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P04-30

THE EFFECT OF THE THYROLIBERIN ON THE SYNTHESES OF THE BETA-ENDORPHIN AND CORTICOTROPHIN ROPIN

Hazar H., Yetkin Y., Yetkin A.

Researching of the regulation of the blood circulation has showed the importance of the opioids. The aim is to study the effect of the thyroliberin on the level of b-endorphin and corticotrophin in blood plasma and spino-cerebellar fluid.

The studies were performed on the cats weighting from 3.5 to 4.0 kg under general anesthesia (Nembutal, 40mg/kg, i.v.). The blood from jugular vein and spinal fluids were withdrawn from thoracic region of the vertebra at the 3rd and 20th minutes before and after the test-substance. Thyroliberin was applied in 1 mg/kg of dose. The level of the b-endorphin and corticotrophin were assessed by the radio-immunologic methods

Thyroliberin did not cause any changes on the level of the beta-endorphin in the plasma and spinal fluid. At 3rd and 20th minutes plasma control level was 47±3.3. pk mol/l. After 3 and 20 minutes, they were found 42±8.2 and 45±10.5 pk mol/l respectively. Control level of spinal fluid was found 1.3±0.4. The levels of beta-endorphin were found 1.8±0.4 and 1.8±0.5 pk mol/l.

In plasma, thyroliberin increased significantly the level of the corticotrophin in the average of %75±1.8 at 3 and 20 minutes, whereas in the spinal fluid thyroliberin decreased the level of the corticotrophin at 3 and 20 minutes %41±1.3 and %63±9.1, respectively.

Thyroliberin did not effect on the beta-endorphin level in plasma and spinal fluid is shown that the effect of the thyroliberin is not over the opioid systems. However, increasing in blood plasma and decreasing in the spinal fluid of the corticotrophin by the effect of thyroliberin can express through the negative feedback. If corticotrophin involve at a high level in the plasma, it makes an inhibitory effect on itself synthesis.

Department of Physiology and Pharmacology, Medical Faculty, Yüzüncü Yil Uni., 65 200, VAN, TURKEY

P04-31

AGEING AND HYPERTENSION DO NOT AFFECT GLUCOSE METABOLISM IN THE RAT

Ruggeri P.*., Cogo C.E. *., Brunori A.*., Natalucci S. °., Burattini R. °

The aim of the study is to investigate whether insulin resistance and impaired glucose effectiveness develop with ageing and exposure to high blood pressure in the spontaneously hypertensive rat (SHR). The minimal model of glucose kinetics was applied to insulinaemia and glycaemia data collected by a 12-sample, 120-minutes intravenous glucose tolerance test (IVGTT) from 36 rats under pentobarbital anaesthesia (40mg/kg, i.p.). These rats were divided into four groups (n=9, each group): two groups of young (12 weeks) spontaneously hypertensive (Y-SHR) and normotensive Wistar Kyoto (Y-WKY) rats and two groups of old (40 weeks) spontaneously hypertensive (O-SHR) and normotensive (O-WKY) rats. The glycaemic metabolism of each group was characterised by the estimates of insulin sensitivity, SI, and glucose effectiveness, SG, obtained from fitting the minimal model to data. The SI index quantifies the ability of insulin to promote glucose metabolism, whereas SG quantifies the ability of glucose to promote its own metabolism. The Y-SHR and O-SHR groups were contrasted to the age matched, Y-WKY and O-WKY groups, respectively, to investigate the possible association between insulin resistance and hypertension. The O-SHR and O-WKY groups were contrasted to the Y-SHR and Y-WKY groups, respectively, to address the issue as to whether abnormalities in glucose metabolism develop with age. No significant differences (p>0.05) were observed in the mean SG and SI estimates between the Y-SHR and the Y-WKY group, as well as between the O-SHR and the O-WKY group. Moreover, no significant differences (P>0.05) were observed in the mean SG and SI estimates between the O-SHR and the Y-SHR group, as well as between the O-WKY and the Y-WKY group. We conclude that, in the genetic model of hypertension considered here, ageing and long-lasting exposure to high blood pressure levels do not necessarily lead to the development of insulin resistance and impaired glucose effectiveness.

*DIMES, University of Genoa °Department of Electronics and Automatics, Polytechnic University of Marche, Ancona, Italy

P04-32

SPATIAL DISCONTINUITIES IN TRANSMISSION OF THE AORTIC PULSE WAVE IN THE ANAESTHETISED RABBIT

Sears T.A., Banks D.

Following the classical studies of Murgo the arterial pulse wave has continued to interest Physiologists and Clinicians for the information it contains about cardiac performance, the peripheral vascular bed and local blood flow. More recently interest has centred on the nature and source of reflected waves that can be the cause of, or exacerbate, hypertension. Arterial pulse waves are usually examined in the frequency domain by Fourier analysis or directly in the time domain by applying appropriate hydrodynamic models to measurements of pulse wave pressure gradients. In the study of nerve impulse transmission the changing spatial distribution of the propagating wave with time reveals spatial discontinuities due to nodes of Ranvier. We thought it of interest to examine the aortic pulse wave in an analogous way by using spatial and temporal sampling frequencies commensurate with the probable conduction times of 1-2ms (conduction velocity 4.0 - 5.0 m/s) between, and the spacing of, the segmental arterial branches (0.8 - 2.0cm). In anaesthetised rabbits we have measured aortic blood pressure successively at multiple sites 10 or 5.0 mm apart along the aorta using a high-fidelity transducer and a sampling rate of 1000Hz or higher. Off-line at each site we derived QRS-event- triggered averages of the pulse wave, usually of 250ms duration, and via an Excel spreadsheet and Origin software transposed the data to create spatial plots of the propagating pulse wave at different times, ms by ms. Such spatial profiles have directly revealed reflection sites and discontinuities that correlate with the sites of major branches of the aorta, such as the renal, and mark the transition from the initial branch-free segment of the descending aorta and the first pair of intercostal arteries.

