Evaluation of diet as a cause of gastric ulcers in horses.

OBJECTIVE: To measure pH, volatile fatty acid (VFA) concentrations, and lactate concentrations in stomach contents and determine number and severity of gastric lesions in horses fed bromegrass hay and alfalfa hay-grain diets. ANIMALS: Six 7-year-old horses. PROCEDURE: A gastric cannula was inserted in each horse. Horses were fed each diet, using a randomized crossover design. Stomach contents were collected immediately after feeding and 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, and 24 hours after feeding on day 14. The pH and VFA and lactate concentrations were measured in gastric juice Number and severity of gastric lesions were scored during endoscopic examinations. RESULTS: The alfalfa hay-grain diet caused significantly higher pH in gastric juice during the first 5 hours after feeding, compared with that for bromegrass hay. Concentrations of acetic, propionic, and isovaleric acid were significantly higher in gastric juice, and number and severity of nonglandular squamous gastric lesions were significantly lower in horses fed alfalfa hay-grain. Valeric acid, butyric acid, and propionic acid concentrations and pH were useful in predicting severity of nonglandular squamous gastric lesions in horses fed alfalfa hay-grain, whereas valeric acid concentrations and butyric acid were useful in predicting severity of those lesions in horses fed bromegrass hay. CONCLUSIONS AND CLINICAL RELEVANCE: An alfalfa hay-grain diet induced significantly higher pH and VFA concentrations in gastric juice than did bromegrass hay. However, number and severity of nonglandular squamous gastric lesions were significantly lower in horses fed alfalfa hay-grain. An alfalfa hay-grain diet may buffer stomach acid in horses.

Synthesis and biological activity of potent, low molecular weight renin inhibitors.

A series of renin inhibitors containing the dipeptide transition state mimics (2R,4S,5S)-5-amino-4-hydroxy-2-methyl-6-cyclohexyl hexanoic acid (Cha-psi[CH(OH)CH2]Ala) and (2R,4S,5S)-5-amino-4-hydroxy-2-isopropyl-6-cyclohexyl hexanoic acid (Cha-psi[CH(OH)CH2]Val) were prepared. A structure-activity study, using pseudopeptide (Boc-Phe-His-Leu-psi[CH(OH)CH2]Val-Ile-His-OH) as our lead structure, led to a new series of inhibitors, which correspond to tripeptides and contain no natural amino acids. For example, R,S-Bpma-Ape-Cha-psi[CH(OH)CH2]Ala-NH2 (IC50 = 1.26 nM against human plasma renin at pH 6.0; molecular weight = 564) has only two thirds of the molecular weight but twice the potency of our original lead. This new class of low molecular weight renin inhibitor displays excellent specificity toward human renin versus the related aspartic proteinase pepsin and angiotensin-1-converting enzyme. Examples are given of selected inhibitors showing encouraging evidence for intestinal absorption after intracolonic and oral administration in male Sprague-Dawley rats.

Design, synthesis, and biological activities of four angiotensin II receptor ligands with gamma-turn mimetics replacing amino acid residues 3-5.

Disulfide cyclization is a powerful method for reducing the conformational space of a peptide. This in turn may enable the study of its bioactive conformation. Several analogues of angiotensin II (Ang II) containing a disulfide bridge between amino acids 3 and 5 have been reported. Among these the cyclic octapeptides c[Hcy3,5]-Ang II, c[Cys3,5]-Ang II, and c[Pen 3,5]-Ang II showed significant activity at Ang II receptors. We have performed conformational analysis studies using theoretical calculations and 1H-NMR spectroscopy on tripeptide model compounds of these cyclic octapeptides which show that the cyclic moieties of c[Cys3,5]-Ang II and c[Pen3,5]-Ang II preferentially assume an inverse gamma-turn conformation. On the basis of these results, we substituted amino acid residues 3-5 in Ang II with two different gamma-turn mimetics giving four diastereomeric Ang II analogues. Interestingly, two of these are equipotent to Ang II in binding to AT1 receptors. In the contractile test using rabbit aorta rings, one of the analogues is an agonist with full contractile activity approximately equipotent to c[Pen3,5]-Ang II but 300-fold less potent than Ang II. This low potency may suggest that Ang II does not adopt a gamma-turn in the 3-5 region when interacting with the receptor.

