultrafiltrate (TF/UF) concentration ratio for magnesium and the tubular fluid-to-plasma (TF/P) concentration ratio for [3H]inulin (r2 = 0·56, n = 17). This indicated that magnesium is concentrated during its passage along the proximal tubule. In contrast, this was not the case for sodium (r2 = 0·11, n = 16). Mean (TF/UF)Mg (1·16 ± 0·07, n = 17) for random punctures was significantly greater than that for sodium ((TF/UF)Na = 1·02 ± 0·02, n = 16). Despite concentration of magnesium in the lumen, significant net reabsorption of magnesium was observed along the length of the tubule (fractional reabsorption, FRMg = 19·4 ± 3·0 %, n = 17). In conclusion, EAAS provides a highly sensitive, reproducible and technically simple method for measuring picomolar quantities of magnesium in renal tubular fluid.

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.

Basic fibroblast growth factor (bFGF) is a potent mitogen which induces growth of collateral vessels in ischaemic and infarcted myocardium. The effect of systemically administered bFGF on left ventricular (LV) function, myocardial hypertrophy and LV remodelling following acute myocardial infarction (MI) have not yet been fully investigated. Thirty Sprague-Dawley male rats were randomized to receive bFGF (0·5 mg) or rat albumin intraperitoneally for 1 week, beginning immediately after the induction of MI. Five animals served as controls and did not undergo any operation. Animals were killed 6 weeks after surgery and the hearts were perfused and fixed at physiological pressure. Transverse cross-sections from infarcted areas were stained with antibodies against proliferating cell nuclear antigen (PCNA) and Masson-trichrome and analysed with a coloured-image analyser for LV area (mm2), LV cavity diameter (mm), infarcted area (%), and wall thickness (mm) in infarcted and non-infarcted regions. LV area was similar in MI rats and in controls (41·7 ± 6·9 and 43·0 ± 1·5 mm2, respectively) and was significantly larger in MI bFGF-treated (MI/bFGF) animals (47·6 ± 7·1 mm2) (P = 0·023). LV cavity diameter was significantly larger in the MI group than in MI/bFGF and control animals (6·0 ± 0·8, 4·9 ± 1·4, and 4·4 ± 0·8 mm, respectively, P = 0·018). Wall thickness in the non-infarcted region was significantly smaller in MI animals (1·4 ± 0·3 mm) than in MI/bFGF animals (1·6 ± 0·4 mm) and the control group (1·6 ± 0·1 mm) (P = 0·015). The ratio between LV cavity diameter/non-MI wall thickness was higher in MI than in control and MI/bFGF groups (4·8 ± 1·6, 2·7 ± 0·6 and 3·3 ± 1·8, respectively, P = 0·03). Proliferating endothelial cells were significantly more abundant in infarcted than in normal areas in both MI and MI/bFGF groups, but with no significant differences between the groups. Intraperitoneal administration of bFGF did not cause any untoward extracardiac effects. Thus, systemic bFGF administration following acute MI in rats prevents dilatation of the LV, induces hypertrophy of the non-infarcted myocardium and exerts no untoward effects on extracardiac organs.

Transmission electron microscopic techniques were used to carry out a quantitative analysis of the density of fenestration in the inner medullary vasa recta of the rat kidney. Measurements were made at 200 µm intervals from the tip to the base of the papilla (1800 µm from the tip). Fenestral diaphragms were estimated to be 65·4 ± 0·78 nm in diameter (mean ± S.E.M.), and were arranged in plaques with a mean interfenestral distance of 114·8 ± 2·6 nm. Near the tip of the papilla there was no correlation between vessel size and degree of fenestration; density of fenestration, however, began to decrease about 1400 µm from the tip. The ratio of fenestrated to non-fenestrated profiles of vasa recta was found to be linear with respect to distance from the tip (r = 0·991), with values ranging from about 40 : 1 near the tip to 2 : 1 near the base of the papilla. We have estimated the proportion of the total surface area of a fenestrated vasa recta occupied by fenestral diaphragms to be 0·057 at 1000 µm from the tip. The total potential conductance (K) of a 200 µm segment of fenestrated vessel at 1000 µm from the tip was calculated to be 0·319 µm3 s-1 cmH2O-1, giving a hydraulic conductivity (Lp) of 0·030 µm s-1 cmH2O-1. We have also examined the reverse question of the conductance of a single fenestra if all the fluid flux across the vessel wall occurred through the fenestrae and none via the intercellular clefts or water channels; single fenestral conductance was estimated to be 1·94 × 10-3 µm3 s-1 cmH2O-1.

