Voltage-dependent calcium currents in vertebrate (Scyliorhinus canicula), protochordate (Branchiostoma lanceolatum), and invertebrate (Alloteuthis subulata) skeletal and striated muscle were examined under whole-cell voltage clamp. Nifedipine (10 µM) suppressed and cobalt (5 mM) blocked striated/skeletal muscle calcium currents in all of the animals examined, confirming that they are of the L-type class. Calciseptine, a specific blocker of vertebrate cardiac muscle and neuronal L-type calcium currents, was applied (0.2 µM) under whole-cell voltage clamp. Protochordate and invertebrate striated muscle L-type calcium currents were suppressed while up to 4 µM calciseptine had no effect on dogfish skeletal muscle L-type calcium currents. Our results demonstrate the presence of at least two sub-types of L-type calcium current in these different animals, which may be distinguished by their calciseptine sensitivity. We conclude that the invertebrate and protochordate L-type current sub-type that we have examined has properties in common with vertebrate 'cardiac' and 'neuronal' current sub-types, but not the skeletal muscle sub-type of the L-type channel. Experimental Physiology (2001) 86.6, 689-694.

Using a genetically modified herpes simplex virus encoding green fluorescent protein we sought to establish if this viral modification could be used in transneuronal tracing studies of the sympathetic nervous system. The herpes simplex virus encoding green fluorescent protein was injected into the adrenal medulla of three hamsters and six rats. After a suitable survival period, neurones in the sympathetic intermediolateral cell column of the thoracolumbar spinal cord, rostral ventral medulla and paraventricular nucleus of the hypothalamus were clearly identified by the presence of a green fluorescence in the cytoplasm of the neurones of both species. Thus, herpes simplex virus encoding green fluorescent protein labelled chains of sympathetic neurones in the hamster and rat and therefore has the potential to be used in transneuronal tracing studies of autonomic pathways in these species. Experimental Physiology (2001) 86.6, 695-702.

Despite minimal model characterisation Langendorff perfused murine hearts are increasingly employed in cardiovascular research, and particularly in studies of myocardial ischaemia and reperfusion. Reported contractility remains poor and ischaemic recoveries variable. We characterised function in C57/BL6 mouse hearts using a ventricular balloon or apicobasal displacement and assessed responses to 10-30 min global ischaemia. We examined the functional effects of pacing, ventricular balloon design, perfusate filtration, [Ca2+] and temperature. Contractility was high in isovolumically functioning mouse hearts (measured as the change in pressure with time (+dP/dt), 6000-7000 mmHg s-1) and was optimal at a heart rate of ~ 420 beats min-1, with the vasculature sub-maximally dilated, and the cellular energy state high. Post-ischaemic recovery (after 40 min reperfusion) was related to the ischaemic duration: developed pressure recovered by 82 ± 5 %, 73 ± 4 %, 68 ± 3 %, 57 ± 2 % and 41 ± 5 % after 10, 15, 20, 25 and 30 min ischaemia, respectively. Ventricular compliance and elastance were both reduced post-ischaemia. Post-ischaemic recoveries were lower in the apicobasal model (80 ± 4 %, 58 ± 7 %, 40 ± 3 %, 32 ± 7 % and 25 ± 5 %) despite greater reflow and lower metabolic rate (pre-ischaemic myocardial O2 consumption (V˙O2,myo) 127 ± 15 vs. 198 ± 17 µl O2 min-1 g-1), contracture, enzyme and purine efflux. Electrical pacing slowed recovery in both models, small ventricular balloons (unpressurised volumes < 50-60 µl) artificially depressed ventricular function and recovery from ischaemia, and failure to filter the perfusion fluid to < 0.45 µm depressed pre- and post-ischaemic function. With attention to these various experimental factors, the buffer perfused isovolumically contracting mouse heart is shown to be stable and highly energised, and to possess a high level of contractility. The isovolumic model is more reliable in assessing ischaemic responses than the commonly employed apicobasal model. Experimental Physiology (2001) 86.6, 703-716.

