5-6 May 1978 DEMONSTRATIONS Bladder electromyograms in the conscious cat BY M. D. CRAGGS and J. D. STEPHENSON. M.R.C. Neurological Prostheses Unit, Departments of Physiology and Pharmacology, Institute of Psychiatry, De Crespigny Park, London, S.E. 5 Electromyograms from the smooth muscle of the detrusor during reflex voiding in anaesthetized cats are best recorded in the 10-40 Hz frequency band (Craggs & Stephenson, 1976). In this band, spiky signals occur just prior to, and during, the intravesical pressure rise. At lower (< 1 Hz) and higher (> 70 Hz) frequencies the electrical activity is poorly correlated with voiding. We demonstrated that the same is true for electromyograms recorded during voiding in a conscious cat. Electromyographic activity was recorded from a pair of platinum electrodes (0-076 mm diameter) chronically implanted in the bladder wall. Recording electrodes were also implanted into other sites including the musculature of the pelvic floor, abdominal wall and urethral sphincters. Intravesical pressure was recorded from an indwelling catheter sewn into the bladder wall and terminating in a Luer connector cemented to the skull. Abdominal pressure was recorded in a similar manner from a silicone rubber balloon placed in the abdomen. The recording electrodes were connected to a socket also on the skull, and led to the main amplifier via a simple field effect transistor differential stage mounted on a plug. A swivel arrangement minimized cable and tube twisting. Voiding, promoted by infusing sterile saline into the bladder via the catheter, was always preceded by characteristic behavioural sniffing and floor scratching. A train of spikes (25-100lYuV) in the 10-40 Hz band always anticipated voiding, and the associated rise in intravesical pressure, by up to 500 msec. The repetition rate of these spikes was maximal (about 5/sec) at the onset of the pressure rise, thereafter declining. Groups of spikes in this frequency band were not seen at other times. The low-frequency band (less than 1 Hz) usually showed large amplitude random fluctuations (500-1 000 uV) but occasionally a 'slow bladder wave' was seen to accompany voiding. In the high-frequency band (> 70 Hz) bursts of activity were often seen, and sometimes these could be related to pelvic floor and abdominal wall contractions. We are currently using these techniques to examine various physiological and pharmacological properties of the bladder, some of which have been previously investigated under general anaesthesia. A selection of results was presented.



The authors thank the Bethlem Royal and Maudsley Hospitals Research Fund for financial assistance.

REFERENCE CRAGGS, M. D. & STEPHENSON, J. D. (1976). Br. J. Urol. 48, 443.

Fate of arachidonic acid in guinea-pig isolated lungs BY FIRIAL AL-UBAIDI, Y. S. BAKHLE, P. J. JOSE and J. P. SEALE. Department of Pharmacology, Royal College of Surgeons, London WC2A 3PN On passage through the pulmonary circulation of guinea-pig isolated lungs, arachidonic acid (AA) is converted, chiefly via the cyclo-oxygenase pathway, to several biologically active metabolites appearing in the effluent (Hamberg, Svensson & Samuelsson, 1976). We have followed the distribution of radioactivity in lung tissue and in effluent following infusion of [14C]AA through the pulmonary circulation of guinea-pig isolated lungs and the effect of stimulating and inhibiting cyclo-oxygenase activity on this distribution. Guinea-pig isolated lungs were perfused via the pulmonary artery with oxygenated Krebs solution (8 ml. min-1; 37 'C) and [14C]AA infused for 3 min (0.1 ml. min-'; final concentration 4/M). Lung effluent was collected during, and after, the infusion in 1 min fractions. Within ten fractions 18-5 % of the infused radioactivity was in the effluent, and in thirty fractions this only increased to 20 %. Only about 9 % of radioactivity in the effluent was unchanged AA and 70 % was associated with cyclooxygenase products and their metabolites. Radioactivity retained in lung was extracted by homogenizing the tissue in cold chloroform: methanol and subsequently analysed by chromatography on columns of silicic acid and Florisil. This analysis showed three major components of the retained radioactivity; phospholipid (72 %), neutral lipid (17 %) and free fatty acid (7 %). Insignificant amounts of radioactivity were associated with cholesterol esters or prostaglandins. This pattern of labelling was observed as soon as 10 min, i.e. 7 min after the end of the infusion. Slow reacting substance of anaphylaxis (SRS-A) stimulates cyclo-oxygenase activity in lungs (Engineer, Piper & Sirois, 1977), utilizing AA from phospholipid. Thirty minutes after an infusion of [14C]AA, partially purified SRS-A was injected into the pulmonary circulation, causing release of active substances (equivalent to 5-10 ng ml.-' PGE2) and a small release of radioactivity (33 x 103 d.p.m.) into the effluent. The SRS-A injections were repeated twice at 30 min intervals; each time, the biological activity released was not diminished but the radioactivity released was decreased (8 x 103 d.p.m. at the third injection). At the end of this procedure the lungs were homogenized and contained about 106 d.p.m. These results suggest that most of the retained radioactivity is not readily available for turnover into cyclooxygenase metabolites. Indomethacin inhibits cyclo-oxygenase action and prevents the activation of AA infused through the pulmonary circulation (Palmer, Piper & Vane, 1973). In our experiments, indomethacin (5 jug ml.-') infused before, during and after infusions of [14C]AA, decreased radioactivity appearing in the effluent (only 7 % in 10 min) and