School of Biomedical Sciences, King’s College, London & Dept Biological Sciences, Open University, Milton Keynes, UK

P04-33

ACTIVE REGULATION OF CORONARY ARTERY DIAMETER IN RESPONSE TO EXTRAVASCULAR PRESSURE ELEVATION.

Azzawi M., Austin C.

Myogenic tone, an important autoregulatory mechanism in vivo, has been demonstrated in vitro using isolated pressurized small arteries by increasing intravascular pressure (IvP). In vivo, however, coronary vessels are subjected to compressive forces of the surrounding cardiac muscle, increasing extravascular pressure (EvP). The effects of increasing EvP on myogenic regulatory mechanisms is unclear. Using novel methodology we studied whether isolated coronary vessels actively regulate their diameter in response to a sustained elevation of EvP. Wistar rats were humanely killed by cervical dislocation. Septal coronary arteries were dissected out and mounted on a modified pressure myograph, superfused with physiological salt solution (pH 7.4, 37oC, 95% air/5% CO2). A secure lid over the myograph chamber allowed EvP to be elevated (via a 95%air/5%CO2 source) over sustained periods. The internal vessel diameter was determined using a video dimension analyser. Data given as mean + SEM. At an IvP of 60 mmHg, coronary arteries (mean diameter 184 mm + 14 mm, n=8) developed myogenic tone. Pressure-diameter relationships were studied over an IvP range of 20-100 mmHg at zero EvP. Active lumen diameters were significantly lower than passive diameters at all pressure increments (p<0.01), demonstrating evidence of active regulation of diameter over an IvP range of 40-60 mmHg. The influence of EvP elevation was also assessed at constant IvP of 60 mmHg (mean diameter 220+ 15 mm, n=5). Elevation of EvP produced an immediate decrease in diameter over an EvP range of 20-100 mmHg. A sustained elevation of EvP led to active regulation of diameter over an EvP range of 40-60 mmHg, stabilising within 1-9 min. Active diameters were significantly lower than passive diameters at 20, 40, 60 (p<0.01), and 100 mmHg (p<0.05) EvP. Thus we demonstrate that coronary vessels show active regulation of coronary artery diameter in response to a sustained elevation of EvP.

Dept Medicine, Manchester Royal Infirmary Oxford road, Manchester M13 9WL, UK

S6 Sensors and effectors in body fluid homeostasis

ORAL SESSION

S6-1

MACULA DENSA CELL SENSING OF DISTAL TUBULAR LOAD AND NITRIC OXIDE RELEASE

Liu R., Persson E.G.

Earlier investigations in our laboratory have indicated that Na,K,2Cl co-transport mechanism is involved in sensing the fluid/NaCl load to the macula densa cells. The luminal NaCl concentration ([NaCl]) at the macula densa (MD) controls both tubuloglomerular feedback (TGF) and renin release. In earlier studies we have found that nitric oxide (NO) inhibits TGF sensitivity potently. The NO concentration in the MD cells is not known.

In the present study we measured NO production rate in MD cells with confocal microscopy in the isolated perfused thick ascending limb using a NO-sensitive fluorophore 4,5-diaminofluorescein (DAF-2). Calcein was used to measure cell volume changes. The loop perfusion fluid was a modified Ringer solution containing 10, 35, or 135 mM NaCl with a constant total osmolarity (290 mOsm), while the bath was perfused with the 135 mM NaCl solution.

The results showed that MD cell volume and NO production increased considerably with increasing luminal [NaCl]. Furthermore, we found that 5 mM L-arginine increased (30%) NO production in the MD cells. 7-nitroindazole, an nNOS inhibitor, could totally inhibit the NO production caused by L-arginine and by increased luminal [NaCl].

In conclusion, we could quantitatively measure the MD cell volume changes caused by the changes of luminal [NaCl], and found that increasing the luminal [NaCl] resulted in an increase in cell volume. We also found that NO formation in macula densa cells could be measured with DAF-2 and that NO production was increased through neuronal NO synthase activation with an increased luminal [NaCl]. An increased NO production will inhibit the vasoconstriction induced by the TGF and at the same time will reduce TGF sensitivity.

Department of Medical Cell Biology, Uppsala, Sweden

S6-2

INTERACTION BETWEEN ANGIOTENSIN II, NITRIC OXIDE AND PROSTAGLANDINS IN THE CONTROL OF RENAL FUNCTION

Salazar F.J., López R., Llinas M.T.

Several studies performed by our group have demonstrated that there is an important interaction between angiotensin II (Ang II), nitric oxide (NO) and prostaglandins (PG) in the acute and long-term regulation of the renal hemodynamic and excretory function. The results obtained suggest that both NO and PG protect the renal vasculature from the hemodynamic and tubular effects of Ang II. Recently we have examined the role of the cyclooxygenase (COX) isoforms in producing the PG involved in modulating the renal vasoconstriction and antinatriuresis induced by acute and prolonged reductions in NO synthesis. It was found that the administration of a non-selective COX inhibitor enhances the renal vasoconstriction and antinatriuresis secondary to a decrease in NO. It has also been found that: A) a reduction in NO synthesis is followed by an stimulation in the production of COX-2-derived metabolites; B) the administration of a selective COX-2 inhibitor enhances the renal vasoconstriction induced by a decrease in NO, and C) this enhancement is similar to that elicited by a non-selective COX inhibitor. These results support the notion that COX-2 play a more important role than COX-1 in producing the PG involved in buffering the renal vasoconstriction secondary to acute and chronic reductions in NO. From the results obtained in our laboratory it can also be proposed that the COX-1 isoform, rather than COX-2, is involved in producing the PG that regulate renal excretory function when endogenous NO synthesis is reduced. In support of this idea, it was found that the administration of a non-selective, but not that of a selective COX-2 inhibitor, enhances the sodium retention elicited by a decrease in NO during acute or prolonged increments in extracelular volume, or during the infusion of a medullary vasodilator (bradykinin). In summary, the results obtained suggest that COX-1 and COX-2 play different roles in the regulation of the renal hemodynamic and excretory function.