We examined the cardiovascular and ventrolateral medullary neuronal responses to muscle contraction in the spontaneously hypertensive rat (SHR) and normotensive Wistar-Kyoto rat (WKY) control. Cardiovascular, respiratory and ventrolateral medullary neuronal responses to muscle contraction evoked by tibial nerve stimulation were recorded. SHRs exhibited significantly larger drops in arterial pressure compared to WKYs in response to muscle contraction (P < 0.05). Basal ventrolateral medulla neuronal discharge rates were similar between the SHR and the WKY groups. A majority of neurons recorded responded to muscle contraction in both the WKY (77 %; n = 53) and the SHR groups (68 %; n = 62). There was no difference in the percentage of neurons that responded with an increase (~60 %) or decrease (~40 %) in firing rate between hypertensive and normotensive rats. Pulse wave-triggered averaging techniques showed that most neurons that responded to muscle contraction also possessed a basal firing rhythm temporally related to the cardiac cycle (85 % in WKYs, 83 % in SHRs). However, decreases in neuronal firing rates in response to muscle contraction were significantly greater in SHRs than WKYs. Therefore, we conclude that muscle contraction unmasks a hyperexcitability of neurons in the ventrolateral medulla of SHRs that parallels the heightened blood pressure responses. Experimental Physiology (2001) 86.6, 717-724.

The mechanisms controlling the coronary vascular responses of vessels perfusing the left ventricular (LV) myocardium that is hypertrophied from chronic volume overload are unclear. We hypothesised that endothelial function is compromised, and receptor-mediated contraction is exacerbated, in coronary resistance vessels from rabbits with LV hypertrophy compared to controls. The mitral valve of 10 rabbits was damaged surgically to cause mitral regurgitation and chronic volume overload, resulting in LV hypertrophy (LV hypertrophy rabbits). Echocardiographic assessment at 12 weeks verified that mitral regurgitation was present in LV hypertrophy but not sham-operated, weight- and age-matched animals (control rabbits; n = 17). Percentage increases from weeks 0 to 12 in LV cross-sectional area (47 ± 7 % vs. 2 ± 8 %), LV volume (47 ± 14 % vs. 7 ± 10 %) and LV mass (27 ± 4 % vs. 3 ± 6 %), were greater (all P < 0.05) in LV hypertrophy vs. control rabbits, respectively. At 12 weeks, coronary resistance vessel (~ 130 µm, internal diameter) reactivity was evaluated using wire myography. Endothelium-dependent (i.e. acetylcholine, 10-8-10-5 M) and -independent (i.e. sodium nitroprusside, 10-9-10-4 M) relaxation, and receptor-mediated vasocontraction (i.e. endothelin-1, 10-11-10-7 M) were similar between groups. However, tension development in response to nitric oxide synthase inhibition (10-6 M N G-monomethyl-L-arginine) was greater (P < 0.05) in LV hypertrophy compared to control rabbits. These results indicate that while coronary resistance vessel function is similar between groups, our estimate of basal nitric oxide production is greater in vessels from LV hypertrzophy than control rabbits. Experimental Physiology (2001) 86.6, 725-732.

Vasoactive intestinal peptide (VIP, 4 µg kg-1 min-1), substance P (3 µg kg-1 min-1) and neurokinin A (2.5 µg kg-1 min-1) were infused intravenously for 30 min in anaesthetized rats and the effects of these peptides on the parotid gland were examined. VIP reduced the numerical density of parotid acinar secretory granules, storing proteins, by 29 % and the glandular amylase activity by 33 %. Substance P reduced the number of secretory granules by 18 % but the amylase activity was unchanged. These results make VIP and substance P likely contributors to the parasympathetic non-adrenergic, non-cholinergic (NANC)-evoked parotid acinar degranulation. Neurokinin A, on the other hand, caused no reduction in granular number but reduced the glandular amylase activity by 19 %, indicating vesicular protein secretion. Experimental Physiology (2001) 86.6, 733-738.