3P PHYSIOLOGICAL SOCIETY, MAY 1978 increased the proportion of unchanged AA. Radioactivity retained in lung increased slightly but the distribution between phospholipid, neutral lipid and free fatty acid pools was not changed compared with untreated lungs. From all these results we conclude that infused AA is either incorporated into lipids and retained in the tissue or metabolized by cyclo-oxygenase and lost in the effluent. Further, the newly incorporated AA does not equilibrate with the pool utilized for subsequent synthesis of cyclo-oxygenase metabolites. We thank the government of Iraq, the M.R.C. and the Asthma Foundation of N.S.W. for grants. REFERENCES

ENGINEER, D. M., PIPER, P. J. & SIROIS, P. (1977). Br. J. Pharmac. 59, 449P. HAMBERG, M., SVENSSON, J. & SAMUELSSON, B. (1976). In Advances in Prostaglandin and Thromboxane Research, vol. 1, ed. SAMUELSSON, B. & PAOLETTI, R., pp. 19-27. New York: Raven Press. PALMER, M. A., PIPER, P. J. & VANE, J. R. (1973). Br. J. Pharmac. 49, 226-242.

A demonstration of the use of autoradiography as a method of exploring afferent autonomic innervation of the respiratory tract BY A. J. BOWER* and KATHERINE PARRYt. *Department of Human Biology and Anatomy, The University of Sheffield, Sheffield 510 2TN, and tDepartment of Veterinary Anatomy, The University of Liverpool, Liverpool L69 3BX One of the problems of exploring the afferent innervation of a system like the respiratory tract is the difficulty of distinguishing between afferent and efferent nerves with the light or electron microscope. The use of autoradiography for the anatomical exploration of the central nervous system is well established (see Cowan & Cuneod, 1977, for review). In 1975 Bower, Molony & Brown published details of a method which showed that autoradiography could be used to explore the peripheral distribution of the autonomic nervous system. The nerve used is the vagus nerve of the domestic hen and the system explored is the respiratory tract. The method relies on the fact that the inferior (nodose) ganglion of the vagus nerve contains only afferent cell bodies. A small amount of tritiated leucine is injected into the inferior ganglion on one side. The neurones incorporate the [3H]leucine into neuronal protein and transport the now radioactive protein to the periphery. Standard autoradiographic techniques are used to display the labelled nerves. Because the efferent nerves passing through the nodose ganglion do not transport the [3H]leucine the only labelled nerves will be afferent ones. Thus this method can differentiate between afferent and efferent nerves. The early technique was to approach the inferior ganglion via the thoracic inlet and then place a wax bath around the nodose ganglion and allow the [3H]leucine to diffuse into the neurones. This method meant that the birds had to be artificially ventilated and there was also the possibility of leakage of [3H]leucine from the bath. A new surgical approach to the nodose ganglion has been developed which allows the birds to survive without the need for artificial respiration. This approach, together with a more refined method of delivering the [3H]leucine, means that the technique is much easier to use and the results are more consistent. The advantage of using autoradiography over other axon transport methods as a

PROCEEDINGS OF THE 4P technique for exploring the afferent innervation is that no prior vagotomy is needed to allow the efferent fibres to degenerate. There is no surgical intervention apart from the delivery of the leucine. Also light microscope autoradiography is easy to do and the results easy to see once the basic techniques have been mastered. The disadvantage is that electron microscopic autoradiography is difficult and time consuming. The method of delivering the leucine and the surgical approach was shown, together with photographs of some results. REFERENCES BOWER, A. J., MOLONY, V. & BROWN, C. M. (1975). Experientia 31, 620-622. CowAN, W. M. & CUNEOD, M. (eds.) (1977). The Use of Axonal Traneportforthe Study of Neuronal Connectivity. Amsterdam: Elsevier.