Department of Physiology, School of Medicine, University of Murcia, SPAIN

OC06-1

INTERACTIONS BETWEEN EPITHELIAL NITRIC OXIDE SIGNALLING AND PDE ACTIVITY IN DROSOPHILA

Davies S., Broderick K., MacPherson M., Regulski M., Tully T., Dow J.

Signalling by nitric oxide (NO) and guanosine 3’, 5’-cyclic monophosphate (cGMP) modulates fluid transport in Drosophila melanogaster. Expression of an inducible transgene encoding Drosophila NO synthase (dNOS) increases both NOS activity in Malpighian (renal) tubules, and DNOS protein in both Type I (principal) and Type II (stellate) cells. However, cGMP content is increased only in principal cells. DNOS overexpression results in elevated basal rates of fluid transport in the presence of the phosphodiesterase (PDE) inhibitor, Zaprinast. Direct assay of tubule cGMP-hydrolysing phosphodiesterase (cG-PDE) activity in wild-type and dNOS transgenic lines shows that cG-PDE is Zaprinast-sensitive and is elevated upon dNOS induction. Zaprinast treatment increases cGMP content in tubules, particularly at the apical regions of principal cells, suggesting localisation of Zaprinast-sensitive cG-PDE to these areas.

Potential cross-talk between activated NO/cGMP and calcium signalling was assessed in vivo with a targetted aequorin transgene. Zaprinast potentiates both neuropeptide- and cGMP-stimulated calcium levels upon dNOS induction. In tubules in which DNOS is overexpressed, the Zaprinast-induced transport phenotype is inhibited by the calcium channel blocker, verapamil.

Molecular genetic intervention in the NO/cGMP signalling pathway has uncovered a pivotal role for cell-specific cG-PDE in regulating the poise of the fluid transporting Malpighian tubule via direct effects on intracellular cGMP concentration and localisation, and via interactions with calcium signalling mechanisms.

Institute of Biomedical and Life Sciences, University of Glasgow & Cold Spring Harbor Laboratory – United Kingdom

OC06-2

THE IMPORTANCE OF ADENOSINE A1-RECEPTORS FOR RENAL SALT EXCRETION

Brown R., Fredholm B., Persson A.

Adenosine serves as an important modulator of a vast array of physiological functions. The present study was performed to investigate the roll of adenosine A1-receptors in regulating blood pressure and electrolyte balance during variations of dietary salt intake and tubuloglomerular feedback (TGF) mechanism in adenosine A1-receptor (A1AR) knockout (ko) mice. A1AR-ko and wild-type (wt) mice were placed on standardized normal-salt (NS)(0.7%) or high-salt (HS)(7%) diets for a minimum of ten days prior to blood pressure and excretion measurements. The animals were chronically implanted with telemetric blood pressure devices for long-term blood pressure measurement. Blood pressure was continuously recorded in the conscious animals during a 2-week period. Mice were placed in metabolic cages and 24-h urine collections were obtained. On a NS-diet mean arterial blood pressure was approximately 20 mmHg higher in the A1AR-ko (109±3 mmHg) compared to the wt mice (92±4 mmHg). On a HS-diet A1AR-ko blood pressure was 107±4 mmHg compared to 104±3 mmHg in the wt mice. Sodium excretion was elevated in the A1AR-ko compared to the wt mice on NS-diet (0,046±0.009 and 0,027±0.004 µmol/min/10gbw, respectively) and was normalized on HS-diet (0.14±0.02 and 0.12±0.03 µmol/min/10gbw, respectively). In a separate set of experiments TGF mechanism was assessed in mice on a NS-diet. The decrease in tubular stop-flow pressure in response to increased distal tubular flow-rate, found in wt mice (11.4±1.1 mmHg), was absent in the A1AR-ko (0.1±0.8 mmHg) mice. In conclusion, on a NS-diet the A1AR-ko animals lack TGF and therefore lose salt. Earlier results from our lab have shown that the A1AR-ko mice have a significant elevated plasma renin concentration on NS-diet and this could explain the significantly increased blood pressure in the A1AR-ko-animals. During high salt intake the TGF is not essential and salt excretion is essentially normalized compared to the controls.

Division of Integrative Physiology, Uppsala University, Uppsala, Sweden

OC06-3

TNBS COLITIS SELECTIVELY ALTERS THE NATURE OF MUCOSAL MICROCIRCULATION TO ALLOW INFLAMMATION

Phillipson M., Henriksnäs J., Antoon J*., Perry M*., Holm L.