The hypothesis has been advanced that synovium offers the main resistance to fluid escape from joints, even though it is under 20 µm thick. To test this, fluid was infused into the knee joint cavity of anaesthetised rabbits to set up a pressure gradient, then the profile of periarticular interstitial fluid pressure (Pif) was measured by advancing a micropipette, connected to a servo-null pressure recorder, in steps through a periarticular tissue 'window' until the joint cavity was entered. With intra-articular pressure (Pj) raised to 15 cmH2O (the pressure of an acute joint effusion) the pressure gradient dPif /dx (where x is distance) across the synovial lining was 0.47 ± 0.04 cmH2O µm-1 (n = 10 joints). This was 23.5-fold greater than the gradient in the subsynovium (0.02 ± 0.01 cmH2O µm-1; P < 0.0001, Student's t test), indicating that the hydraulic resistivity of the subsynovium is <=<>4 % of that of the synovium. The pressure profile was not altered by circulatory arrest. To test the hypothesis further, the effect of a stab perforation of the synovial lining on fluid drainage rate ((·Qs) was studied. Perforation raised both ·Qs and the conductance term d ·Qs/dPj more than 10-fold (n = 6 joints; P < 0.0001, ANOVA). The results thus support the view that, despite its thinness, the synovial lining offers the main hydraulic resistance to fluid drainage from a synovial joint. Experimental Physiology (2001) 86.6, 739-747.

To determine the functional role of nitric oxide (NO) in regulating vascular conductance during high intensity dynamic exercise in skeletal muscles composed of all major fibre types, female Wistar rats (277 ± 4 g; n = 7) were run on a motor-driven treadmill at a speed and gradient (60 m min-1, 10 % gradient) established to yield maximal oxygen uptake (V˙O2,max). Vascular conductance (ml min-1 (100 g)-1 mmHg-1), defined as blood flow normalised to mean arterial pressure (MAP), was determined using radiolabelled microspheres during exercise before and after NO synthase (NOS) inhibition with N G-nitro-L-arginine methyl ester (L-NAME; 10 mg kg-1, I.A.). The administration of L-NAME increased MAP from pre-L-NAME baseline values, demonstrating that NOS activity is reduced. The administration of L-NAME also reduced vascular conductance in 20 of the 28 individual hindlimb muscles or muscle parts examined during high speed treadmill exercise. These reductions in vascular conductance correlated linearly with the estimated sum of the percentage of slow twitch oxidative (SO) and fast twitch oxidative glycolytic (FOG) types of fibres in each muscle (Δconductance = -0.0082(%SO + %FOG) - 0.0105; r = 0.66; P < 0.001). However, if the reduction in vascular conductance found in the individual hindquarter muscles or muscle parts was expressed as a percentage decrease from the pre-L-NAME value (%Δ = (pre-L-NAME conductance - post-L-NAME conductance)/ pre-L-NAME conductance × 100), then the reduction in vascular conductance was similar in all muscles examined (average %Δ = -23 ± 2 %). These results suggest that NO contributes substantially to the regulation of vascular conductance within and among muscles of the rat hindquarter during high intensity exercise. When expressed in absolute terms, the results suggest that the contribution of NO to the regulation of vascular conductance during high intensity exercise is greater in muscles that possess a high oxidative capacity. In contrast, if results are expressed in relative terms, then the contribution of NO to the regulation of vascular conductance during high intensity exercise is similar across the different locomotor muscles located in the rat hindlimb and independent of the fibre type composition. Experimental Physiology (2001) 86.6, 749-757.

Very brief and intense exercise triggers a biphasic metabolic and respiratory response with a second phase that occurs after the cessation of the muscular activity. The effects on minute ventilation (V˙E) produced by manipulation of the peripheral circulation in metabolically active muscles could thus be studied without the confounding effects of painful contractions. The second phase of breath-by-breath V˙E and pulmonary gas exchange responses to a brief change in work rate (400 W for 12 s) were studied in six healthy male subjects on four occasions (24 tests). An upper thigh cuff inflation was randomly applied either above or below the systolic blood pressure (200 or 90 Torr, respectively) for 90 s just after the cessation of the contractions prior to the delayed rise in pulmonary gas exchange (eight tests in each subject). Total occlusion produced a significant reduction in the delayed rise in V˙E (-29 ± 3 %) which normally occurred 20-25 s after the cessation of the contractions. In contrast, cuff inflation at a level predominantly impeding venous return while partially maintaining the arterial supply reduced the rise in pulmonary gas exchange in similar proportion to that during total obstruction but with a slight but not significant reduction in ventilation (-9 ± 5 %). V˙E during partial occlusion was if anything higher than in control tests with similar oxygen uptake (280 W), despite a higher blood pressure (BP) during occlusion (+7 Torr). It is concluded that the factors resulting from a reduction in venous return or from the involvement of the arterial baroreflex are not responsible for the changes in V˙E produced by the obstruction of the circulation to and from metabolically active muscles. It is proposed that factors related to the level of the perfusion pressure in hyperaemic muscles, possibly located at the venular end of the microcirculation, could account for the changes in V˙E observed. Experimental Physiology (2001) 86.6, 759-768.