Therapeutic, but not low-dose, angiotensin-converting enzyme inhibition causes regression of cardiovascular changes in spontaneously hypertensive rats.

Therapy with angiotensin II-converting enzyme (ACE) inhibitors has been suggested to prevent cardiovascular hypertrophy in hypertension even in doses that are subantihypertensive. We investigated the effects of two different ACE inhibitors on blood pressure and cardiovascular changes during as well as after discontinuation of treatment in spontaneously hypertensive rats (SHR). SHR were treated with either enalapril (ENA) or ramipril (RAM) from age 12 to age 20 weeks. Each drug was given in either an antihypertensive (ENA 15 mg center dot kg-1, RAM 3 mg center dot kg-1) or a subantihypertensive (ENA 50 mu g center dot kg-1, RAM 10 mu g center dot kg-1) dose. Mean arterial pressure (MAP) was reduced with antihypertensive doses of ENA (26%) as well as RAM (21%). Regression of cardiovascular changes occurred as reduction in left ventricular (LV) weight/body weight ratio (25 and 21% for ENA and RAM, respectively), reduction in perfusion pressure at maximal vasodilation of the perfused hindquarter (PPdil, 17 and 17%), and reduction in maximal developed pressure (PPmax, 13 and 17%). These effects partly persisted 10 weeks after treatment was discontinued. However, treatment with subantihypertensive doses of ENA and RAM had no effect on MAP, LV/body weight ratio, PPdil, or PPmax. Overall, regression of cardiovascular parameters correlated closely to the decrease in MAP. Similarly, no changes in MAP, LV weight/body weight ratio, PPdil, or PPmax were noted when young SHR were treated with subantihypertensive doses of RAM from age 6 to age 12 weeks, during which time hypertension becomes established. At doses having equal effects on blood pressure, plasma concentrations of RAM were considerably lower than those of ENA. Skeletal muscle concentrations were very low or undetectable in comparison to plasma concentrations for both drugs. Therefore, both RAM and ENA caused regression of cardiovascular changes that could be explained by a concomitant reduction in blood pressure. This regression persisted for a considerable time after discontinuation of treatment. On the other hand, no specific antitrophic effects in the absence of blood pressure reduction was evident with either drug. Furthermore, despite substantial differences in plasma concentrations, RAM, and ENA administered chronically appeared to affect cardiovascular parameters equally in the adult SHR.

Raised plasma concentrations of endothelin-1 and -3 in marmosets with acute aortic stenosis: no relation to the renin-angiotensin system.

Plasma levels of endothelin (ET), plasma renin activity (PRA) and angiotensin II (Ang II) were measured in anaesthetized marmosets exposed to acute aortic stenosis proximal to the renal arteries. In vehicle experiments, ET rose from 5 +/- 2 to 38 +/- 4 pg ml-1, PRA from 5 +/- 2 to 99 +/- 21 ng ml-1 h-1 and Ang II from 21 +/- 4 to 213 +/- 76 pg ml-1. Administration of renin inhibitor and angiotensin converting enzyme inhibitor reduced PRA and Ang II to control levels, while the plasma levels of ET increased further (51 +/- 10 and 71 +/- 16 pg ml-1, respectively). During aortic stenosis the two isoforms ET-1 and ET-3 appeared in the circulation, while in conscious control animals only ET-1 was found. It is concluded that the increased plasma levels of ET in our primate model could not be ascribed to the increased circulating levels of PRA and Ang II.

Attenuation of renal ischaemic injury by felodipine.