Inflammatory Bowel Diseases (IBD) cause severe gastrointestinal injury and inflammation of the mucosa. Interaction of leukocytes with endothelial adhesion molecules may initiate the signal, inducing inflammation. We have found that the blood vessels in the superficial gastric mucosa are resistant to leukocyte adhesion and inflammation. Here we studied leukocyte-endothelial (L-E) interactions in the colonic mucosal venules to test the hypothesis that the onset of IBD depends on a breakdown of the anti-inflammatory properties of the superficial mucosal microcirculation. Rats were treated with saline or TNBS (50mg/ml in 50% ethanol, intrarectally) 7-14 days prior to the experiments, then anesthetized with Inactin and L-E interactions in the different colonic layers was assessed either with the dual label antibody technique (ICAM-1 and P-selectin expression) or intravital microscopy. In mucosal venules in the saline treated group, almost no rolling or adherent leukocytes could be detected. However, in the TNBS treated group, rolling in the mucosal venules was 6.9±2.0/min/50µm. In the submucosal and muscularis venules, rolling and adherent leukocytes were observed in both saline and TNBS treated groups without any significant difference between the groups. There was no expression of P-selectin in the control colonic mucosa and only a low expression of ICAM-1 (23% of that expressed per gram in the submucosa and muscularis). After TNBS, P-selectin was significantly increased only in the mucosa, while ICAM-1 was not upregulated. Conclusion: In control animals there is no leukocyte rolling or adhesion in colonic mucosa. However, after induction of colitis, p-selectin is selectively activated in the mucosal venules causing leukocyte rolling, which could be responsible for the onset of inflammation.

Dept of Medical Cell Biology, Uppsala, Sweden and *Dept of Physiology and Pharmacology, UNSW, Sydney, Australia

OC06-4

RENAL HYALURONAN ACCUMULATION AND HYALURONAN SYNTHASE EXPRESSION AFTER ISCHEMIA-REPERFUSION INJURY

Hansell P., Johnsson C., Jacobson A., Heldin P., Hällgren R., Göransson V.

Hyaluronan (HA) is a connective tissue component with unique water binding and pro-inflammatory properties. In anaesthetized rats we investigated if renal cortical HA accumulation and the intrarenal distribution and expression of HA synthases (Has 1,2,3) correlate with renal dysfunction after renal ischemia-reperfusion (IR) injury. After 20, 30 or 45 min of unilateral renal ischemia and 72h of reperfusion, renal function and cortical HA content were measured. Has 1, 2 and 3 mRNA were determined using RT-PCR in control and IR kidneys subjected to 45 min ischemia and 72h reperfusion. IR-kidneys had reduced urine concentrating ability, potassium excretion, glomerular filtration rate (GFR) and renal blood flow. On average, IR-kidneys had more than ten times higher amounts of cortical HA than the contralateral control kidney and their water content was elevated while papillary HA was largely unaffected. Has 2 expression in the cortex was heavily upregulated in IR kidneys while Has 3 remained at control levels. Has 1 could not be detected. There was a direct correlation between the amount of cortical HA and the time period of ischemia and also between the cortical amount of HA and depression of functional parameters. In conclusion, IR injury depresses parameters of renal function which coincides with an elevated cortical HA content and Has 2 expression. The enhanced Has 2 expression indicates that the cortical HA accumulation is primarily dependent on increased HA synthesis and not impaired degradation/elimination. The water binding and proinflammatory properties of HA may contribute to renal dysfunction after IR.

Unit of Physiology, Depts of Medical Cell Biology, Biochemistry, Surgery, Medicine, Uppsala Univ. - Sweden

S6-3

THE NEURAL REGULATION OF SODIUM HANDLING BY THE KIDNEY.

Johns E.J.

The kidney receives a very dense innervation from the sympathetic nervous system which represents the autonomic control of its function. The efferent renal sympathetic nerves enter the kidney and pass in close proximity to both vascular and tubular structures. As activity within the nerves increase there is a progressive recruitment of functions; at low levels renin secretion only is raised; thereafter tubular sodium reabsorption increases while it is only at higher levels of activity that renal blood flow and glomerular filtration rate are reduced. In terms of dealing with fluctuating levels of sodium intake, the dynamic responses at the kidney will be exerted via neural influences on renin release and the level of sodium reabsorption. The sensory information flowing into the central nervous system arises from the cardiovascular baroreceptors, somatonsensory receptors, visceral receptors as well as from higher cortical centres. It is therefore important to understand what factors may alter the ability of the central nervous system to sense changes in extracellular fluid balance and thereby determine renal sympathetic nerve activity. It is evident that the brain renin-angiotensin system acts in a neuro-modulatory fashion to determine the sensitivity of the reflex neural regulation of kidney function. Moreover, it is important to be aware of how paracrine and autocrine factors may influence the impact of the sympathetic nerves on the epithelial cells mediating fluid reabsorption. Thus at the cellular level, nitric oxide, superoxide anions and the degree of oxidative stress can influence the effectiveness of transmission at the neuroeffector junction. An understanding of these interactions is important in order to appreciate mechanism underlying pathophysiological states, such as hypertension, where the neural control of the kidney is often abnormal.

Department of Physiology, University College Cork, Ireland.

S6-4

LONG-TERM CONTROL OF TOTAL-BODY SODIUM: PRESSURE ESCAPE, RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM AND NO

Seeliger E., Reinhardt H.W.

