Ionic dependency of membrane potential and autorhythmicity in the atrium of the whelk Busycon canaliculatum

• 1.1. Calcium-free media usually caused a cessation of all electrical and mechanical activity of the Busycon atrium. Where any electrical activity survived, the action potential consisted of a pre-and plateau-like potential devoid of the usual terminal spike.
• 2.2. High Ca salines induced tonic force, membrane depolarization and reduction in generation of spontaneous action potentials. The Ca ionophore A23187 enhanced contractions and the SR CaATP-ase inhibitor cyclopiazonic acid induced slight depolarization, tonic contractures and increased action potential firing.
• 3.3. The inorganic Ca antagonist Co²⁺ was without effect on the preparations, although the lanthanide Gd³⁺ inhibited contractions and spontaneous action potentials as well as inducing membrane potential depolarization.
• 4.4. The organic Ca entry-blocker nifedipine enhanced both spontaneous action potential amplitude and the phasic contractions they generated.
• 5.5. High K salines considerably depolarized atrial preparations with accompanying large tonic contractures and suppression of action potentials. The K channel-blocker 4AP enhanced action potential amplitude with slight increase in contractions, and TEA depolarized the atrium, and enhanced action potentials and rhythmic contractions.
• 6.6. Sodium-free salines strongly hyperpolarized atrial preparations and abolished spontaneous action potentials and, on washout, the membrane potential became temporarily unstable. In 2 preparations, low chloride and chloride-free media induced significant membrane potential hyperpolarization.
• 7.7. It is concluded that, in the atrium, the resting membrane potential is largely determined by the transmembrane K gradient, but with significant conductances to Na and C1 though probably not Ca. The action potential spike appears to be a Ca-dependent event and the plateau-like phase may be a Na-dependent event.

     

GABA affects the glutamate receptor-chloride channel complex in mechanically isolated and internally perfused Aplysia neurons.

The effects of gamma-aminobutyric acid (GABA) on the glutamate receptor chloride ion (Cl-) channel complex were examined in mechanically isolated and internally perfused Aplysia neurons using a concentration clamp technique. GABA at concentrations of 3 x 10(-6) M or more, concentration dependently delayed the recovery of the glutamate response from desensitization. This effect was independent of the GABA response and Cl- redistribution. Muscimol (10(-4) M) mimicked the effect of GABA. However, this was not the case for baclofen (10(-3) M). In some isolated neurons, GABA at concentrations of more than 10(-4) M clearly induced an additional Cl- current, the current kinetics of which were different from those induced by lower concentrations of GABA. Even in the continued presence of 10(-4) M GABA, which desensitized the fast GABA response, higher concentrations of GABA (3 x 10(-4) M to 10(-2) M) elicited the additional current in a concentration-dependent manner. The presence of 10(-4) M glutamate completely abolished this current, indicating cross-desensitization between the glutamate and slow GABA responses. High concentrations of GABA (3 x 10(-2) M) did not activate the glutamate receptor coupled to the large cation channel. The results suggest that, in Aplysia neurons, the glutamate receptor-Cl- channel complex has some similarities to the GABA receptor-Cl- channel complex.     

Modulatory effect of neuropeptide Y on acetylcholine-induced oedema and vasoconstriction in isolated perfused lungs of rabbit.

1. The modulatory role of neuropeptide Y (NPY) on pulmonary oedema induced by acetylcholine and capsaicin was investigated. The effects of NPY on the haemodynamic response to acetylcholine, phenylephrine and substance P were also investigated. 2. Isolated, ventilated, exsanguinated lungs of the rabbit were perfused with a constant flow of recirculating blood-free perfusate. The double/arterial/venous occlusion method was used to partition the total pressure gradient (delta Pt) into four components: the arterial gradient (delta Pa), the pre- and post-capillary gradients (respectively delta Pa' and delta Pv') and the venous pressure gradient (delta Pv). Endothelial permeability was evaluated by measuring the capillary filtration coefficient (Kf,c). 3. Acetylcholine (10(-8) M to 10(-4) M) and substance P (SP, 10(-10) M to 10(-6) M) induced a concentration-dependent increase in the Kf,c. Capsaicin (10(-4) M) and 5-hydroxytryptamine (5-HT) (10(-4) M) also increased this parameter. NPY (10(-8) M) completely inhibited the effects of acetylcholine and capsaicin on the Kf,c, without preventing the effects of substance P and 5-HT. 4. Acetylcholine induced concentration-dependent vasoconstriction in the precapillary segment. The effect was inhibited by NPY and aspirin, an inhibitor of cyclo-oxygenase, while ketanserin, a 5-HT2 receptor antagonist, and SR140333, a new NK1 antagonist, had no protective effect. Phenylephrine increased delta Pa at high concentration, an effect also inhibited by NPY and aspirin. Substance P had no significant haemodynamic effect. When injected together with NPY, substance P (10(-6) M) induced a significant increase in the total pressure gradient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Applicability of the isolated perfused rat brain for studying central cholinergic mechanisms

