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PubMed1997

Trimetazidine prevents renal injury in the isolated perfused pig kidney exposed to prolonged cold ischemia.

BACKGROUND: Ischemia caused by cold storage (CS) and reperfusion of the kidney is often responsible for delayed graft function after transplantation. Significant attention has been focused on the cascade of events involved in ischemia-reperfusion injury, with the objective of identifying drugs to ameliorate the functional damage that occurs. METHODS: The purpose of this study was to evaluate the renal function of isolated perfused pig kidneys after 48 hr of CS with Euro-Collins (EC) solution plus trimetazidine (EC+TMZ), standard EC solution, or University of Wisconsin (UW) solution. Normothermic isolated perfused pig kidneys were randomized into five experimental groups: (A) control group (cold flush with cold heparinized saline and immediately reperfused; n=6); (B) cold flush with cold heparinized saline with TMZ (10(-6) M), n=6; (C) 48 hr of CS with EC and reperfusion (n=8); (D) 48 hr of CS with EC+TMZ alone and reperfusion (n=8); (E) 48 hr of CS with UW and reperfusion (n=8). Proton nuclear magnetic resonance spectroscopy and biochemical studies were performed for the functional evaluation during reperfusion. Lipid peroxidation was also determined. Histological examination (optical and electron microscopy) was performed after CS and reperfusion. RESULTS: Using TMZ, the renal perfusate flow rate as well as the glomerular filtration rate and proximal tubular function were significantly improved. This improvement of renal function during reperfusion was correlated with a less significant cellular and interstitial edema. In addition, tubular injury markers were significantly lower in the group preserved with EC+TMZ, and TMZ reduced lipid peroxidation dramatically during reperfusion. CONCLUSIONS: The addition of TMZ to the EC solution increased the preservation quality and renal tubular function, and gave protection from reperfusion injury better than EC alone or UW. These results strongly suggest that TMZ has a cytoprotective effect and may therefore be useful for kidney preservation.

Adenosine↗
PubMedDate not supplied

31P nuclear magnetic resonance and saturation transfer studies of the isolated perfused rat kidney.

Previous studies using 31P nuclear magnetic resonance (NMR) saturation transfer techniques to quantitate the energy metabolism of the kidney have often resulted in estimates of adenosine triphosphate (ATP) turnover which are much lower than those predicted from the renal oxygen consumption and reasonable values of the P/O ratio. We measured the ATP turnover in isolated perfused kidneys of rats, using 31P NMR saturation transfer and a new procedure for quantitation of the intracellular Pi concentration. The estimated turnover rates of ATP were higher than previously reported. The P/O ratios calculated on the basis of these rates of ATP turnover and rates of renal oxygen consumption reported in the literature were within the range of theoretically possible values. Thus, 31P NMR saturation transfer can be used to quantitate the ATP turnover in the isolated perfused rat kidney.

Adenosine Triphosphate↗
PubMed1988

Moment analysis of drug disposition in kidney: transcellular transport kinetics of p-aminohippurate in the isolated perfused rat kidney.

The mean renal epithelial cell residence time, Tcell, was defined as a model-independent characteristic of the transcellular transport process in the isolated perfused kidney. The transcellular transport process includes transport at the basolateral membrane, diffusion in the cytosol, and transport at the brush border membrane. The parameter Tcell represents the mean time for the drug secreted from the tubules to pass through the renal epithelial cells, and is calculated as the difference of the mean urinary transit time between secreted drug and inulin in the single-pass perfusion system. Therefore, the urinary excretion rate-time course is indispensable to evaluate Tcell. p-Amino-hippuric acid was used as a model compound. The bovine erythrocytes in the perfusate kept the isolated kidney in an almost constant physiological condition, including secretion function. The renal vein outflow curves were also analyzed by the use of moments. The dispersion in the catheter was corrected by a deconvolution. The apparent secretion intrinsic clearance and the apparent volume of distribution were calculated from the moments. The present method will be useful for analysis of the transcellular transport mechanism and the effect of disease states on renal transport of drugs.

Aminohippuric Acids↗
PubMed1997

[Discordance of increase in stress proteins in right and left myocardium of perfused isolated rat heart].