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 experiments were designed to elucidate the mechanism of thiopentone-induced inhibition of contractile responses in isolated human epigastric arteries. Segments of human epigastric arteries were obtained from patients who underwent elective or emergency caesarean section, placed in standard physiological salt solution (PSS), cut into rings at 3 mm intervals and suspended in organ baths for recording of isometric contractions at 37¡C, and pH 7·4. Three protocols were employed to examine the inhibitory effect of thiopentone: (a) concentration-dependent effect on 10-7 M noradrenaline (NA)- or high-K+ (40 mM)-induced contractions; (b) effect on NA-induced extra- and intracellular Ca2+-dependent contractions and (c) effect on the dose-response curve when Ca2+ is restored to Ca2+-depleted rings in Ca2+-free 40 mM K+-depolarizing medium. Thiopentone (1 × 10-6-4 × 10-3 M) caused concentration-dependent relaxation of both NA- and high-K+-induced contractions. The magnitudes of both precontractions were not significantly different but the IC50 values for thiopentone relaxation of high-K+ contractions were significantly lower than for NA contractions. Thiopentone (10-4 M) significantly attenuated the phasic (intracellular Ca2+ dependent) contractile responses to 10-5 M NA in Ca2+-free PSS as well as the tonic (extracellular Ca2+ dependent) contractions upon restoration of Ca2+. In contrast, nifedipine (1 µM) did not modify the phasic response but significantly attenuated the tonic response. Thiopentone (10-4 M) also almost completely abolished concentration-dependent Ca2+-induced contractions in K+-depolarized Ca2+-depleted rings. The results suggest that in the smooth muscle of human epigastric arteries, thiopentone-induced relaxation is non-specific and is associated with impairment of Ca2+ supply from both extracellular and intracellular pools.

The aim of this study was to evaluate whether long-term administration of L-arginine, a physiological substrate for the production of nitric oxide, improved blood pressure, heart rate, cardiac hypertrophy and particularly structural changes in the coronary and carotid artery of spontaneously hypertensive rats (SHR). The experiments started with three groups of 10-week-old animals: control Wistar rats, untreated SHR and SHR treated with L-arginine (SHR + L-arginine). L-Arginine was administered to SHR in a daily dose of 300 mg kg-1 intraperitoneally for 6 weeks. Blood pressure and heart rate were recorded each week. At the end of the experiment in one-half of each group heart weight and body weight were determined and the heart weight/body weight index was calculated. In the other animals, the cardiovascular system was perfused via the left ventricle with a glutaraldehyde fixative at 120 mmHg and the coronary and carotid arteries were processed for transmission electron microscopy. The inner diameter and wall thickness (tunica intima and tunica media) were measured on semithin sections. The reliability of the genetic feature in the SHR group was proved by the increased heart weight, heart weight/body weight index, wall thickness and wall thickness/inner diameter ratio of coronary and carotid arteries in comparison to the group of control Wistar rats. Long-term administration of L-arginine did not significantly influence blood pressure and heart rate in comparison with untreated SHR. Neither were any differences found in cardiac hypertrophy or the geometry of the coronary and carotid arteries (thickness of arterial wall, inner diameter, wall/diameter ratio). In conclusion, the changes in the cardiovascular system in SHR were not reversed, or even alleviated, by chronic treatment with L-arginine.