Felodipine is a vasodilating calcium channel blocker of the dihydropyridine type. The effects of felodipine on post-ischaemic renal function were evaluated in rats subjected to bilateral renal artery occlusion for 30 or 60 min. In a first set of experiments the recovery of renal function after 30 or 60 min of renal artery occlusion was followed intermittently for 16 days by endogenous creatinine clearance. Renal function was better preserved in rats given felodipine (45 nmol/kg i.v.) during the occlusion period than in vehicle-treated control rats. The survival rate after 60-min occlusion was 11% in controls but 70% in the felodipine-treated rats. After occlusion for 30 min the survival rate was similar in the two groups, but renal function recovered faster in the felodipine group than in the controls. In a second series, acute renal damage was evaluated by the extent of erythrocytes trapped in the kidney after 30-min reperfusion following 60-min renal artery occlusion. Felodipine administration (45 nmol/kg) during the occlusion reduced renal damage compared with vehicle controls. Kidney weight and systemic haematocrit were also better maintained in the felodipine-treated rats. Furthermore, renal damage was reduced by the t-butyl analogue or felodipine. H 186/86, which is devoid of vasodilatory effects. The results demonstrate that treatment with the vasodilator calcium channel blocker felodipine protects the kidney from ischaemic/reperfusion injuries. The tissue protection is not related to the haemodynamic effects alone, since the haemodynamically inactive dihydropyridine H 186/86 also reduced the extent of renal damage. An additional antiperoxidant or scavcnger-like effect inherent in the dihydropyridine molecule is suggested.

The contribution of ultrafiltration pressure for glomerular hyperfiltration in young nephrectomized rats.

UNLABELLED: Ablation of renal tissue in infancy is followed by increased ultrafiltration pressure, enlarged filtering area and decreased hydraulic conductivity of the glomerular capillaries. This study examines the effect of a calcium channel blocker, felodipine, on ultrafiltration pressure and renal function in rats uni-nephrectomized at 5 days of age. The rats were treated with felodipine from 24 to 60 days of age or left untreated. Sham-operated untreated rats were used as controls. Arterial blood pressure levels were similar in the three groups of rats. Glomerular filtration rate and renal plasma flow in the left kidney were significantly higher in treated and untreated rats than in sham-operated untreated rats; significantly higher values were also found in treated than in untreated rats. Single nephron glomerular filtration rate was significantly higher in treated and in untreated than in sham-operated untreated rats, but was not different in treated and untreated rats. Ultrafiltration pressure was significantly higher in untreated rats than in treated and in sham-operated untreated rats. Ultrafiltration coefficient (Kf) was significantly higher in treated rats than in untreated and sham-operated untreated rats. Hydrolic conductivity of the glomerular capillaries was significantly lower in untreated than in treated and in sham-operated rats, but was not different in treated and sham-operated rats. Glomerular volume was higher in treated and untreated rats than in sham-operated rats. CONCLUSION: high ultrafiltration pressure is not an absolute requirement for the hyperfiltration that follows infant nephrectomy.

Microelectrode studies of D-glucose- and K+-induced changes in membrane potential of electrofused insulin-producing cells.

In the present study electrical field-induced fusion has been applied to both normal pancreatic islet cells isolated from obese hyperglycemic mice and clonal insulin-producing cells (RINm5F) derived from a transplantable rat insulinoma. The fused cells were then punctured with microelectrodes to measure changes in membrane potential after exposure to stimulatory concentrations of D-glucose or K+. Fused cells of normal islet cellular origin revealed a resting membrane potential of -60 mV and were depolarized by 24 or 27 mV after exposure to 11 mM D-glucose or 30 mM K+. Although D-glucose induced depolarization, it was not possible to establish the existence of an oscillating burst pattern superimposed by action potentials. The resting membrane potential of the fused RINm5F cells was also -60 mV and decreased to -30 mV after exposure to 30 mM K+. As judged from the membrane potential measurements, the reconstitution of the plasma membrane subsequent to electrical breakdown is essentially the same whether the giant cells originated from normal islet cells or RINm5F cells.

pH sensitivity of the basolateral membrane of the rabbit proximal tubule.