Summary The ACh content of the isolated perfused rat brain as well as that of the rat brain in vivo was determined by gas chromatography. After a perfusion period of 30 min the endogenous ACh content of the isolated brain was significantly higher than that of the brain in vivo. Physostigmine caused a rise in the ACh concentration of both the isolated brain and the brain in vivo. Oxotremorine produced an increase in the ACh content in the brain in vivo, but not in the isolated brain after appropriate dosage.The concentration of the drugs in the perfusion medium of the isolated brain was so that distinct EEG changes could be observed but spontaneous electrical activity could be maintained during the whole perfusion period.     

Renovascular actions of adenosine in the isolated perfused rat kidney: possible underlying mechanisms

We studied the renovascular action of adenosine on isolated perfused rat 10 min after drug injections. Adenosine was applied intraarterially as a single bolus injection in logarithmically increasing doses (0.3-30 microg). Adenosine treatment induced a biphasic vascular-response, namely, an initial vasoconstriction followed by a long-lasting vasodilation. Pretreatment with 0.1. 0.3, or 1.0 mM theophylline or quinidine (2 microg/ml) significantly depressed both components of the adenosine response. The vasoconstrictor response to adenosine was not affected by either 0.5 or 1.0 microg/ml dihydroergocristine. whereas the vasodilatory response was dose-dependently reduced. The biphasic response to adenosine was markedly depressed by 10 microg/ml indomethacin and was augmented by combining this agent with quinidine. We studied the possible roles of the platelet activating factor (PAF) and nitric oxide-cGMP systems in the renovascular actions of adenosine. Tebokan (a PAF antagonist) antagonized both components of the response, but methylene blue (MM) reduced only the pressory part Electron-microscopic examination of kidneys exposed for 15 min to MM showed some acute degenerative alterations and constriction in the glomeruli. From these findings, we conclude that the P1/A1, and P2x purinoceptors, the prostaglandins, PAF, and the NO-cGMP systems have a share in the renovascular actions of adenosine.     

Potential mechanisms involved in the absorptive transport of cadmium in isolated perfused rabbit renal proximal tubules

Lumen-to-cell transport, cellular accumulation, and toxicity of cadmium as ionic cadmium (Cd²⁺) or as the l-cysteine (Cys) or d,l-homocysteine (Hcy) S-conjugate of cadmium (Cys-S-Cd-S-Cys, Hcy-S-Cd-S-Hcy) were studied in isolated, perfused rabbit proximal tubular segments. When Cd²⁺ (0.73 μM) or Cys-S-Cd-S-Cys (0.73 μM) was perfused through the lumen of S₂ segments of the proximal tubule, no visual evidence of cellular pathological changes was detected during 30 min of study. Cd²⁺-transport was temperature-dependent and was inhibited by Fe²⁺, Zn²⁺, and elevated concentrations of Ca²⁺. Luminal uptake of Cys-S-Cd-S-Cys was also temperature-dependent and was inhibited by the amino acids l-cystine and l-arginine, while stimulated by l-methionine. Neither l-aspartate, l-glutamate, the synthetic dipeptide, Gly-Sar nor Zn²⁺ had any effect on the rate of Cys-S-Cd-S-Cys transport. Conclusions: When delivered to the luminal compartment, Cd²⁺ appears to be capable of utilizing certain transporter(s) of Zn²⁺ and some transport systems sensitive to Ca²⁺ and Fe²⁺. In addition, Cys-S-Cd-S-Cys and Hcy-S-Cd-S-Hcy appear to be transportable substrates of one or more amino acid transporters participating in luminal absorption of the amino acid l-cystine (such as system b^0,+). These findings indicate that multiple mechanisms could be involved in the luminal absorption of cadmium (Cd) in proximal tubular segments depending on its form. These findings provide a focus for future studies of Cd absorption in the proximal tubule.     