Long-term control of mean arterial blood pressure (MABP) is closely related to control of total-body sodium (TBS). The renin-angiotensin-aldosterone system (RAAS), pressure natriuresis, and nitric oxide (NO) are thought to be important elements of TBS control. Standardized balance studies were performed in freely moving dogs to elucidate their individual contributions as well as their interactions. Long-term 20% reduction of renal perfusion pressure (rRPP) in dogs on high Na intake results in Na retention on day 1 via stimulation of the RAAS, which augments TBS, thus increasing MABP. On the following days, 24-h Na balances become equilibrated again. This resetting of 24-h balances on an elevated level of TBS was termed Pressure Escape in analogy to mineralocorticoid escape. Pressure Escape is mainly accomplished by suppression of aldosterone; low-dose aldosterone infusion during rRPP results in ongoing Na retention. After accomplishing Pressure Escape, the TBS surplus is defended by the body: reduction in Na intake does not reduce surplus of TBS and elevation of MABP, because of a renewed RAAS stimulation. Long-term infusion of the NO-inhibitor L-Nitroarginine (LN; non-pressure dose to prevent pressure effects on renin release and natriuresis) results in TBS deficit via aldosterone suppression. LN does not alter Na retention during rRPP, nor does it compromise the accomplishment of Pressure Escape. A significant contribution of pressure natriuresis to TBS control could only be demonstrated during long-term 20% elevation of RPP as induced by sustained elevation of TBS. Here, pressure natriuresis facilitates Na excretion, which prevents further Na accumulation, but does not restore TBS to normal. A 20% reduction of RPP does not induce Na retention by the putative mechanism of (low-) pressure (anti-)natriuresis, yet only via RAAS stimulation. It is suggested that pressure natriuresis is not operative at lowered, normal, or moderately elevated pressures.

Institute of Physiology, Charite Berlin, Germany

S6-5

INITIAL DYNAMICS OF WHOLE BODY SDOIUM CONTROL: A PHYSIOLOGICAL WHODUNIT

Bie P.

Normally long term control of sodium metabolism does not imply changes in arterial blood pressure (BP). Therefore, an increase in sodium intake will elicit either an elevation in BP which subsides over time or a neurohumoral tuning of renal function at constant BP. The acute response to sodium loading mimicking sodium intake may help to determine whether regulatory sodium excretion (NaEx) can increase markedly over hours without change in BP. However, the quantitative performance of normal NaEx is easily distorted by drugs leaving conscious, unstressed organisms as the only sources of reliable data.

Recent studies in normal dogs and volunteers have confirmed that slow infusions of saline (0.006-0.04 mmol/min/kg b.wt.) may be performed over hours without changes in arterial blood pressure, but with gradually increasing NaEx to 5-10 times control. Even in subjects on low-salt diet (0.5 mmol/kg/d) a slow infusion of saline (0.02 mmol/min/kg) elicited an immediate natriuresis rising to a 7-fold increase in NaEx over a few hours. The renal response is explainable by the concomitant decrease in renin system activity (PRA, Ang II and aldosterone). However, BP, glomerular filtration rate, plasma hormones (atrial natriuretic peptide, vasopressin, oxytocin), and intrarenal humoral events (as reflected by excretion rates of nitrates, cGMP, and endothelin) remained constant. Other results indicate that NO generation may be a powerful controller of NaEx capable of overriding the renin system. Taken together, the results leave open several explanations for the sensory mechanism(s) by which the renin system normally seem to dominate the regulation of NaEx: (i) low-pressure receptors, (ii) osmoreceptors, or (iii) concentration receptors responding to filtered load of sodium. In any case, relatively fast components of sodium metabolism exist, demonstrate exquisite sensitivity, operate at constant BP, and may change NaEx by 1-2 orders of magnitude in a matter of hours.

University of Southern Denmark, Odense, Denmark

POSTER SESSION

P06-01

ACUTE EFFECTS OF ACIDOSIS ON PROTEIN AND AMINO ACID METABOLISM IN PERFUSED RAT LIVER

Holecek M., Safranek R., Rysava R., Kadlcikova J., Sprongl L.

Acidosis is frequently associated with protein wasting and derangements in amino acid metabolism. As its effect of on protein metabolism is significantly modulated by other abnormal metabolic conditions caused by specific illness, it is difficult to separate out the effects on protein metabolism solely due to acidosis.

The aim of the present study was to evaluate using a model of isolated perfused rat liver the direct response of hepatic tissue to acidosis.

We have compared the hepatic response to perfusion with solution of pH 7.2 and pH 7.4 (controls). Parameters of protein and amino acid metabolism were measured using both recirculation and single pass technique with 4,5-[3H]leucine, [1-14C]leucine and [1-14C]ketoisocaproate (ketoleucine) as tracers and on the basis of difference of amino acid levels in perfusion solution at the beginning and the end of perfusion. Statistical analysis was performed using Mann-Whitney test.

In the liver perfused with solution of pH 7.2 we observed higher rates of proteolysis, protein synthesis, amino acid utilization, and urea production. Furthermore, the liver perfused with solution of pH 7.2 released a higher amount of proteins to perfusate than the liver perfused with solution of pH 7.4. Enhanced decarboxylation of ketoisocaproate in liver perfused by solution of a lower pH indicates increased catabolism of branched-chain amino acids (leucine, valine, and isoleucine), decreased reamination of branched-chain keto acids to corresponding essential amino acids, and increased ketogenesis from leucine.

The study was supported by a grant of the Grant Agency of the Czech Republic No. 305/01/0578.

Charles University, Faculty of Medicine, Hradec Kralove, Czech Republic

P06-02

THE EFFECT OF AMILORIDE DURING INFUSION OF OXYTOCIN IN MALE SPRAGUE-DAWLEY RATS

Nordquist L., Isaksson B., Sjöquist M.

A possible natriuretic mechanism of action of intravenously infused oxytocin was investigated in male Sprague-Dawley rats. The effects of an intravenous bolus injection of amiloride (3.0 mg/kg BW) on urine volume, potassium and sodium excretion and omolality were measured with and without an intravenous infusion of oxytocin in saline solution (1200 ng/h/kg BW). Control values were obtained during infusion of saline solution (0.9% NaCl). To simulate experimental conditions control animals were given an injection of saline solution identical in volume to the injection of amiloride and the following rinsing volume.