Conventional microelectrodes were used to study the effects of bath pH and bicarbonate concentrations on the basolateral membrane potential (Vbl) of cells from the superficial proximal convoluted (PCT) and proximal straight (PST) tubules of the rabbit kidney perfused in vitro. Bathing solution pH was varied over the range of 5.9-7.4 using either control (22-25 mM) or low bicarbonate (5.0-6.6 mM) Ringer solutions and the appropriate CO2 tensions. The results show a strong pH dependence of the steady-state values of Vbl in both the convoluted and straight tubule segments. The pH-dependent depolarization was approximately 35 mV/pH unit change of the bathing solution in the acid direction and could be demonstrated in CO2-free HEPES-buffered solutions. A depolarizing response to increased bath potassium concentration (HK) was observed that was linearly related to the absolute value of the Vbl under control conditions. Under acidotic conditions, reduced HK depolarizations indicate that a decrease in the relative potassium permeability of the basolateral membrane is the principle mechanism underlying the effects of bath pH on Vbl.

Electrophysiology of basolateral bicarbonate transport in the rabbit proximal tubule.

Conventional microelectrodes were used to examine the electrogenic pathways for bicarbonate transport across the basolateral membranes of proximal convoluted (PCT) and straight (PST) tubule segments of the rabbit kidney perfused in vitro. When bath bicarbonate concentration was reduced from 22 to 6.6 mM at a constant pH, transient depolarizations lasting several seconds with a peak value of approximately 15 mV were seen in both tubule segments. Acetazolamide (0.1 mM) in the lumen and bath solutions reduced the magnitude and increased the duration fo this response. The final pH of the bathing solution influenced both the peak height and steady-state values of the intracellular potential when bicarbonate concentration was reduced either with constant CO2 or with an increase in CO2. Reducing bath sodium concentration by replacement with either tetramethylammonium or N-methyl-D-glucamine resulted in a sustained depolarization of both PCT and PST cells. This response was inhibited by the addition of 10(-4) M 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS) in the bathing solution. By analogy with bicarbonate transport in rat and amphibian proximal tubules, these data suggest that bicarbonate exit across the basolateral membrane of the rabbit proximal tubule is electrogenic and coupled to sodium and that basolateral bicarbonate exit can be inhibited by both acetazolamide and SITS in the bathing solution.

Is 125I iothalamate an ideal marker for glomerular filtration?

The triiodinated angiographic contrast medium, iothalamate, has (usually labelled 125I) been used extensively as a marker for glomerular filtration. We have studied the renal handling of 125I iothalamate (IOT) in vivo and in vitro in several species. In renal cortical slices from chicken, rabbit, rat, and monkey, the tissue-to-medium ratio of IOT was twice that of 51Cr-EDTA (EDTA) at 37 degrees C; a difference that was abolished at 0 degree C and markedly reduced by added o-iodohippurate or iodipamide. In five chickens the steady-state renal clearance of IOT (CIOT) was twice (P less than 0.05) that of EDTA (CEDTA) or 3H inulin (C1); a difference that was abolished by administration of 100 mg/kg/hr of novobiocin, an organic anion transport inhibitor. CEDTA was similar to C1 before as well as after transport inhibition. Utilizing the Sperber technique the mean apparent tubular excretion fraction (ATEF) of IOT was 8%, while that of EDTA was 1% (P less than 0.01; N = 10). After novobiocin coinfusion (new steady-state) ATEFIOT was significantly reduced (P less than 0.01) and not different from that of EDTA (-1%). In the same animals the total urinary recovery of IOT was 84 and 57% (P less than 0.01) before and after novobiocin, respectively, while corresponding values for EDTA was unchanged by the inhibitor. In seven rats the renal extraction of IOT was reduced from 29 to 17% (P less than 0.05) by coinfusion of probenecid (5 mg/kg/hr). Corresponding extractions were 82 to 34% (P less than 0.005) and 22% (unchanged) for PAH and EDTA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermodynamical aspects of the determination of bicarbonate in urine.