Transcutaneous aortovelography as a measure of central blood flow BY A. H. BRTON*, J. BROTHERHOODt, G. C`Ross:, G. C. HANSON*, L. H. LIGHTt and R. F. SEQUEIRA§. *Intensive Therapy Unit, Whipps Cross Hospital, London, tBioengineering Division, Clinical Research Centre, Harrow, and §Cardiology Department, Royal Infirmary, Bristol Transcutaneous aortovelography (TAV), a non-invasive ultrasound Doppler technique specifically designed to give reproducible quantitative measurements of mainstream blood velocity in the human aortic arch, has been demonstrated previously (Cross & Light, 1971, 1974; Light, 1977). TABLE 1

Deviation from exact agreement or

Trial Reproducibility (4 trials): Within-observer agreement Between-observer agreement

Proportionality against

proportionality (S.D. as % of mean) 6-6

7.3 11

acetylene cardiac output (c.o.) during exercise

Proportionality against intravascular blood velocity (VPk) measurement Proportionality against green dye measurement of stroke volume (s.v.) Proportionality against thermodilution cardiac output in the critically ill




Total range covered Forty-two subjects (3-67 years) Fourteen observers (eight inexperienced) c.o. = 7-22 1./min (13 subjects)


= 48-120 cm/sec (8 patients)

s.v. = 20-160 ml. (20 patients) c.o. = 2 5-10 I./min

(10 patients) W

peak intravascular velocity.

t Present address: N.I.M.D. Hampstead.

PH YSIOLOGICAL SOCIETY, MAY 1978 5P Reproducibility trials on resting normal subjects in several centres (e.g. Frazer et al. 1976) have given virtually identical results, within-observer and betweenobserver variability both being 7 % (S.D.). What is known about the stability of the transverse flow profile and the limited compliance of the aorta (which is probably relatively tightly constrained at the site of TAV observations) suggests that serial TAV measurements should show close proportionality to volumetric blood flow in any one subject (Sequeira et al. 1976). Comparisons against acetylene uptake during exercise (Light, 1974) and against dye dilution measurements of stroke volume during diagnostic catheterization (Sequeira et al. 1976) bear out this expectation. They have now been supplemented by studies of the changes resulting from disease progression and therapeutic interventions affecting blood volume, respirator pressures and inotropic state in ten critically ill patients who required haemodynamic monitoring. The standard deviation from exact proportionality between serial thermodilution, cardiac output and the recorded blood velocity (averaged over 6 beats) in a total of 62 observations was 8-5 %. These results, summarized in Table 1, suggest that changes in cardiac output can be non-invasively followed by TAV with useful accuracy over a considerable range of conditions. Inadequate recordings, as were given by 2 out of 12 patients in the intensive care study, are clearly distinguishable on the on-line spectral display which is provided on the instrument. In addition to giving a measure of relative flow in any one patient, mean blood velocity also gave at least a rough indication of the absolute level of central blood flow in the various patients. The instantaneous velocity wave form often gives further information on left ventricular function and sympathetic drive. REFERENCES

CROSS, G. & LIGHT, L. H. (1971). J. Physiol. 217, 5-7P. CROSS, G. & LIGHT, L. H. (1974). Bioengineering 9, 464-471. FRAZER, C. B., LIGHT, L. H., SHINEBOURNE, E. A., BUCHTAL, A., HEALY, M. J. R. & BEARDSHAW, J. A. (1976). Eur. J. Cardiol. 4, 181-189. LIGHT, L. H. (1974). In Cardiovascular Applications of Ultrasound, ed. RENEMAN, R. S., p. 340. Amsterdam: North Holland. LIGHT, L. H. (1977). In Echocardiology, ed. BoM, N., pp. 233-243. The Hague: Martinus Nyhoff. SEQUEEIRA, R. F., LIGHT, L. H., CROSS, G. & RAFTERY, E. B. (1976). Br. HeartJ. 38, 442-451.

A new method for running computer models of physiological systems BY F. L. PRATT and 0. E. PRATT. Department of Neuropathology, Institute of Psychiatry, De Crespigny Park, London SES 8AF The programmes for most models of physiological or metabolic processes are written for use on large computers, on which many users share available time. This sharing of time has inherent disadvantages for research and for teaching because there is, inevitably, congestion leading to delays which are unpredictable. If the best use is to be made of such models by research workers and keen students it is essential that there be access without arbitrary limits. Virtually unlimited access is available if programmes can be modified to run on a local computer.