Discordance of functional and structural changes in right and left myocardium has been considerably discovered in various physiologically stimulated hearts and patients with cardiopathy, but the reasons for that have not yet been known. In the present study, induction of stress proteins, i.e. heat shock proteins, was analyzed and compared between the left and right myocardium of normoxic and hypoxic perfused isolated rat hearts by the methods of two dimensional electrophoresis and silver staining. The results showed that three Hsp70 isoforms (molecular weight 68, 70 and 72 k mu) with pI ranging from 6.3 to 7.3 were increased distinctly in both right and left myocardium under the conditions of perfusion as mentioned above. Moreover, the amount of the stress proteins increased in the right myocardiums was higher than that in the left myocardium, indicating that the response of right heart to the stimulus of hypoxic or normoxic perfusions is different from the reaction of left heart, and the extent of protection in left and right heart by stress proteins is unlike. In addition, the activity of catalase was found to be obviously declined in all perfused isolated hearts. It was suggested that the different increase in stress proteins may be due to different structure and status of right and left ventricle and the oxidative stress may be one of the important reasons to induce or enhance the synthesis of stress proteins.

Animals↗
PubMed1996

The lysine-1 analog of guanylin induces intestinal secretion and natriuresis in the isolated perfused kidney.

Guanylin is an endogenous peptide synthesized by several mammalian species that mimics the effects of a thermostable enterotoxin of Escherichia coli (STa: NTFYCCELCCNPACAGCY) in the gut. We have cloned a lysine-1 derivative of rat guanylin (Lys-1-NTCEICAYAACTGC) and tested its effects on ileal tissue membranes in Ussing chambers and in the isolated perfused rat kidney. Rabbit ileal mucosa membranes were mounted into a Ussing chamber and the effects of Lys-1 guanylin (Lys-1 G) and STa enterotoxin peptide on chloride secretion were determined by changes in short-circuit current (Isc). Lys-1 G (10 to 100 nM) showed a dose-dependent effect on chloride secretion with a maximal response estimated to be 52 microA/cm2. Lys-1 G mimics the effect of STa peptide, but the enterotoxin elicited a greater maximal effect of 120 microA/cm2 (P < 0.01). Lys-1 G (2.5 micrograms/ml) promoted an increase in both urine flow (from 0.13 +/- 0.07 to 0.40 +/- 0.01 ml g-1 min-1, N = 4; P < 0.05) and glomerular filtration rate (from 0.68 +/- 0.02 to 0.85 +/- 0.00 ml g-1 min-1, N = 4; P < 0.01) in the isolated perfused kidney and a reduction of the fractional reabsorption of sodium (from 76.0 +/- 0.03 to 59.5 +/- 0.85%, N = 4; P < 0.01). These maximal effects were accompanied by intense natriuretic effect observed 30 and 60 min after drug administration. The Lys-1 G analog similar to STa enterotoxin elicited intestinal chloride secretion and a natriuretic effect. These data demonstrate that the cloned peptide analog retains the biological activity of the native hormone and presents activity similar to STa. The properties of Lys-1 G resemble those of a factor formed during perfusion of the hypoxic rabbit kidney and named by us factor natriureticus similis (FNS).

Animals↗
PubMed1992

Effects of R 56865 and phenytoin on mechanical, biochemical, and morphologic changes during ouabain intoxication in isolated perfused rabbit heart.

The Na+/Ca2+ overload inhibitor R 56865 (N-[1-[4-(4-fluorophenoxy)-butyl]-4-piperidinyl)-N-methyl-2- benzothiazolamine) has been reported to prevent or attenuate ischemia- as well as ouabain-induced cellular sodium and calcium load. We investigated the potency of this compound in preventing mechanical, biochemical, and ultrastructural consequences of ouabain (OUA) intoxication in isolated rabbit heart. The protective effect of the digitalis antidote phenytoin (PHT) on the consequences of ouabain intoxication was examined for comparison. In isolated perfused rabbit heart, OUA (0.4 microM) caused an increase in left ventricular end-diastolic pressure (LVEDP) that was accompanied by depletion of high-energy phosphates (80% less than in control), accumulation of tissue lactate (12-fold) and damage of contractile elements and mitochondria. Accumulation of lactate was associated with a decrease in oxygen consumption by the isolated perfused heart. R 56865 (1.0 microM) and phenytoin (60 microM) prevented increase in LVEDP, breakdown of the energy-rich phosphates creatine phosphate (CrP) and ATP, accumulation of lactate, and morphologic changes induced by OUA. The above-mentioned toxic effects of OUA are interpreted as consequences of mitochondrial failure finally leading to breakdown of the oxidative phosphorylation. Thus, we conclude that the protective action of both compounds, R56865 and PHT, may be attributed to prevention or attenuation of mitochondrial failure due to OUA-induced disturbance of ion homeostasis.