The effect of amiloride on urinary flow after administration of oxytocin was an 11-fold increase (from 4.289±0,577 µL/min to 48.827±60694 µL/min), thereby contributing to a 660-time increase in sodium excretion (from 0.025±0,007 to 16.621±2.074 µmol/min). In amiloride-only treated animals, the flow after the bolus dose was 17.731±1.757uL/min and the sodium excretion 4.482±0.795 µmol/min. Administration of oxytocin only resulted in a flow of 8.468±1.555uL/min and a sodium excretion of 1.191±0.317 µmol/min.

Nor was the amiloride-caused change in potassium excretion inhibited by oxytocin. The potassium excretion after treatment with amiloride decreased to 13% of that in the control group (from 3.220±0.387 µmol/min to 0.172±0.080 µmol/min) In the group receiving amiloride and oxytocin both, the decrease was to 4% of control group values (from 3.220±0.387 µmol/min to 0.054±0.019 µmol/min).

Hence, the effects of amiloride were not inhibited by the actions of oxytocin, but rather enhanced. In summary, amiloride administrated after reaching a near steady state effect of oxytocin was found to give rise to an effect far greater than that after administration of oxytocin or amiloride alone. It's therefore concluded that the intrarenal natriuretic mechanisms of oxytocin are likely not to emanate from the amiloride sensitive sodium channels.

Inst. for Medical Cell Biology, Dept. for Integrative Physiology – Uppsala, Sweden

P06-03

THE EFFECT OF CHANGES IN INTESTINAL TRANSIT ON REMOTE SECRETO-MOTOR REFLEXES

Timar-Peregrin A., Revesz* D.

Introduction: The involvement of nervous system in local control of secreto-motor reflexes (SMR) to various secretagogues has already been studied to some extent as well as the importance of enteric nervous system regulating local motility. However, the underlying mechanisms for enteric reflexes to changed intestinal transit (IT) remain to be elucidated. We have therefore designed an experimental model allowing us to study the effects of changes in IT on SMR in vivo.

Methods: Anaesthesia was induced with Nembutal and maintained by intra-arterial infusion with chloralose in rats. Arteria and venae femoralis were catheterised. A proximal jejunal and a distal ileal segment were isolated, cannulated and connected to a pressure transducer and the rest of the intestine was extirpated. In some experiments the extrinsic nervous supply to the segments was cut. The proximal segment was perfused at various perfusion rates (PR).

Results: Increased PR at 6, 64 resp 2ml/h caused a significant reduction of motility Hz in the perfused segment of normal (ND) resp denervated (D) animals. PR over 32ml/h elevated the Hz of the propagating contractions in the distal segment of D animals (p<0.05). In the stimulated intestinal segment, PR exceeding 32ml/h augmented the secretion in over 75% of both ND and D rats. An increase of secretion was observed in the more distal segment with PR over 2ml/h in 80% of the ND animals. On the other hand, no secretion appeared in the D animals.

Conclusions: We can conclude that denervation caused an increase in motility of the distal segment indicating a descending inhibitory effect of the nervous system to increased IT. These changes in transit rate result in an elevation of motility by other pathways e.g. hormones. On the other hand, the observed increase in secretion in the more distal segment disappeared after severing the nerves suggesting the involvement of nervous pathways in the development of such secretion to faster IT proximally in the gut.

The Baker Medical Research Institute, Melbourne, Australia and *Sahlgrens Academy, Gothenburg, Sweden

P06-04

EARLY CHANGES IN TUBULOGLOMERULAR FEEDBACK IN HYPERTHYROID RATS

Hultström M., Sandgren A., Källskog Ö.

The aim of the present study was to evaluate the tubuloglomerular feedback mechanism (TGF) in hyperthyroid rats. The TGF resets in hypertension. Whether the change in TGF is caused by hypertension or hypertension develops as an effect of changed renal autoregulation is not completely understod. Changes of the TGF mechanism before the onset of hypertension might be indicative of the cause of hypertension in hyperthyroid rats.

Methods: Male Sprague-Dawley rats were treated with either 1000 µg/kg triiodothyroxine (T, n=8) or vehicle (C, n=8) for two days before surgery. Renal blood flow (RBF) and glomerular filtration rate (GFR) was measured for 20 minutes following a one hour stabilization period. Thereafter TGF measurements were performed using micropuncture.

Results: There was no difference in blood pressure but a clear difference in GFR (C:3.48±0.15ml/(min*kg BW) vs. T:3.86±0.13ml/(min*kg BW) P<0.05) and RBF (C:6.106±0.52ml/min vs. T:7.77±0.48ml/min P<0.05). Micropuncture results show an increase in stop flow pressure (C:38.91±1.27 mmHg vs. T 43.02±1.02 mmHg P<0.05) and a decrease in TGF reaction at maximal stimulstion (C: 11.82±0.91mmHg vs.T: 7.017±0.77mmHg P<0.05). The turning point was not significantly altered.

Discussion: The increase in RBF and GFR are in accordance with what others have found. The TGF result indicates that an increased proximal absorbtion reduces the signal to the macula densa. This leads to a lower TGF activity and afferent dilation, which is seen as an increase in stop flow pressure. The change in macula densa activation will also trigger the renin angiotensin system that is considered to be the cause of hyperthyroid hypertension.

Conclusion: Hyperthyroidism causes early changes in renal autoregulation which may be responsible for the development of hypertension in hyperthyroid rats.

Department of Medical Cellbiology, Uppsala University, Sweden

P06-05

CHANGE OF BODY FLUID PARAMETERS BY SODIUM INTAKE IN TRAINED CONSCIOUS DOGS

Kjolby M.J., Wamberg S., Bie P.