PROCEEDINGS OF THE We have devised such a system for a local computer (System 80, Trivector Systems Ltd., Hitchin, Herts) which is based on an 8-bit microprocessor (Intel 8080) a 'floppy' (flexible magnetic) disk unit, a Basic interpreter and a programme assembler. Both the latter make full use of the disk storage. The major part of most programmes for running computer models is concerned, of necessity, with interaction between the operator and the programme, e.g. commands to initiate a run, or reporting to the operator changes in the state of the patient and seeking, in consequence, further instructions. We have used the Basic language for these interactive parts of programmes, because this language is better suited than Fortran for interactive work. It also provides greater flexibility for adaptation and modification of the model, or for its extension into related fields. However, if Basic is used throughout, a large program will run rather slowly. This difficulty has been overcome by compiling into microprocessor code those sections of the programme which are used most frequently and which do not involve communication with the operator. We have made minor modifications to the Basic interpreter (which allows the user to add to it special instructions to perform nonstandard operations). Our modifications enable the interpreter to accept commands (which are stored in the Basic programme) to load machine language routines into the computer store from the disk at the same time as it loads a Basic programme which uses these routines. One of the models for which this system has been used is that of human respiration, gas transport and hydrogen ion regulation which, in its published form, comprises some 1600 executable Fortran statements (Dickinson, 1977). Another, which we have devised, is concerned with the regulation of glucose and of amino acid metabolism, and shows responses to fasting or to glucose loads, as well as to the effects of endocrine disorders or inborn errors of metabolism. Once the equipment for our system has been bought (major items: working store, floppy disk unit), running costs are minimal, much less than those of extended time for interactive work on a large computer, which can be prohibitive. An individual microprocessor-based system can emulate, in principle, the large computer in everything except speed of operation. However, because time on a machine such as ours is freely available, the speed of operation is not of importance. Systems such as the one which we have used will become cheaper as microprocessor-based equipment decreases in cost. They will become increasingly important both for teaching and research. 6P

Supported by the Kennedy Trust. REFERENCE DIcKiNSON, C. J. (1977). A Computer Model of Human Respiration. London: MTP Press.

Removal of ATP by lead acetate BY N. AMBACHE, I. A. JOHNSON, S. W. KILLICK and J. VERNEY. M.R.C., Physiology Department, Royal College of Surgeons, London WC2A 3PN The method is based upon the extremely low water-solubility of lead phosphates, e.g. Pb3(PO4)2:0-14 jug/ml. (0 'C), which is considerably lower than that of other metal phosphates.

PH YSIOLOGICAL SOCIETY, MAY 1978 7P The following bland procedure removes ATP (and ADP) almost completely from solutions by precipitation, and is suitable for samples intended for biological assays. Assays depend on the sharp transient ATP relaxation of carbachol-contracted guinea-pig caecal taeniae, selected for non-tachyphylaxis and rendered catecholamine-insensitive with phenoxybenzamine + pindolol; 2 ml. baths, Krebs-Henseleit, 30 'C; for fuller details see Ambache, Daly, Killick & Woodley (1977). Sensitivity: 10-100 ng ATP/ml. ATP removal has been confirmed in some assays on guinea-pig vas deferens and by UNh. spectrophotometry (259 nm). Lead acetate treatment (0 'C). ATP disodium salt (adenosine 5'-triphosphate, Sigma; mol. wt. 551'2), freshly dissolved and diluted in distilled water to between 0 01 mM (pH 6-1) and 4 mM (pH 3.4), is subdivided into 1 ml. aliquots. Assuming 1 mole ATP (tetrabasic) to require 2 moles lead, add excess Pb(CH3COO)2.3H20 (A.R.) in 5-50 ,A. to give molar ratios Pb: ATP = 8 to 200. Precipitation may produce only haziness, but is confirmed by a greyish sediment after centrifugation. The unreacted excess lead in the decanted supernatant is precipitated as PbCO3 by adding 10-50 % excess NaHCO3 (A.R.) in 5-50 ,tu., to give molar ratios NaHCO3: initial Pb = 2-2 to 3. This procedure results in 1-10 % dilution and leaves in solution, after recentrifugation, calculable amounts of sodium acetate and bicarbonate, with traces of PbCO3 (solubility, 1t1 ,ug/ml. (20 °C), i.e. 1/18 that of BaCO3). Such traces prove harmless to the taenia muscle, probably because they are mopped up by the 1 18 mM inorganic phosphate in the bath fluid. Indeed, parallel reagent blanks have been inactive and non-toxic to taeniae when administered, either neat in 01 ml. volume (containing < 0 1 ,ug = 0-4 n-mole PbCO3) or, when necessary, suitably diluted so as not to exceed the bicarbonate and acetate tolerances of the taeniae. Care is taken to ascertain the maximum volume of blank (neat or diluted) tolerated; and not to exceed this volume when the treated samples are assayed. Effectiveness. Provided sufficient excess lead is used, ATP removal is very thorough. With a molar ratio Pb: ATP = 4 (i.e. 100 % excess lead), inactivation can be incomplete, e.g. 65 and 90 % in two experiments; but with a ratio of 8 it exceeds 99 % (two experiments). With Pb: ATP ratios of 20-200 the reduction in biological activity is consistently 99-99 9 % (11 experiments). It is particularly necessary to use the larger excesses of lead when chloride, sulphate and inorganic phosphate are present (e.g. in tissue extracts), because these anions compete with ATP for Pb2+. An almost total removal of ATP can also be achieved, without dilution, by adding the lead acetate and NaHCO3 in solid form. Thus, for 1 ml. ATP, 0 2 mm (110 ,tg/ml.), add 3 mg lead acetate (molar ratio Pb/ATP = 40); centrifuge; to decanted supernatant (pH 5.8) add 146 mg NaHCO3 (10% excess); recentrifuge. Final pH, 6-0; ATP removal, 99 5 % or more (3 experiments). These procedures do not remove PGE2 (a carboxylic acid) at all. The possibility arises that the immobilization of ATP demonstrated above contributes to the toxicity of lead. REFERENCE

AMBACHE, N., DALY, S., KILLICK, S. W. & WOODLEY, J. P. (1977). Br. J. Pharmac. 61, 113-114P.