Adenosine Triphosphate↗
PubMedDate not supplied

Clara cell 10-KDA protein inhibits endotoxin-induced airway contraction in isolated perfused rat lungs.

Clara cell 10-kDa protein (CC10) is a major component of bronchoalveolar lavage fluid and is suggested to be a natural regulator of airway inflammation, possibly through its effects on the proinflammatory enzyme(s), phospholipase A2. We examined the effect of recombinant human (rh) CC10 on endotoxin-induced airway contraction and cytokine release in isolated perfused rat lungs. We found that rhCC10 added to the lung perfusate abolished the endotoxin-induced airway contraction, and that it inhibited both the release of interleukin-1 beta and interleukin-6 into the lung perfusate and the release of tumor necrosis factor-alpha into the pulmonary lavage fluid. By contrast, the levels of interferon-gamma were unaffected by CC10 administration. Rutin, a phospholipase A2 inhibitor, and N omega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, also attenuated the contraction induced by endotoxin. These findings demonstrate that rhCC10 inhibits endotoxin-induced airway contraction and the release of proinflammatory cytokines (interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha) in isolated perfused rat lungs. The results also indicate that phospholipase A2 and nitric oxide are involved in the airway contraction in this model, possibly through their influence on the production of eicosanoids.

Acetylcholine↗
PubMed1994

Effects of hexoses and their derivatives on glucagon secretion from isolated perfused rat pancreas.

Although it has been firmly established that D-glucose inhibits glucagon secretion from pancreatic A cells, the regulatory mechanism of glucagon secretion by D-glucose has not been elucidated. To study this regulatory mechanism by D-glucose, the effects of hexoses and their derivatives on glucagon secretion from the A cells of isolated perfused rat pancreas were investigated. When these cells were perfused with D-glucose, D-fructose, D-sorbitol, D-galactose, 2-deoxy-D-glucose, D-gluconic acid sodium salt and D-glucosamine HCl salt, glucagon secretion was significantly inhibited. None of the hexoses or their derivatives tested were found to stimulate glucagon secretion. The effects of these sugars on glucagon secretion were independent of their metabolism in the cells. From the findings that the sugars both metabolized and unmetabolized in the cells demonstrated comparable inhibition of glucagon secretion from the isolated perfused rat pancreas, it is speculated that the recognition system for these sugars may be probably present on the A cell membrane and responsible for mediating these inhibitory effects of glucagon secretion.

Animals↗
PubMed1982

Metabolism of acetaminophen by the isolated perfused kidney.

Acetaminophen (APAP) produces proximal tubular necrosis in the Fisher 344 rat. This lesion may result from the covalent binding of reactive intermediates of APAP to cellular macromolecules when glutathione (GSH) is sufficiently depleted. Experiments were designed to evaluate the ability of the kidney to convert APAP to reactive electrophilic metabolites capable of depleting renal GSH by quantifying GSH concentrations in isolated perfused kidneys perfused with APAP. Perfusion without APAP reduced (3 X 10(-5) -3 X 10(-5) M) to the perfusion medium further reduced renal GSH content. Treatment of rats with polybrominated biphenyls enhanced the ability of 3 X 10(-8) M APAP to deplete GSH. In contrast, treatment with piperonyl butoxide reduced the depletion of GSH produced by 3 X 10(-5) M APAP. At 3 X 10(-5) M APAP, the glucuronic acid, sulfate and the mercapturic acid conjugates were excreted by the isolate perfused kidneys. After treatment with polybrominated biphenyls, mercapturic acid excretion increased 4-fold, whereas the glucuronic acid and sulfate conjugate excretions were unaffected. These data suggest that the kidney can produce an electrophilic metabolite of APAP which can combine with and deplete renal GSH. An electrophilic metabolite of APAP produced by the kidney may initiate APAP induced renal necrosis.

Acetaminophen↗
PubMed1981

Effect of phenobarbital pretreatment on benzene biotransformation in the rat. II. 9,000 g supernatant and isolated perfused liver versus living rat.