Background. The effects of daily sodium intake (NaInt) on renal and cardiovascular parameters were measured under steady state conditions. Dogs were fed a commercial low-salt diet plus NaCl to 8 levels of NaInt.

Methods. NaInt were 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 8.0 mmol/kg/d for 7 days. Potassium intake was 2.79±0.03 mmol/kg/d. Measurements were made 20 h postprandially after 9 h dehydration. Systolic (SBP), diastolic (DBP) and mean arterial blood pressure (MABP) were measured invasively. Clearance of exogenous creatinine provided glomerular filtration rate (GFR). Plasma volume (PV) was measured by dye dilution and blood volume (BV) determined from PV and arterial hematocrit. Plasma hormones were determined by radioimmunoassay techniques.

Results. SBP, DBP and MABP remained constant irrespective of NaInt at 136.7±1.0, 88.9±0.4 and 107.9±0.4 mmHg, respectively. Heart rate (HR) was constant at 63±1 min-1. PV increased by 0.47±0.04 ml per kg body weight per unit increase in NaInt (p<0.01), i.e., 0.47 (ml/kg)/(mmol/kg/d); BV increased by 0.66±0.07 (ml/kg)/(mmol/kg/d) (p<0.001). Plasma sodium was constant at 145.2±0.2 mmol/l. Plasma potassium decreased linearly with increasing NaInt by -0.038 (mM)/(mmol/kg/d) (p=0.001) while plasma renin (PRA), angiotensin II (AngII) and aldosterone (Aldo) decreased exponentially (=a*exp(k*NaInt)+b, kPRA=-5.5, kAngII=-4.6 and kAldo=-2.4, respectively, all p<0.05). Plasma atrial natriuretic peptide, angiotensinogen and vasopressin did not change (69±7 pg/ml, 1008±56 ng/ml and 1.16±0.06 pg/ml, respectively). GFR was constant at 39.1±2.6 ml/min.

Conclusions. Large increases in sodium intake were associated with (i) constancy of blood pressures and HR, (ii) exponential decreases in PRA, AngII and Aldo concentrations, (iii) increase in BV, and (iv) a linear decrease in plasma potassium concentration. BV and plasma potassium may work together to inhibit the activity of the renin system activity during increases in sodium intake.

Dept. of Physiology and Pharmacology, IMB, University of Southern Denmark, DK-5000, Odense, Denmark.

P06-06

THE EFFECTS OF ULTRASOUND ON SYNOVIAL FLUID ZINC LEVEL IN PATIENTS WITH RHEUMATOID ARTHRITIS

Akcil E., Seckin B., Ergun A.

Recently, trace element levels such as serum zinc and copper in rheumatoid arthritis patients became important. In some studies synovial fluid zinc levels of rheumatoid arthritis patients were found to be increased.

In this study, the effect of ultrasound on synovial fluid zinc level and synovial fluid leukocyte count was investigated in classical and/or definite rheumatoid arthritis patients, with the application of a dose of 2 watt/cm2 for 5 minutes on the knee joints of twenty patients. Synovial fluid zinc levels were estimated by atomic absorption spectrophotometer.

Synovial fluid zinc levels immediately after application of ultrasound was significantly decreased when compared with that before ultrasound application (p<0.05). When the synovial fluid zinc level immediately after ultrasound application was compared with that 24 hours after ultrasound application, the difference was found to be statistically significant (p<0.05).

Synovial fluid leukocyte count immediately after ultrasound application was statistically significantly increased when compared with synovial fluid leukocyte count before ultrasound application (p<0.05). The decrease of synovial fluid zinc level and increase of synovial fluid leukocyte count immediately after ultrasound application seems to be related to the increase of inflammatory activity due to the ultrasound application.

Ankara University, School of Medicine, departments of Pathophysiology, Physical Medicine, Physiology, Ankara, TURKEY

P06-07

SUPPRESSED NITRIC OXIDE MEDIATED ARTERIAL DILATION IN RATS WITH ENHANCED RBC AGGREGATION

Yalcin O., Ozdem S., Armstrong J.K., Meiselman H.J., Baskurt O.K.

The effects of enhanced red blood cell (RBC) aggregation on nitric oxide (NO) dependent vascular control mechanisms have been investigated in a rat model. Rats were exchange-transfused with the suspensions of rat RBC coated with specially designed and produced co-polymers, resulting in significantly enhanced RBC aggregation during the five day follow-up period. Mean arterial blood pressure increased gradually in five days. Arterial segments of 300 micrometers were isolated from gracilis muscle of rats on the fifth day and mounted between two glass micropipettes in a special chamber equipped with pressure servo control system. Both dose dependent dilation by acetylcholine and flow-mediated dilation of arterial segments pressurized to 30 mmHg and pre-constricted to 50% of the original diameter by phenylephrine were significantly blunted in rats with enhanced RBC aggregation, compared to the control group. Both responses were totally abolished by non-specific NOS inhibitor (L-NAME) treatment of arterial segments, indicating that the responses were NO-related. Additionally, expression of eNOS protein was found to be decreased in muscle samples obtained from hyperaggregating rats. These results imply that enhanced RBC aggregation may result in suppressed expression of NO synthesizing mechanisms, leading to altered vasomotor tonus. This effect can be explained by the decreased wall shear stress due to increased axial accumulation of hyperaggregating RBC.

Depts. Physiology and Biophysics, Akdeniz Un. Fac. Med., Antalya, Turkey and USC Keck Sc. Med., Los Angeles, CA, USA

P06-08

EFFECTS OF SELECTIVE OPIOID ANTAGONISTS ON HEMODYNAMIC RESPONSES TO HEMORRHAGE

Frithiof R., Hjelmqvist H., Ullman J., Eriksson S., Rundgren M.