Lymphatic drainage from the liver in biliary obstruction BY D. BLOOM, P. M. DANIEL, 0. E. PRATT* and E. SPARGO.* Department of Applied Physiology and Surgical Science, Royal College of Surgeons of England, Lincoln's Inn Fields, London WC2A 3PN, and * Department of Neuropathology, Institute of Psychiatry, De Crespigny Park, London SE5 8AF During the course of work on the effect of bile on the blood-brain barrier, in which the bile duct of the rat was ligated, we observed that the lymphatics draining the liver became distended and yellow in colour. In order to get some idea of what proportion of the bile leaving the liver, after the bile duct is tied, is carried in the lymphatics, we cannulated the cisterna chyli and collected lymph before and after the operation. Under ether anaesthesia the cisterna chyli was cannulated with polythene tubing (Portex) about 150 mm long and led out through a stab incision in the flank. A fine needle, no. 36 gauge, 200 mm long (Cooper's Needle Works, Aston Lane, Birmingham), was threaded through the catheter so that a steady flow of heparin could be infused in order to prevent clotting in the tube. In some animals the bile duct was tied after the insertion of the catheter. An arterial catheter was also inserted. The animal was now placed in a Bollman (1948) restraining cage and allowed to recover. Collection of lymph was continued from 2 to 12 hr. Within a few minutes of tying the bile duct the lymph flow increased several-fold, approaching the normal flow of bile of 0.5-1.0 ml. hr-'. The alkaline phosphatase concentration in the lymph rose by about 25 % within 2-3 hr of tying the bile duct. This rise occurred before there was any appreciable increase in the level of the enzyme in the blood. Within 1 hr of tying the bile duct the lymph became visibly yellow and gave a strongly positive test for bilirubin. In another series of animals (eight rats and two rabbits), under sodium pentobarbitone anaesthesia, the proximal end of the bile duct was cannulated with polythene tubing connected to a pressure transducer (Elcomatic EM 750). During the operation a side tap was left open so that there was no obstruction to the bile flow. When this tap was closed the pressure in the bile duct rose in about 10 see to between 1-5 and 2 kPa and remained steady at this level for at least 2 hr. It is clear that in biliary obstruction the lymphatics provide a pathway through which protein, bilirubin and other protein-bound substances can reach the bloodstream. This observation may be of value in the treatment of patients with biliary obstruction. REFERENCE

BOLLMAN, J. L. (1948). J. Lab. din. Med. 33, 1348.

Partial and total circulatory support in the rat using a small heart-lung machine BY E. PROCTOR. Department ofApplied Physiology and Surgical Sciences, Royal College of Surgeons, Lincoln's Inn Fields, London WC2A 3PN For some years we have used the dog as our main model for cardio-pulmonary bypass and circulatory studies, but recently, for a variety of surgical, physiological,

PHYSIOLOGICAL SOCIETY, MAY 1978 9P and financial reasons, we came to examine the possibility of using the rat for some of this work where size was not a prerequisite. For our purpose we required a heart-lung machine (or pump-oxygenator) which was (a) capable of total circulatory support (200-280 ml./kg. min) at normal temperature, (b) had a haemolytic index sufficiently low to permit of 1-2 hr cardiopulmonary bypass with consistent survival, (c) had a priming volume no greater than the blood volume (20-30 ml.) of the young adult Wistar rat, in order to reduce the need for homologous blood, and (d) had heat-exchange capacity sufficient for rapid core cooling and rewarming of the rat. Heat

exchanger Anti-foam wool


Arterial T line

_Venous return

Diffuser Gas

Fig. 1. Diagram of oxygenator showing annular foam column for gas exchange and heat exchange, with defoaming cylinder.

The subsequent design (Fig. 1) was based on an experimental range of dog pumpoxygenators described several years ago (Proctor & Fernando, 1973), in which an annular column is used to combine gas and heat exchange in order to reduce priming volume. Relevant dimensions and values: height and width of foam column (18 x 1-2 cm); width of foam annulus (0 35 cm); gas diffuser (50 x 0415 mm holes); length and width of defoaming cylinder (75 x 2-0 cm); defoaming agent (Antifoam A, Dow Corning Ltd); volume of reservoir (15-20 ml.); priming volume, including lines and 1O ml. in reservoir (25-30 ml.); blood flow (260 ml./kg. min); gas flow (100 ml./min 95:5 oxygen/carbon dioxide); filter (stainless steel 150 ,um pore size). The apparatus is made in acrylic plastic (Perspex), and the heat exchanger in stainless steel.