The Physiological Society hosts a number of Special Interest Groups whose task it is to foster lively debate among members within a specific area of topical research. Inevitably there are many topics that straddle these sometimes arbitrary divides where fresh understanding may be derived from two or more groups focussing attention on a specific topic. Vagal control of the cardiovascular system is a good example where an exchange of ideas may be of mutual benefit among the Comparative section (utilising the diversity of form resulting from natural selection) and the Cardiovascular/Respiratory Control section (concentrating on the intricacies of the mammalian system). The title of the joint symposium held at the Oxford meeting recognised the evolutionary theme that was used to highlight the various patterns of sympathetic control that are found among the vertebrates. The first contribution highlights the presumptive ancestral nature of vagal control, where it predated sympathetic innervation of the heart (Taylor et al., pp. 771-776). The role of the vagus in unusual situations is examined by use of examples from periodic feeding in reptiles (Wang et al., pp. 777-784), cardiac shunting in crocodiles (Axelsson, pp. 785-789), and diving bradycardia in birds (P. J. Butler, delivered at the meeting but not included here). The central role of the vagus in mammalian homeostasis is emphasised with respect to hibernation (Milsom et al., pp. 791-796), while complexities of the mammalian system are illustrated by the rat heart (Jones, pp. 797-801) and perinatal development in lambs (Lalani et al., pp. 803-810). We conclude with overviews of the controversies in human studies regarding vagal control during exercise (Coote & Bothams, pp. 811-815) and its utility in the clinical setting (Casadei, pp. 817-823). We hope this collection of short articles will help stimulate further debate and interaction. Experimental Physiology (2001) 86.6, 769-769.

STUART EGGINTON Convenor, Comparative Special Interest Group

TERESA THOMAS Convenor, Cardiovascular/Respiratory Control Special Interest Group

It seems that a dual location for vagal preganglionic neurones (VPNs) has important functional correlates in all vertebrates. This may be particularly the case with the central control exerted over the heart by cardiac VPNs (CVPNs). About 30 % of VPNs but up to 70 % of CVPNs are in the nucleus ambiguus (NA) of mammals. There is a similar proportional representation of VPNs between the major vagal nuclei in amphibians and turtles but in fish and crocodilians the proportion of VPNs in the NA is closer to 10 % and in some lizards and birds it is about 5 %. However, the CVPNs are distributed unequally between these nuclei so that 45 % of the CVPNs are located in the NA of the dogfish, and about 30 % in the NA of Xenopus and the duck. This topographical separation of CVPNs seems to be of importance in the central control of the heart. Cells in one location may show respiration-related activity (e.g those in the dorsal vagal nucleus (DVN) of dogfish and in the NA of mammals) while cells in the other locations do not. Their different activities and separate functions will be determined by their different afferent inputs from the periphery or from elsewhere in the CNS, which in turn will relate to their central topography. Thus, CVPNs in the NA of mammals receive inhibitory inputs from neighbouring inspiratory neurones, causing respiratory sinus arrythmia (RSA), and the CVPNs in the DVN of the dogfish may generate cardiorespiratory synchrony (CRS).

Experimental Physiology (2001) 86.6, 771-776.

The vagus is clearly of primary importance in the regulation of reptilian cardiorespiratory systems. Vagal control of pulmonary blood flow and cardiac shunts provides reptiles with an additional means of regulating arterial oxygen levels that is not present in endothermic vertebrates (birds and mammals). Within a given species, there exists a clear correlation between withdrawal of vagal tone on the cardiovascular system and elevated metabolic rate. Undisturbed and resting reptiles are normally characterised by high vagal tone, low pulmonary blood flow and large right-left (R-L) cardiac shunts. The low oxygen levels that result from the large R-L shunt may serve to regulate metabolism. However, when metabolism is increased by temperature, exercise or digestion, the R-L cardiac shunt is reduced, which serves to increase oxygen delivery. This response is partially elicit ed by reduction of vagal tone. Interspecies comparisons reveal a similar pattern. Thus, species that are able to sustain the highest metabolic rates possess the highest degree of anatomical ventricular separation and, therefore, less cardiac shunting. It is interesting to note that when cardiac shunts occur in mammals, due for example to developmental defects, they are associated with reduced maximal metabolic rates and impaired exercise tolerance. It appears, therefore, that full separation of ventricular blood flows was a prerequisite for the evolution of high aerobic metabolic rates and exercise stamina in mammals and birds.