Renal excretion mechanisms of the antiviral nucleoside analogue AM 188 in the rat isolated perfused kidney

1. AM 188 is an antiviral guanosine analogue that undergoes extensive renal excretion in humans. The present study was designed to investigate the disposition of AM 188 over a range of concentrations in the rat isolated perfused kidney (IPK) to explore the mechanisms involved in its renal handling. 2. Right kidneys of male Sprague-Dawley rats (n = 23) were isolated and perfused in recirculating mode with Krebs'-Henseleit (pH 7.4) buffer containing 0.65% bovine serum albumin, 3.6% dextran, amino acids and glucose. [14C]-Inulin was added to the perfusate reservoir to permit estimation of glomerular filtration rate (GFR). [3H]-AM 188 and unlabelled AM 188 were added to the perfusate as a bolus initially, followed by a constant rate of infusion at 5, 25, 125, 500 or 1000 microg/min to achieve initial target perfusate concentrations of 1, 5, 25, 100 or 200 microg/mL, respectively. During the 130 min over which AM 188 was infused, urine was collected in 10 min intervals (commencing 10 min after the bolus dose) and perfusate was collected at the mid-point of these intervals to permit calculation of the renal clearance (CLR) of AM 188. Binding of AM 188 in perfusate, measured using ultrafiltration, was negligible. 3. The bolus dose and infusion regimen produced relatively stable AM 188 concentrations in perfusate in the 5, 25 and 125 micro g/min groups and progressively increasing concentrations in the 500 and 1000 microg/min groups. High-pressure liquid chromatography analysis of IPK perfusate and urine suggested that there was no or negligible metabolism of AM 188 in the kidney. The CLR/GFR ratio for AM 188 (mean+/-SD) was 5.76 +/- 1.57, 5.99 +/- 0.52, 6.02 +/- 1.47, 3.38 +/- 0.26 and 1.08 +/- 0.42 in the 5, 25, 125, 500 and 1000 microg/min groups, respectively, showing significant reductions at the two highest infusion rates (P < 0.05). Although there was no difference between the five groups in the distribution of AM 188 between kidney tissue and perfusate (KT/P), at the end of perfusion the corresponding urine-to-tissue concentration ratio declined significantly in the 1000 microg/min group. 4. AM 188 undergoes substantial net renal secretion over a wide range of perfusate concentrations. A reduction in renal clearance at perfusate concentrations above 25 microg/mL could be due to saturation of carrier-mediated transport at the brush border membrane and/or a solubility limitation leading to precipitation of AM 188 in tubular cells and/or tubular urine.     

Wall stress-induced dysrhythmias in the isolated working rat heart perfused through a cannula placed in the left ventricle via aorta

The purpose of the present study was to determine if our recently introduced novel working rat heart preparation could be used to study wall stress-induced dysrhythmias. A double cannula, which consisted of an outer cannula that, was inserted in the aorta and an inner cannula that was advanced into the left ventricle was used. The perfusion flowed through the inner cannula into the left ventricle and was ejected from there into the aorta. Afterload was changed suddenly from 60 to 160 Hg of pressure by turning a valve so that the fluid was diverted to a column set at a different height. A sudden increase of aortic pressure that lasted for 10 sec caused cardiac ectopic beats. Wall stress-induced dysrhythmias were more sustained during perfusion with low potassium and low magnesium Krebs-Henseleit solution. Bradykinin (1 microg) or epinephrine (10 microg) was injected as a bolus via an in-line injection port placed at the inner cannula. Bradykinin significantly reduced the incidence of ectopic beats and epinephrine increased the incidence of nonsustained runs of VT. This "working" heart preparation is a convenient tool to study wall stress-induced dysrhythmias.     