PROCEEDINGS OF THE The perfusate can be blood, a colloid/electrolyte solution, or most commonly a mixture of both. Circulatory support can be carried out either by the closed-chest method (venous return via the jugular vein), or the open-chest method (venous return via right atrial basket). Arterialized blood is returned via one femoral artery. This heart-lung machine has been used successfully for over 50 survival experiments, including both partial and total circulatory support in the closed-chest model and hypothermia at 10 'C with total blood replacement (haematocrit < 1) by asanguineous solutions. REFERENCE

PROCTOR, E. & FERNANDO, A. R. (1973). J. Surg. Res. 15, 112-116.

The use of electrical impedance to monitor changes in blood flow in man BY H. M. ARENSON, J. P. PAYNE and T. G. C. SMITH. Research Department of Anaesthetics, Royal College of Surgeons of England, London, W.C.2 In patient monitoring there is a need for a sensitive non-invasive technique to assess circulatory function, and electrical impedance cardiography has evolved as a promising clinical tool to supply this need. In this investigation, measurements of changes in central and peripheral blood flow were carried out in twenty-six healthy conscious subjects and in twelve anaesthetized patients subjected to a Valsalva manoeuvre so that well-Inown trends of changes in blood flow could be ascertained by the impedance technique. As arterial blood pressure decreased and heart rate increased during the 'active forceful expiration', there was a parallel and significant decrease in the peak height of the first derivative dz/dt of the impedance wave form (Fig. 1). Both stroke volume and cardiac output derived from a standard formula (Kubicek, Karmegis, Patterson, Witsoe & Mattson, 1966) fell within known ranges. Passive increase of the intra-thoracic pressure of anaesthetized subjects produced similar trends although the fall in mean arterial blood pressure was excessive whereas heart rate changes were minimal. Calf blood flow decreased significantly for all subjects during the strain phase of the Valsalva manoeuvre. After release of the airway pressure, a rebound in blood flow equivalent to the classical blood pressure 'overshoot' (Hamilton, Woodbury & Harper, 1936) was observed for conscious subjects while for anaesthetized patients responses were slow with a gradual return to control values. This different pattern is very suggestive of a 'blocked response to the Valsalva manoeuvre' reported in patients with impaired autonomic reflexes (Corbett, 1969). Hence, the peak height of the first derivative dz/dt seems to follow the circulatory changes induced by the Valsalva manoeuvre and has proved to be a sensitive index which is reproducible and easily recorded. H. M. Arenson was supported by CNP9-BRASIL.

REFERENCES CORBETT, J. L. (1969). D.Phil. Thesis, Oxford University. HAMILTON, W. F., WOODBuRY, R. A. & HARPER, H. T. (1936). J. Am. med. Ass. 107, 853-856. KulBICEK, W. G., KARMEGIS, J. N., PATTERSON, R. P., WITSOE, D. A. & MATTSON, R. H. (1966). Aerospace Med. 37, 1208-1212.











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1H I


I I I I I I I I i A A I i IIa i I 1

jjjjjjmW"'II.V -1.1. I .1

F r I ;j AAMANA" 111IM 111"1114111111 q 4 H 1111ilm-111-.111111111 i I I i 11II I I 11PI-1 H il



I I ill



I. - I dzldt





E 150-



< AA-E


-, w

75 40

I 0

Time (sec)


@ ,tI t





* 'Ia


' !

; '

'ar I

Fig. 1. Conscious subject. As the airway pressure was increased, the four classical phases of the Valsalva manoeuvre are indicated by the responses of the heart rate and arterial blood pressure. Accordingly, the peak height of the dz/dt was reduced during the 'airway strain' and increased after the release.

Computer determination of oxygen uptake in man BY M. SLAZENGER. Research Department of Anaesthetics, Royal College of Surgeons of England, London, WC 2A 3PN A computer program which determines oxygen uptake by a subject breathing normally was described. Data are acquired from a mass spectrometer and a pneumotachograph. The program examines flow data, determines the change-over point between inspiration and expiration and integrates the flow to generate tidal volumes. Concurrently the concentration of oxygen determined by the mass spectrometer is multiplied by the tidal volume to generate a value for the volume of oxygen. The expired volume of oxygen is subtracted from the inspired volume of oxygen to give the volume of oxygen taken up. This method has an advantage over the Benedict Roth method (Roth, 1922) in

PROCEEDINGS OF THE 12P that it can be used in a non-rebreathing circuit. It is also less susceptible to irregular breathing patterns and is independent of changing carbon dioxide concentrations. REFERENCE

RoTr, P. (1922). Bo8ton med. 8urg. J. 186, 457-501.