Experimental Physiology (2001) 86.6, 777-784.

There are large differences in the morphology of the vertebrate heart; from the fish heart with its single atrium and single ventricle to the crocodilian, bird and mammalian hearts with two fully separated atria and ventricles (Van Mierop & Kutsche, 1985). It is only in crocodilians, birds and mammals where the heart has a complete interventricular septum, that a full intracardiac separation of blood pressure and flow in the systemic and pulmonary circulations can occur. In birds and mammals the left ventricle gives rise to the aorta supplying the body with oxygenated blood and the pulmonary arteries arise from the right ventricle carrying deoxygenated blood to the lungs. In these two animal groups no intra- or extra-cardiac mixing of blood (shunting) is possible, and no shunting of blood between the pulmonary and systemic circulations occurs in healthy adults. The crocodilians are unique in comparison to other reptiles and also to birds and mammals. In comparison to other reptiles (snakes, lizards and turtles) the crocodilian heart is unique in that the ventricle is fully divided into a left and a right ventricle whereas the non-crocodilian reptile heart is subdivided into three intraventricular compartments that are interconnected (no morphological subdivision of the ventricle) allowing intracardiac mixing of oxygenated and deoxygenated blood. The crocodilian heart is also unique compared to the bird and mammalian heart in that shunting of blood away from the pulmonary circulation is still possible as the right ventricle gives rise not only to the pulmonary arteries but also to the left aorta (Fig. 1). This allows deoxygenated blood from the right ventricle to bypass the lungs and to be recirculated into the systemic circulation (pulmonary-to-systemic shunt). Apart from the 'extra' left aorta from the right ventricle, three other morphological features of the crocodilian cardiovascular system have been the focus of discussion over recent years. (1) The foramen of Panizza; an opening between the right and left aorta situated in the common wall of the left and right aorta (Fig. 1A). (2) The subpulmonary conus situated in the pulmonary outflow tract of the right ventricle (Fig. 1B). (3) The aortic anastomosis that connects the two aortic arches just posterior to the heart (Fig. 1C; van Mierop & Kutsche, 1985). The subpulmonary conus contains connective tissue nodules protruding into the outflow tract that acts as an 'extra' and unique valve mechanism (Fig. 1B). These three areas have received much attention from comparative physiologists interested in the function of the crocodilian/reptile circulation, and the evolution of the cardiovascular system and its regulation. There is increasing evidence for a close regulation of the three unique structures in the crocodilian cardiovascular system and their importance for the normal function of the crocodilian circulation. This short overview will give a summary of the latest findings.

Experimental Physiology (2001) 86.6, 785-789.

Autonomic events associated with entrance into hibernation are primarily mediated by the parasympathetic nervous system while those associated with arousal from hibernation are primarily sympathetically mediated. During deep hibernation, both sympathetic and parasympathetic tone are greatly reduced or absent. Within this context, the pattern of autonomic influence on cardiorespiratory control is intriguing. The dramatic fall in heart rate exhibited by mammals entering hibernation begins before there is any noticeable fall in body temperature and is due to a cyclic vagal activation that induces skipped beats and regular asystoles, and also slows the even heart beat. As body temperature subsequently falls, the vagal influence is progressively withdrawn and periods of vagal and sympathetic dominance alternate and give rise to regular periods of arrhythmia (tachycardia followed by bradycardia). Superimposed on this is a vagally mediated, respiratory sinus arrhythmia. As metabolic rate and body temperature fall, breathing frequency slows, depending on species, either by a prolongation of the pause between breaths, or by a waxing and waning of breathing frequency giving rise to discrete periods of apnoea interspersed by periods of continuous breathing. In such cases, the waxing and waning, and resulting episodic pattern, appear to be due to alternating positive and negative influences, possibly arising from vagal-pontine interactions acting on the medullary respiratory centres. The respiratory sinus arrhythmia is accentuated in these species. While advances have been made in describing the overt changes in autonomic control of cardiorespiratory processes during hibernation, the mechanisms behind, and the biological significance of cardiac and respiratory changes mediated through orchestrated arrhythmias and apnoeas, remain enigmatic.