Short chain fatty acids (SCFAs) and CO2 have been shown to stimulate net Mg2+ efflux from the isolated reticulorumen in vivo. To investigate the underlying mechanisms of Mg2+ transport we performed Ussing chamber and microelectrode experiments and measured 28Mg2+ fluxes across sheep rumen epithelium in vitro. In the presence of SCFAs mucosal-to-serosal Mg2+ flux (JMgm-s) amounted to 82·3 ± 7·8 nmol cm-2 h-1 and serosal-to-mucosal Mg2+ flux (JMgs-m) to 3·2 ± 0·7 nmol cm-2 h-1. Replacing SCFAs with gluconate caused a 50 % reduction of JMgm-s, whereas JMgs-m was not affected. Among the SCFAs, n-butyrate was more effective in stimulating JMgm-s than acetate, propionate or iso-butyrate. Eliminating HCO3--CO2 from SCFA-containing solutions did not affect Mg2+ fluxes, whereas the same replacement in SCFA-free solutions led to a further reduction in JMgm-s. JMgm-s decreased after the addition of ethoxyzolamide to SCFA-free, bicarbonate buffered solutions. Decreasing mucosal pH from 6·4 to 5·4 increased JMgm-s in SCFA-free, bicarbonate buffered solutions. SCFAs had no effect on the apical membrane potential of rumen epithelial cells. The experiments show that both SCFAs and CO2 stimulate Mg2+ transport through an increase in JMgm-s, most probably via stimulation of a Mg2+-2H+ exchange mechanism. SCFAs may have additional metabolic effects on Mg2+ transport.

The acinar cells of the ferret parotid gland are supplied with parasympathetic nerve fibres containing vasoactive intestinal peptide (VIP) and substance P. In this study intracarotid infusions of the two peptides (0·5-1·0 µg kg-1 min-1 of each for 40 min) in the pentobarbitone-anaesthetized ferret, treated with atropine and adrenoceptor antagonists, induced a loss of acinar secretory granules from this gland, by 32 % in response to VIP and by 46 % in response to substance P. Stimulation of the parasympathetic auriculo-temporal nerve (40 Hz, in the presence of adrenoceptor antagonists) caused a larger loss of acinar granules from the gland than stimulation of the sympathetic superior cervical ganglion (intermittently, 50 Hz for 1 s every 10th second, in the presence of atropine) over 40 min (52 % versus 10 %). A 27 % granular loss in response to parasympathetic stimulation followed upon atropinization. The parasympathetic response was not further diminished by the tachykinin antagonist Spantide ((D-Arg1, D-Pro2, D-Trp7,9, Leu)-substance P). Thus, despite the large exocytotic response to the infusion of substance P, the parasympathetic non-adrenergic, non-cholinergic secretion of storage granules seemed, under the present experimental conditions, to occur independently of the action of substance P.

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.

Phloridzin-insensitive D-glucose uptake into enterocytes isolated sequentially from along the crypt-villus axis showed the majority of transport activity to reside in cells from the upper third of the villus. In contrast, total postnuclear glucose transporter 2 (GLUT2) protein content of the cells was high even close to the crypt and was almost constant for the upper 80 % of the villi. A 4 h lumenal perfusion in vivo with 100 mM D-glucose prior to harvesting the enterocytes produced a 2- to 3-fold increase in phloridzin-insensitive D-glucose uptake which extended down 70 % of the villus. Vascular infusion in vivo with either 800 pM gastric inhibitory polypeptide (GIP) or glucagon-like peptide-2 (GLP-2) prior to harvesting enterocytes produced the same response as lumenal glucose, while glucagon like peptide-1 (GLP-1) had no effect. Inclusion of 30 µM brefeldin A (BFA), an inhibitor of protein trafficking, in the lumenal perfusate produced a small, but not significant, increase in the control uptake profile along the villus in isolated enterocytes. However, BFA significantly reduced the upregulation induced by lumenal glucose and vascular GIP and blocked the stimulation produced by vascular GLP-2. Biotinylation of surface proteins and isolation with protein A indicated that there was no change in the membrane abundance of GLUT2 after GLP-2 treatment. These results are discussed in relation to the role of gastrointestinal peptide hormones in controlling intestinal hexose transport and the possibility of protein trafficking being involved in mediating the upregulation of GLUT2 activity in the enterocyte basolateral membrane.