Despite the fact that the composition of urine varies a lot during the day, this has essentially been neglected as a factor of importance in determinations of urine bicarbonate. The investigations reviewed in combination with an own study shows that qualitative and quantitative factors in urine composition impacts the solubility of carbon dioxide as well as the dissociation constant for the bicarbonate buffer system. These two "constants" are of outstanding importance in the determination of bicarbonate, using the total carbonic acid method as well as the carbon dioxide equilibration method. Nomograms are presented to quantify the influence of different urine compositions on the determinations of bicarbonate in final urine and tubular fluid.

The determination of ultrafiltrable calcium and magnesium in serum.

Ultrafiltrate of human serum was investigated in order to evaluate the serum content of calcium and magnesium. The acid and base concentrations and pH of the serum was altered through titration with HCl- or NaOH-solutions. The Pco2 was varied in the titrated serum using different carbon dioxide tensions. This was performed when serum was filtered in a recycling system. It is shown that the analysis of calcium and magnesium have to be done under anaerobic conditions or at standardized pH and Pco2 situations, as the concentrations vary with both pH and Pco2. The concentration ratio between ultrafiltrate and serum for calcium and magnesium was found to be 0.56 and 0.74 respectively at pH=7.41 and Pco2=40 mmHg.

Intracellular potassium activity in the rabbit proximal straight tubule.

Double-barreled liquid ion-exchanger microelectrodes were used to measure basolateral membrane potential (VBL) and intracellular potassium activity (aiK) in superficial proximal straight tubules (sPST) of the rabbit perfused in vitro. The mean +/- SE (number of cells in parentheses) value of VBL was -37.8 +/- 2.49 (20) vM and aiK was 48.6 +/- 2.27 (20) mM. The calculated Nernst equilibrium potential (EK) across the basolateral membrane was -68 mV. Lowering both potassium concentration to 0.1 mM reversibly decreased both VBL and aiK to -12.2 +/- 1.21 (19) mV and 11.3 +/- 1.29 (19) mM, respectively. Bath ouabain (10(-5) resulted in similar changes. These results demonstrate that intracellular potassium is actively accumulated in sPST perfused in vitro and that accumulation results primarily from Na-K-ATPase activity in the basolateral membrane. During recovery from low K bath, the temporal relationship between VBL and aiK and the effects of ouabain and high K bath on recovery are used to demonstrate directly electrogenic pumping. Lowering bath pH to 6.7 (HCO-3 = 5 mM) and the presence of 0.5 mM BaCl2 in the bath resulted in a large and rapid depolarization of VBL with little or no change in aiK. These results suggest that the response of VBL to both maneuvers is caused by a decrease in potassium permeability of the basolateral membrane.

Intracellular potentials in rabbit proximal tubules perfused in vitro.

Conventional microelectrodes were used to measure the basolateral membrane potential (VBL) in isolated perfused superficial proximal convoluted (sPCT) and superficial proximal straight (sPST) tubules of the rabbit kidney. Stable recordings for periods up to 2 h can be obtained. The mean +/- SE (n = number of cells) values of VBL were sPCT = -51.0 +/- 1.63 (24) and sPST = -47.0 +/- 0.97 (94) mV. Inhibitors of active transport, ouabain (10(-5) M) and low bath potassium (0.1 mM), caused a significant depolarization of VBL in sPST. In contrast, short-duration bath cooling (10 degrees C) had no significant effect. Removal of luminal glucose caused a larger hyperpolarization in sPCT (-13.9 +/- 1.77 (9) mV) than in sPST (-3.8 +/- 1.02 (5) mV). Removal of luminal glucose and alanine resulted in an even larger hyperpolarization of VBL in sPCT (-19.0 +/- 0.44 (6) mV). Perfusion of the lumen with a solution resembling late proximal tubular fluid in sPST resulted in hyperpolarization of VBL (-4.3 +/- 0.85 (4) mV). Reducing bath pH to 6.7 depolarized VBL (39.9 +/- 1.77 (13) mV). This effect can be associated with a decrease in the relative potassium permeability of the basolateral membrane. These results demonstrate the feasibility of using intracellular electrical measurements to determine both luminal and basolateral membrane characteristics in isolated proximal tubular segments.