One-way stimulation of the large fibres of spinal roots through chronically implanted electrodes BY G. S. BRINDLEY and M. D. CRAGGS. M.R.C. Neurological Prostheses Unit, Institute of Psychiatry, London SES 8AF It has long been known (Katz, 1939) that, in electrical excitation of nerve, (1) impulses arising near the cathode may be blocked near the anode; (2) impulses may be generated near the anode when an electrical pulse ceases, especially if the structure stimulated has a connective tissue sheath (as peripheral nerves have but spinal roots have not); and (3) such 'anode break excitation' can be minimized by causing the pulse to cease gradually. Kuffler & Vaughan Williams (1953) used fact (1) to achieve, by rectangular pulses passed between a single pair of electrodes, stimulation of large fibres in one direction only but of small fibres in both directions. Their technique worked on freshly exposed spinal roots, but not on peripheral nerves, doubtless because of fact (2). Burke & Ginsborg (1956) and Accornero, Bini, Lenzi & Manfredi (1977) used fact (3) to achieve similar one-way stimulation of large fibres with two-way stimulation of small fibres in freshly exposed peripheral nerves. In the practical problem of artificially emptying the urinary bladder by means of electrodes implanted on sacral anterior roots (Brindley, 1977) it would be useful if impulses could be sent towards the spinal cord in all fibres of the roots (thereby blocking by collision and other means the outgoing traffic responsible for detrusorsphincter dyssynergia), but towards the periphery in the small parasympathetic fibres only. A spinal root with electrodes implanted on it becomes embedded in dense fibrous tissue, which probably behaves electrically like the sheath of peripheral nerve; certainly in our hands the technique of Kuffler & Vaughan Williams (1953) does not work on trapped mammalian roots. The demonstration showed, however, that the technique of Burke & Ginsborg (1956), with minor modifications, does work. REFERENCES L. ACCORNERO, N., Byu, G., LENZI, G. & MANFREDI, M. (1977). J. Phy8iol. 273, 539-560. BRINDLEY, G. S. (1977). J. Neurol. Neuro8urg. Psychiat. 40, 358-369. BURKE, W. & GINSBORG, B. L. (1956). J. Phy8iol. 132, 586-598. KATZ, B. (1939). Electric Excition of Nerve. Oxford University Press. KuFFLFaR, S. W. & VAuGrHAN.WnLrIAMs, E. M. (1953). J. Phy8iol. 121, 289-317.



Characterization of a histamine-independent vascular permeabilityincreasing factor generated on exposure of rabbit plasma to zymosan BY P. J. JOSE, M. J. PECK, C. ROBNSON and T. J. WILLIAMS. Department of Pharmacology, Institute of Basic Medical Sciences, Royal College of Surgeons of England, Lincoln's Inn Fields, London WC2A 3PN Incubation of rabbit plasma with zymosan resulted in the generation of a vascular permeability-increasing factor (PAPA) whose activity was only slightly reduced in the presence of the antihistamine, mepyramine (Williams, 1978). This paper describes the preliminary characterization of this factor. Zymosan (1 mg/ml.) was added to fresh rabbit plasma (containing 10 u./ml. of heparin) and incubated at 37 'C for 30 min. The zymosan was then removed by centrifugation. Plasma samples were assayed in rabbit skin; plasma exudation was measured as the accumulation of intravenously injected [131I]albumin over a 30 min period following intradermal injection (Williams, 1978). Little exudation was observed following the intradermal injection (0.1 ml. volumes) of plasma samples treated with zymosan. However, addition of prostaglandin (PGE, 100 ng/0 1 ml.) before injection resulted in large potentiated responses, by a mechanism which has been previously investigated (Williams & Peck, 1977). Therefore, to facilitate measurement, PGE1 was routinely added to all samples before injection. Plasma not exposed to zymosan, assayed in the same manner, had little activity. The inhibitors of kinin formation, soya bean trypsin inhibitor and Trasylol, had no effect on the generation of PAPA (Williams, 1978). The generation of PAPA was inhibited by heating plasma at 56 0C for 30 min before addition of zymosan. EDTA (10 mM) but not EGTA (10 mm) prevented the generation of PAPA. No loss of activity was observed if EDTA or EGTA were added after the generation of PAPA. Column chromatography of activated plasma using Sephadex G-100 gel in saline at 4 0C produced a single peak of activity which showed a similar degree of insensitivity to mepryamine as the crude samples. The peak of activity corresponded to a molecular weight of approximately 20,000. No activity was detected in fractions of plasma not exposed to zymosan. The active fractions from the Sephadex G-100 column were unstable when heated at 100 0C for 30 min, at pH 7*4, and were unstable to incubation at 37 0C for 30 min at pH 1.0. Thus, exposure of rabbit plasma to zymosan results in the generation of a factor, PAPA, which increases vascular permeability in rabbit skin. This activity is largely independent of histamine release. PAPA produces little plasma exudation in the absence of a vasodilator substance such as PGE1, as appears to be the case with permeability-increasing mediators generated in vivo (Williams & Peck, 1977). PAPA generation is dependent on a heat (56 0C) labile step, and is cation- (but not calcium-) dependent. It is proposed that PAPA, probably a complement component, may be important in mediating vascular permeability changes in acute inflammatory reactions in the rabbit. This work was supported by the Medical Research Council.