Interaction of formaldehyde with glutathione in the isolated/ventilated perfused lung and the isolated perfused liver

The interaction of formaldehyde (CH2O) with reduced glutathione (GSH) was evaluated in aqueous solution and in isolated perfused lungs and livers. Addition of CH2O (0-4.9 mM) to a solution of 0.17 mM GSH in 2 mM EDTA, pH 7.4, resulted in a time- and concentration-dependent depletion of GSH. Perfusion of livers with fortified Krebs-Ringer bicarbonate buffer containing 0.3-4.9 mM CH2O resulted in a dose-dependent depletion of GSH. Perfusion of isolated ventilated lungs with perfusate containing 4.9 mM CH2O resulted in a depletion of GSH to 75% of controls. However, lower concentrations of CH2O in the lung perfusate did not result in depletion of GSH. These results demonstrate that exposure to CH2O in aqueous solution or in the perfused lung and liver is capable of depleting endogenous GSH. However, the concentrations of CH2O required to yield a significant depletion of endogenous GSH exceed those encountered in vivo. Thus, it is unlikely that depletion of GSH by CH2O is a causal factor in formaldehyde-induced toxicity.     

Modulatory roles of GABAergic mechanisms in post-rotatory nystagmus in the rabbit

1. To determine how the GABAergic mechanism operates in the generation of post-rotatory nystagmus, an experiment was performed with GABAergic drugs in rabbits. 2. Subconvulsive doses of picrotoxin (0.3-0.6 mg/kg, i.v.) and bicuculline (0.1 mg/kg, i.v.) decreased the number of post-rotatory nystagmus beats, whereas strychnine sulphate, at a subconvulsive dose (0.1 mg/kg i.v.), increased it. 3. Diazepam (1 mg/kg, i.v.) remarkably increased the number of post-rotatory nystagmus beats. Pretreatment with picrotoxin (0.45 mg/kg, i.v.), bicuculline (0.1 mg/kg, i.v.) or semicarbazide-HCl (180 mg/kg, i.v.) antagonized the effects of diazepam (1 mg/kg, i.v.). 4. GABAergic mechanisms may play a modulatory role in the production of nystagmus rhythm. Strychnine-sensitive neurons involved in the vestibular mechanism may behave in a different manner from picrotoxin-sensitive neurons.     

Dissipation mechanisms for 5-hydroxytryptamine in the coronary circulation of the isolated perfused heart of the rat.

1. The contribution of uptake into vascular endothelial cells, of neuronal uptake and of extraneuronal uptake in the dissipation of 5-hydroxytryptamine (5-HT) perfused through the coronary circulation of the rat heart was examined. 2. Hearts from reserpine-pretreated rats were isolated and perfused in vitro with 5-HT, in the absence or presence of inhibitors, and rates of appearance of the deaminated metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in the venous effluent were measured using an h.p.l.c. assay. 3. The steady-state rates of 5-HIAA appearance in the venous effluent in hearts perfused with 1 microM 5-HT (422 +/- 8.48 pmol g-1 min-1, n = 12) were reduced by pretreatment of the rats with 6-hydroxydopamine (22% inhibition), and by inclusion in the perfusion solution of 30 microM cocaine (28% inhibition), 100 microM 3-O-methylisoprenaline (64% inhibition), 100 microM corticosterone (58% inhibition), or 30 microM cocaine and 100 microM 3-O-methylisoprenaline (87% inhibition). 4. The extraneuronal deamination of 5-HT in the heart was saturable (Km = 101 microM, Vmax = 31.2 nmol g-1 min-1). The neuronal deamination of 5-HT was saturated by about 50 fold lower concentrations of 5-HT than was extraneuronal deamination, but Km and Vmax values could not be determined. 5. In the coronary circulation of the rat heart, 5-HT was dissipated by the uptake processes for catecholamines, extraneuronal uptake (predominantly) and neuronal uptake, and subsequent metabolism by monoamine oxidase. There was no evidence that a cocaine-sensitive uptake of 5-HT into vascular endothelial cells made any significant contribution to 5-HT dissipation in the heart.     