Factors responsible for different quantitative effect of phenobarbital (PB) pretreatment (sodium phenobarbital, 50 mg kg-1 day-1 for 3 days) on benzene metabolism to phenol in vivo and in vitro were studied in male Wistar rats. A more than 4-fold increase of benzene metabolism was observed wih 9,000 g supernatant of liver homogenate, 2.8- to 4-fold increase with isolated perfused liver; phenol formation in vivo after oral benzene was increased by PB 2-fold, but only shortly following benzene administration and the enhancement rapidly diminished to 1.15-fold increase in the total excreted phenol. Benzene concentrations i 9,000 g supernatant incubations were 2 mM, those with isolated perfused livers were up to 4 mM, but those in blood in vivo were below 0.3 mM; the effect of PB induction in vivo disappeared along with decreasing benzene and increasing phenol blood concentrations which surpassed benzene 2-3 h after oral benzene administration. The effect of benzene concentration on the manifestation of PB induction is also supported by almost a 2-fold increased phenol formation in PB rats over controls in vivo after repeated administration of benzene. The elimination of radioactive metabolites of orally administered benzene-14C, 3 mmoles kg-1, in urine was markedly inhibited by intraperitoneal administration of phenol (1.2 mmol kg-1), but not by pyrocatechol, resorcinol or hydroquinol (0.6 mmole kg-1, respectively) suggesting that phenol might inhibit benzene metabolism in vivo especially when its concentration exceeds that of benzene.

Animals↗
PubMed1975

Bicarbonate and sodium reabsorption by the isolated perfused kidney.

The role of bicarbonate reabsorption and of transtubular chloride gradients in the bulk reabsorption of sodium and water by renal tubules can be tested in the isolated perfused kidney by perfusing with a medium from which bicarbonate has been omitted. Perfusion of the isolated rat kidney with an artificial medium in which bicarbonate is replaced by chloride results in a fall in fractional reabsorption of sodium from 97 to 84%. Stepwise restoration of bicarbonate concentration in the perfusion medium to 25 meq/liter is associated with a parallel recovery of sodium reabsorption to control levels. Inhibition of bicarbonate reabsorption with acetazolamide produces a slightly smaller reduction in sodium reabsorption (97-89%), an effect not seen in the absence of bicarbonate. Acetazolamide greatly increases phosphate excretion and free water clearance in a way consistent with suppression of proximal tubular reabsorption. By contrast, simple omission of bicarbonate from the perfusing medium does not alter phosphaturia or free water clearance. Reabsorption of bicarbonate appears to account for a fraction of sodium reabsorption roughly equivalent to the proportion of sodium associated stoichiometrically with bicarbonate in the glomerule filtrate. The data do not support the hypothesis that the development of a transtubular chloride gradient is critically important for the reabsorption of a large fraction of the glomerular filtrate.

Acetazolamide↗
PubMed2003

S-nitrosothiol detection in isolated perfused rat heart.

Nitrogen monoxide (NO) has important cardiovascular actions, and it has been suggested that they may be partly mediated by the reaction with protein sulfhydryl groups to produce S-nitrosothiols. In this work we describe and test a method that allows S-nitrosothiol detection in crude membrane preparations obtained from isolated perfused rat hearts. Isolated rat hearts were perfused under control conditions or in the presence of the NO donors SIN-1 and isosorbide dinitrate. Additional hearts were subjected to 10-20 min of ischemia followed or not by 10-20 min of reperfusion. At the end of perfusion a crude membrane fraction was prepared, and S-nitrosothiol concentration was assayed fluorometrically, on the basis of 2,3-naphthotriazole production from 2,3-diaminonaphthylene. The sensitivity of the method, as evaluated using S-nitrosoalbumin, was on the order of 1-2 pmol/mg of protein. S-nitrosothiols were undetectable under control conditions, as well as after ischemia or ischemia-reperfusion. On the other hand, significant S-nitrosothiol formation was observed after infusion of SIN-1 or isosorbide dinitrate (26.4 +/- 7.4 and 19.9 +/- 5.6 pmol per mg of protein, respectively). In conclusion, S-nitrosothiol production was observed in rat heart membranes after exposure to NO donors, while S-nitrosothiol concentration was below the sensitivity limits of the assay either under baseline conditions or after acute ischemia and reperfusion.

Animals↗
PubMed2002

Dose optimization of a doxorubicin prodrug (HMR 1826) in isolated perfused human lungs: low tumor pH promotes prodrug activation by beta-glucuronidase.