Experimental Physiology (2001) 86.6, 791-796.

Cardiac vagal preganglionic neurones are found in two locations in the medulla oblongata. The neurones of the ventral group near the nucleus ambiguus (NA) have a discharge pattern which reflects strong respiratory and baroreceptor inputs. In contrast the neuronal discharge of the dorsal group near the dorsal vagal motor nucleus (DVMN) is not modulated by either of these inputs. Electrophysiological analysis of these groups has shown that the DVMN group possesses C-fibre axons (conduction velocity, < 2 m s-1) whilst the NA group has B-fibre axons (conduction velocity, 10-30 m s-1). Therefore, selective stimulation of preganglionic inputs to cardiac ganglia is possible using anodal block techniques. The results of these kinds of experiments have shown that both populations have similar functions (related to cardiac chronotropy, dromotropy and inotropy), although the magnitude and time course of the effects differ substantially. Anterograde tracing experiments from brainstem to heart have confirmed these striking differences, as the potent NA group of neurones has a greater divergence of innervation. Both populations project to similar clusters of ganglion cells on the atrial epicardium suggesting functional overlap. On-going levels of sinus arrhythmia produced by vagus activity may depend on the recruitment and relative combination of these two populations of neurones. Much work remains to be done to investigate preganglionic convergence from neurones of the NA and the DVMN to their postganglionic targets on the heart.

Experimental Physiology (2001) 86.6, 797-801.

Although vagal afferents are active during intrauterine life, vagal denervation does not lead to gross changes in the occurrence of fetal breathing movements. However, lack of volume feedback has profound and deleterious effects on pulmonary compliance and impairs gas exchange during the early neonatal period. Intrathoracic vagal denervation, which spares vocal cord paralysis and upper airway obstruction in spontaneously breathing unanaesthetised neonatal lambs, leads to reduced pulmonary compliance and persistent hypoxaemia. The underlying mechanisms of these adverse effects include slow breathing frequency, prolonged expiratory times, decreased augmented breaths and the inability to maintain dynamic functional residual capacity leading to progressive atelectasis and regional ventilation- perfusion mismatch. Our data suggest that intrathoracic vagal denervation does not lead to pulmonary oedema in newborn lambs which most probably occurred because of upper airway obstruction in previous studies. We provide evidence that the vagal afferent fibre-receptor system provides critical feedback for the maintenance of normal breathing patterns and pulmonary gas exchange during the early neonatal period.

Experimental Physiology (2001) 86.6, 803-810.

There is much evidence showing that the rapid rise in heart rate at the onset of exercise is due to a withdrawal of cardiac vagal tone. This short review discusses the main afferent mechanisms involved in this effect. In addition to signals from central motor command it is shown that muscle mechanoreceptors of group III afferent fibres also play a significant role. Recent studies in man demonstrating this by stretching muscles such as the triceps surae or by direct compression of muscles are briefly reviewed. The evidence also supports the idea that these small fibre mechanoreceptors inhibit the baroreceptor-heart rate reflex. Several studies suggest that skeletal muscle metaboreceptors (mainly group IV) are more important for increasing cardiac sympathetic and vasoconstrictor nerve activity. At the conclusion of exercise the cessation of mechanoreceptor stimulation is an important factor in determining the rapid return of heart rate to resting level.

Experimental Physiology (2001) 86.6, 811-815.

Until about 40 years ago, it was thought that the parasympathetic innervation of the mammalian heart was confined to supraventricular structures. Hence, neither the vagus nor its primary neurotransmitter, acetylcholine (ACh), were believed to have significant effects on the inotropic state of the ventricles or on their excitability. However, it is now well-established that vagal/muscarinic stimulation prolongs ventricular refractoriness in humans and has a small but distinct negative inotropic effect on the left ventricle, which is accentuated in the presence of elevated sympathetic activity (Löffelholz & Pappano, 1985).

Experimental Physiology (2001) 86.6, 817-823.

Experimental Physiology (2001) 86.6, 825-825.

Experimental Physiology (2001) 86.6, 826-826.