WILLIAMS, T. J. (1976). J. Phy8iol. 254, 4-5P. WILLIAMS, T. J. (1978). J. Phy8iot. 281, 44-45P. WILLIAMS, T. J. & PECK, M. J. (1977). Nature, Lond. 270, 530-532.

The quantitative measurement of white thrombus formation and arteriolar diameter in the hamster cheek pouch BY G. P. LEWIS, HELEN WEBB and J. WESTWICK, Department of Pharmacology, Royal College of Surgeons, Lincoln's Inn Fields, London WC2A 3PN

Statistical triggering: a new way of looking at sigmoidal kinetics of voltagedependent ionic channels BY KENNETH A. RUBINSON, University Chemical Laboratory, Cambridge CB2 lEW A characteristic feature of Na, K, and some Ca currents is their sigmoidal activation in response to a step depolarization. Hodgkin & Huxley (1952) suggested that these kinetics might arise from a co-operative process in which more than one particle is required to create an open channel. An alternative is to assume that the channels are heterogeneous with a finite lag between the depolarization and the voltagedependent process that permits a detectable current. Because of the heterogeneity the length of the lag is statistically distributed in time about a mean delay value. For the potassium current it is possible to generate families of sigmoidal curves which fit closely those determined experimentally (Fig. 1A) with the assumption B

I10XiA I A










30S X


j X~~~~~~~~~ 20









3 4 Time(msec)











Time (msec)

Fig. 1. For legend see opposite.




15P PHYSIOLOGICAL SOCIETY, MAY 1978 that the lag is followed by a single exponential (first-order kinetic) process. For the sodium currents, again close fits can be obtained assuming heterogeneity and having the kinetics follow a simple sequential form: A -- C -> D with C conducting. All resulting kinetic variables vary monotonically with voltage. AO is a function of the number of channels, their conductivity, and the electro-chemical potential. A simple physical explanation of the lag may be that it is a time needed to make or break a number of hydrogen bonds. This could explain the pronounced effect on the time course when replacing H20 by D20. Even though the appearance of the conducting form, C, is heterogeneous, its disappearance in response to a step repolarization can be simple exponential; for instance if C is not stabilized in the same way as A. REFERENCE

HODGKIN, A. L. & Huxixy, A. F. (1952). J. Phyaiol. 117, 500-544.

COMMUNICATIONS Effect of sodium removal on tension and membrane potential after inhibition of the sodium pump in dog Purkinje fibres BY E. CORABOEUF*, P. GAUTIERt and P. GUIRAUDOUt. t Centre de Recherches ClinMidy, 34082 Montpellier Cedex, and * Laboratoire de Physiologie compare, 91405 Orsay Cedex, France In cardiac tissues low-Na media increase Na efflux, inducing a fall in [Na]i (Ellis, 1977) while contracture develops as a consequence of the Na-Ca exchange. In nerve this exchange is suppressed by Mn at high concentrations (Baker, 1972) and acid media (Baker & McNaughton, 1977). We measured changes in mechanical tension and membrane potential during perfusion with low Na (13.7 mM) or Na-free media in dog Purkinje fibres in which the sodium pump had been previously inhibited by K-free media or ouabain 10-4 M. In K-free media replacement of Na with Tris, Li, choline or sucrose produced a contracture (C) and a hyperpolarization (AEm). Both C and AEm show a maximum after 1-2 min. Fig. 1. A, Potassium currents of squid under voltage clamp. Holding potential -60 mV. Temperature 6 'C. Depolarization is by a step change to the potential in mV shown at the , the best fit with a function which is right of each curve. 0O experimental points; the same as for the sodium currents but with k2 set at zero. Data courtesy of Dr Y. Pichon. B, Sodium currents from squid, 1'5 'C. Data courtesy of Dr J. Kimura. Holding potential - 100 mV with a step depolarization to the voltage shown near each curve. The equation used to fit the currents is

1(t) =

(27ro02)-i[k1/(k2-kl)] .A0.

{exp [-k (t-u- At)]

-exp -k2 (t- U-At)]}. exp [- U2/2o-2] du.

This is simply the equation for C in the sequential reaction A k, C with a Gaussian function having its centre shifted to a time At.

k) D convoluted

Proceedings of the Physiological Society. Royal College of Surgeions Meeting 5--6 May 1978. Bladder electromyograms in the conscious cat [proceedings].

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