Introduction

During a continuous hemorrhage a paradoxical sympathoinhibition causes bradycardia and hypotension. The aim of this study was to investigate the contribution of central opioid mechanisms in initiating and prolonging this decompensated phase in conscious sheep.

Methods

Adult conscious ewes were continuously bled (0.7 ml/kg/min) from a jugular vein until mean arterial blood pressure reached 50 mm Hg. Starting 30 min before hemorrhage either artificial cerebrospinal fluid (aCSF) or one of the following selective opioid receptor antagonists were infused intracerebroventricularly (ICV); ICI 174,864 (delta-rec antagonist, 0.24mg/h), nor-BNI (kappa-rec antagonist, 2.4 mg/h), CTOP (my-rec antagonist, 0.12 mg/h). The infusion was terminated and the shed blood retransfused 60 min after hemorrhage was completed. Cardiovascular parameters were monitored via ultrasonic flow probes and carotid and pulmonary catheters. Data are expressed as mean ± SEM.

Results

Infusion of nor-BNI significantly increased the blood loss necessary to initiate the decompensated phase compared to aCSF controls (18.9 ± 1.0 n=6 vs 13.9 ± 0.5 n=7). Neither ICI 174,864 (16.9 ± 0.9 n=3) nor CTOP (14.2 ± 1.3 n=7) infusion affected the onset of hypotension. There were no apparent differences between experimental groups regarding other measured cardiovascular parameters before, during and after hemorrhage.

Conclusion

ICV nor-BNI delays but does not fully prevent the onset of hypotension and bradycardia during a continuous hemorrhage. This suggests that endogenous opioid kappa-receptor agonists in the CNS are partly involved in initiating the decompensated phase of hemorrhage in conscious sheep. However, studies using localized injections into specified anatomical areas in conscious animals are needed to further investigate the involvement of CNS pathways in the decompensated phase.

Karolinska Institutet, Dept of Physiology & Pharmacology, Stockkholm, SWEDEN

P06-09

ANGIOTENSIN II AT2 RECEPTORS AND DUODENAL MUCOSAL BICARBONATE TRANSPORT IN THE S-D RAT

Ewert S., Fändriks L.

Background: Activation of the angiotensin II receptor type 2 receptor (AT2R) has been associated with increased duodenal mucosal alkaline secretion in previous experiments using Sprague-Dawley rats. This effect was absent after changing to another line of S-D rats. The present investigation was undertaken to evaluate if the magnitude of expression of AT2R determined the duodenal mucosal alkaline secretory response to the AT2R agonist CGP42112A.

Methods: Duodenal mucosal alkaline secretion was measured in anaesthetised rats by means of in situ pH-stat titration. Real time PCR and Western blot were used to determine the AT1R and AT2R RNA and protein expression, respectively.

Results: In the previous S-D line CGP42112A elicited a significant 45(8)% net increase of the duodenal mucosal alkaline secretion. In the current line a similar dose of CGP42112A did not elicit any change in duodenal mucosal alkaline secretion (n=11). The response to luminal PGE2 (10-5 M) was similar in the two lines of S-D rats. The RNA expression of AT1aR and AT1bR were significantly lower in tissue from the previous line. The AT2R RNA expression was significantly higher in the previous line. The protein expression of AT1R protein did not differ between the previous and the current line. The AT2R protein expression was significantly higher in tissue from the previous compared to current line. The calculated individual AT1R to AT2R ratios (RNA and protein) were significantly higher in the current line compared to the previous line S-D rats.

Conclusion: A low AT2R expression explains the absence of secretory response to the AT2 agonist CGP42112A.

Dept of Gastroresearch, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden

S7 Cellular and Molecular aspect of renal physiology

ORAL SESSION

S7-1

TUBULAR CELL FUNCTION : FROM HEALTH TO DISEASE

Prie D., Terzi F., Silve C., Friedlander G.

The general purpose of our group aims to identify the molecular and cellular mechanisms underlying pathological states of renal function. These studies combine in vitro and in vivo approaches, both in aninals and humans, from cell culture to experimental models of renal injury and to clinical investigation. Two examples are given.

The first one concerns the role of vimentin, an intermediate filament which is expressed by mesenchyme-derived cells, but not by epithelial cells, under normal conditions. However, vimentin is re-expressed by proximal tubular cells in culture or, in vivo, during the recovery phase after ischemic injury. Using mutant mice in which the vimentin gene has been invalidated by homologous recombination, we explored the role of this re-expression and showed that vimentin expression affects selectively the activity of Na-glucose cotransporters. In the absence of vimentin, ischemia-induced glycosuria persists for a longer period of time. These data support an important role of vimentin in tubular function after ischemia.

The second example concerns the molecular basis of renal phosphate leak, a defect responsible for hypophosphatemia, nephrolithiasis, and bone demineralization. In numerous patients, this syndrome is not accounted for by endocrine disorders such as hyperparathyroidism. In 20 of these patients with persistent idiopathic hypophosphatemia associated with a decrease in maximal renal phosphate reabsorption, we looked for mutations of type 2a Na-phosphate cotransporter (NPT2a). Two patients, one with recurrent urolithiasis and one with bone demineralization, were heterozygous for two distinct mutations. Phosphate-induced current and sodium-dependent phosphate uptake were impaired in Xenopus oocytes expressing the mutant NPT2a. Coinjection of oocytes with wild-type and mutant RNA indicated that the mutant protein had altered function.

These examples illustrate the interest of combining different approaches in renal pathophysiology.

INSERM U426 & Dept. of Physiology, Faculte X. Bichat, University Paris 7, Paris, France



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