HMR 1826 (N-[4-beta-Glucuronyl-3-nitrobenzyl-oxycarbonyl]doxorubicin) is a nontoxic glucuronide prodrug from which active doxorubicin is released by beta-glucuronidase. Preclinical studies aimed at dose optimization of HMR 1826, based on intratumoral pharmacokinetics, are important to design clinical studies. Using an isolated perfused human lung model, the uptake of doxorubicin into normal tissue and tumors after perfusion with 133 microg/ml (n = 6), 400 microg/ml (n = 10), and 1200 microg/ml (n = 6) HMR 1826 was compared. Extracellular tissue pH was measured, and enzyme kinetic studies were performed in vitro to investigate the effect of pH on the formation of doxorubicin. Extracellular pH was lower in tumors than in healthy tissue (6.46 +/- 0.35, n = 8 versus 7.30 +/- 0.33, n = 10; p < 0.001). In vitro, beta-glucuronidase activity was 10 times higher at pH 6.0 than at neutral pH. After perfusion with HMR 1826, there was a linear relationship between HMR 1826 concentrations in perfusate and normal lung tissue. After perfusion with 133, 400, and 1200 microg/ml HMR 1826, the final doxorubicin concentrations in normal and tumor tissue were 2.7 +/- 0.9, 11.1 +/- 5.4, and 21.8 +/- 8.4 microg/g (p < 0.05 for all comparisons), and 0.7 +/- 0.3, 8.6 +/- 2.0 microg/g (p < 0.01 versus 133 microg/g), and 8.7 +/- 4.9 microg/g, respectively. This agrees with the enzyme kinetic observations of saturation of beta-glucuronidase at 400 microg/ml HMR 1826 in the acidic environment of the tumor. Therefore, the escalation of the HMR 1826 dose most likely results in higher circulating concentrations than 400 microg/ml but does not increase the uptake of doxorubicin into tumors and, subsequently, antitumor efficacy. The isolated perfused human lung is an excellent model for preclinical investigations aimed at optimization of tissue pharmacokinetics of tumor-selective prodrugs.

Adult↗
PubMed1989

Furazolidone-induced injury in the isolated perfused chicken heart.

The Langendorff isolated heart preparation was adapted to determine the effect of furazolidone (0.5 and 2 micrograms/ml of perfusate) on hearts of 3-week-old broiler chickens. Following 115 minutes of perfusion, both concentrations of furazolidone caused approximately a two-fold increase in myocardial vascular resistance and a six-fold increase in lactate dehydrogenase release into the effluent fluid, compared with a control perfused group of isolated hearts (P less than 0.01). Ultrastructural alteration differences were not found between the drug-treated and control groups. It was concluded that: (i) furazolidone, at concentrations only moderately above therapeutic plasma concentrations, caused detrimental changes in myocardial vascular resistance and lactate dehydrogenase release and (ii) the isolated chicken heart preparation is an example of a cost-effective, reliable laboratory tool for screening potential cardiotoxins.

Animals↗
PubMed2004

Metabolism of apigenin by rat liver phase I and phase ii enzymes and by isolated perfused rat liver.

The metabolism of apigenin, a low estrogenic flavonoid phytochemical, was investigated in rat using liver models both in vitro (subcellular fractions) and ex vivo (isolated perfused liver). In vitro, phase I metabolism led to the formation of three monohydroxylated derivatives: luteolin which was the major metabolite (K(m) = 22.5 +/- 1.5 microM; V(max) = 5.605 +/- 0.090 nmol/min/mg protein, means +/- S.E.M.), scutellarein, and iso-scutellarein. These oxidative pathways were mediated by cytochrome P450 monooxygenases (P450s). The use of P450 inhibitors and inducers showed that CYP1A1, CYP2B, and CYP2E1 are involved. In vitro studies of phase II metabolism indicated that apigenin underwent conjugation giving three monoglucuronoconjugates and one monosulfoconjugate. Luteolin led to the formation of four monoglucuronoconjugates, two sulfoconjugates, and one methylconjugate identified as diosmetin. Ex vivo during the apigenin perfusion of an isolated rat liver, none of the phase I metabolites could be recovered. In contrast, two monoglucuronoconjugates and one of the sulfoconjugates of apigenin already identified in vitro were recovered. Moreover, two new derivatives were isolated and identified as a diglucuronoconjugate and a glucuronosulfoconjugate. This work provides new data about the metabolism of apigenin and shows the interest value of using various experimental models in metabolic studies.

Animals↗
PubMed1998

Leukotoxin (9, 10-epoxy-12-octadecenoate) impairs energy and redox state of isolated perfused rat lung.