A phenomenologic evaluation of CO2-diffusion restriction in kidney tubules studied in an artificial membrane system.

The chemical course in a multi-membrane system with interacting H+ and HCO3 ions has been described phenomenologically as an analogy of the neutralisation reaction between secreted H+ and filtered HCO-3 ions in the proximal tubules of the kidney. It was shown that the produced CO2 gave the highest PCO2 in the asymmetrically placed reaction centre, which favours a build-up of a high intratubular PCO2. The CO2 transport was dependent on the rate-limiting permeation of the reacting ions, and the permeation could be increased by the influence of solutions of macromolecules such as carbonic anhydrase, albumin and dextran.

PCO2 of the proximal tubular fluid and the efferent arteriolar blood in the rat kidney.

Recordings in vivo of the carbon dioxide tension of the proximal tubular fluid and of the efferent arteriolar blood were performed with PCO2 microelectrodes in the rat kidney. The buffer lines of the efferent arteriolar blood and systemic arterial blood were determined with an ultramicro equilibration system and the acid-base status of the systemic arterial blood was measured. The intratubular PCO2 was significantly higher than the PCO2 of the arterial blood, and the PCO2 of the efferent arteriolar blood was significantly lower than that of the arterial blood. The buffer capacity was higher and the bicarbonate concentration slightly lower for the efferent arteriolar blood than for the arterial blood. It is concluded that a PCO2 difference exists across the tubular wall and that the high intratubular PCO2 favours a chemical equilibrium of the carbonic acid-bicarbonate system in the proximal tubular fluid. It is supposed that the slightly lowered bicarbonate concentration in the efferent arteriolar blood is an effect of the glomerular ultrafiltration process.

CO2 along the proximal tubules in the rat kidney.

The proximal intratubular pH of the rat kidney was measured in vivo with an antimony electrode system. PCO2 and bicarbonate concentration of the proximal tubular fluid were determined with an ultramicro equilibration system. The tubular fluid to plasma inulin concentration ratio was evaluated by a microscope fluorometric method. The acid-base parameters and the inulin concentrations were determined under control conditions and during acetazolamide treatment. The intratubular PCO2 was higher than the PCO2 of the systemic arterial blood under control conditions and the difference in PCO2 was increased during acetazolamide treatment. In acetazolamide treated rats the rate of fractional bicarbonate reabsorption was decreased in the early part of the proximal tubule, while it was of about the same in the middle and late parts as compared with control rats. The total bicarbonate reabsorption in the proximal tubule was reduced by 50% due to the carbonic anhydrase inhibition. It seems possible that the bicarbonate is still reabsorbed as CO2 after carbonic anhydrase inhibition, as hydrogen ion secretion is not totally stopped by this treatment. The increase in intratubular PCO2 after acetazolamide treatment is assumed to be due to an inhibition of the carbonic anhydrase facilitating effect on outward diffusion of CO2 from the tubular lumen across the cell wall.

Electrochemical forces for chloride transport in the proximal tubules of the rat kidney.

The electrochemical forces for chloride transport in the proximal tubule of the rat kidney were studied using micropuncture techniques. Electrical transmembrane potentials were recorded in randomly punctured tubules with Ling-Gerhard electrodes. Chloride activities in the luminal, cellular and interstitial compartments were measured with ion selective micro-electrodes. Electrical potential measurements between cell to interstitium and lumen to interstitium were -72.1 +/- 2.6 mV and +0.5 +/- 1.4 mV (mean +/- S.D.) respectively. The calculated chloride concentrations for lumen, cell and interstitium were 133.0 +/- 10.3 mM, 8.5 +/- 1.0 mM and 99.1 +/- 3.2 mM (mean +/- S.D.) respectively. The net electrochemical forces, qualitatively, offer a passive chloride ion pathway through the tubular wall and a chloride equilibrium over the luminal membrane seems to exist.