We investigated the perturbation of energy balance and redox state in leukotoxin (9, 10-epoxy-12octadecenoate) (Lx)- and endothelin-1 (ET-1)-induced lung injury, using isolated perfused rat lungs. To examine any relationship between these parameters, intracellular levels of adenine nucleotides, pyridine coenzymes and glutathione were determined by reversed-phase high-performance liquid chromatography (HPLC) in the freeze-dried tissues of isolated rat lungs. The tissue samples were perfused with a physiological salt solution containing either Lx only, Lx plus NG-monomethyl-L-arginine (L-NMMA), Lx plus NG-monomethyl-D-arginine (D-NMMA), Lx plus superoxide dismutase (SOD) or ET-1 only. In isolated perfused lung tissue, 10 mol of Lx caused permeability-increased lung injury, and 10 nM of ET-1, which caused a comparable increase in wet lung weight, evoked pulmonary capillary hypertensive lung injury. Lx-injured lungs showed decreases in the contents of ATP, NADPH, NADH, reduced glutathione (GSH), (2ATP + ADP)/2(ATP + ADP + AMP) ratio (energy charge) and NADH/NAD+ ratio, and increased the contents of ADP and AMP compared with the vehicle control and ET-1-injured lungs. Such effects of Lx were significantly attenuated by pretreatment with 0.4 mM L-NMMA or 500 units/ml of SOD, but not with 0.4 mM D-NMMA. On the other hand, the ET-1-injured lung evidenced decreased tissue GSH. These findings indicate that Lx shifted the lung redox state toward oxidation and that Lx-induced lung injury was involved in the imbalance of the energy and redox state via production of nitric oxide and/or superoxide anion.

Adenine Nucleotides↗
PubMed1996

An in vitro-in vivo validation of the isolated perfused tumor and skin flap preparation as a model of cisplatin delivery to tumors.

The isolated perfused tumor and skin flap (IPTSF) is a unique model system in which drug disposition is evaluated in tumor tissue and surrounding normal tissue, both of which are supplied by the same vascular system. We compared tissue Pt concentrations obtained following systemic administration of cisplatin (CDDP) to whole pigs bearing tumored skin flaps with data obtained from IPTSF treated similarly. During the in vivo study, CDDP was administered intravenously to six pigs that had tumor and skin flaps. IPTSF were created in four pigs and isolated in a perfusion chamber and perfused with medium containing CDDP for 180 min. Venous plasma or perfusate samples were serially collected throughout perfusion. Tissue samples were collected after perfusion was complete. All samples were assayed for Pt by atomic absorption spectroscopy. Area under the curve of Pt profiles from IPTSF and in vivo perfused flaps were not significantly different. Pt concentrations were significantly higher in tumor samples from in vivo perfused flaps than in samples from IPTSF. Pt concentrations in skin and subcutaneous tissue were not significantly different. When consideration is given to all of the potential variables that were operative in these experiments, the results of this study demonstrate that Pt distribution within the IPTSF was comparable to that obtained in vivo.

Animals↗
PubMedDate not supplied

Inositol-1,4,5-trisphosphate mass content in isolated perfused rat heart during alpha-1-adrenoceptor stimulation.

Inositol-1,4,5-trisphosphate (IP3) has been proposed to be a second messenger in response to alpha-1-adrenoceptor stimulation also in myocardial cells. We studied the effect of alpha-1-adrenoceptor stimulation (5 x 10(-5) mol/l phenylephrine or 5 x 10(-5) mol/l noradrenaline both in the presence of 10(-6) mol/l timolol) on IP3 mass content in isolated perfused rat hearts. IP3 content was determined by a specific receptor-binding assay-kit (TRK 1000, Amersham) after validating the method. For comparison also the effect of muscarinic stimulation (10(-4) mol/l carbachol in the presence of 10(-6) mol/l timolol) on IP3 content was measured in corresponding preparations. A basal IP3 level of about 75 pmol/mg protein was found. There were no prominent effects of alpha-1-adrenoceptor stimulation on total IP3 content in isolated perfused rat hearts. Phenylephrine gave a statistically significant increase of about 40% at 1/4 min and a statistically significant decrease of about 25% at 4 min after start of exposure. Noradrenaline, however, gave no statistically significant change of IP3 at the time-points studied. Muscarinic stimulation caused a slight, statistically insignificant, increase of IP3 at 1/4 min. The results are compatible with an assumption that agonist stimulation evokes a localized increase of IP3 which may be masked by a relatively high total IP3 mass content. The IP3 peak after phenylephrine coincided with the early positive inotropic phase of the response reported earlier in perfused rat hearts for alpha-1-adrenoceptor stimulation by phenylephrine. Although this might be compatible with a role for IP3 in this early and transient phase, a mediator function of IP3 in the inotropic response is not established.

Animals↗