Multiple mechanisms are involved in the acute vasodilatory effect of 17beta-estradiol in the isolated perfused rat heart.

The purpose of this study was to define the dose-dependent effects of 17beta-estradiol on coronary flow and cardiac function in isolated rat hearts and to identify the mechanisms involved in its vasodilator action. Hearts from female and male Wistar rats were perfused at constant pressure (100 mm Hg). Stereoisomer specificity and the mechanism of vasodilation by 17beta-estradiol were examined in female rat hearts. Function was measured by a left ventricular (LV) balloon and coronary flow (CF) with an ultrasonic flowmeter. 17Beta-estradiol at 10(-6), 5 x 10(-6), and 10(-5) M increased CF in female hearts by 5 +/- 2, 27 +/- 4 (p < 0.05 vs. baseline), and 40 +/- 4% (p < 0.05 vs. baseline), respectively. The effect of 17beta-estradiol in hearts from male rats was similar but less pronounced compared with females [deltaCF 8 +/- 3, 19 +/- 3 (p < 0.05 vs. baseline)] and 25 +/- 7% (p < 0.05 vs. baseline; p < 0.05 vs. female 17beta-estradiol). Maximum vasodilation by the stereoisomer 17alpha-estradiol was significantly smaller [deltaCF 5 +/- 3, 4 +/- 3 (p < 0.05 vs. female 17beta-estradiol) and 14 +/- 1% (p < 0.05 vs. baseline; p < 0.05 vs. female 17beta-estradiol)] for 10(-6), 5 x 10(-6), and 10(-5) M. Pretreatment with the NO-synthesis inhibitor Nomega-methyl-L-arginine (10(-4) M) had no effect on the maximal vasodilator response to 17beta-estradiol (10(-5) M) [deltaCF 36 +/- 6% (p < 0.05 vs. baseline)]. When hearts were pretreated with the prostaglandin-synthesis inhibitor diclofenac (10(-6) M), the maximal vasodilator effect of 17beta-estradiol was partially attenuated [deltaCF 12 +/- 7% (p < 0.05 vs. female 17beta-estradiol)]. Similarly, pretreatment with the K+ATP-blocker glibenclamide (10(-6) M) partially inhibited the maximal vasodilator effect of 17beta-estradiol [deltaCF 22 +/- 6% (p < 0.05 vs. baseline; p < 0.05 vs. female 17beta-estradiol)]. Pretreatment with the Ca2+ channel antagonist nifedipine (7.2 x 10(-8) M) completely blocked the vasodilator effect. In isolated perfused rat hearts, 17beta-estradiol induced marked acute coronary vasodilation; this effect is in part gender specific, and in female hearts, largely stereoisomer specific. The dilator effect is mediated predominantly by calcium channel blockade, but prostaglandin release and K+ATP channel activation also are involved. In the isolated perfused rat heart, NO production does not contribute to the acute vasodilator effect of 17beta-estradiol.     

Vascular resistance in the perfused isolated rat tail

1. A constant flow perfusion system using the isolated rat tail has been developed to facilitate the study of resistance vessel behaviour and the action of vasoactive drugs.2. Baseline resistance remains stable for several hours and dose response curves to bolus injections of pressor agents are reproducible when dialysed bovine serum albumen is used in the perfusion medium to maintain osmotic pressure.3. Noradrenaline, adrenaline, serotonin, vasopressin, angiotensin II, high potassium concentrations and sympathetic nerve stimulation constricted the vascular bed.4. Angiotensin I, bradykinin, histamine, acetylcholine and isoprenaline did not alter vascular resistance under baseline conditions.5. Maximal sensitivity to noradrenaline occurred at 32 degrees to 34 degrees C. Below 30 degrees C, resting tone increased and the pressor effect of noradrenaline was prolonged.6. Low concentrations of (+/-)-propranolol in the perfusate enhanced adrenaline and noradrenaline vasoconstriction, high concentration of (+/-)-propranolol had a direct pressor effect and did not affect catecholamine responses.7. The preparation is a simple and relatively inexpensive adjunct to established methods of studying resistance vessel behaviour under varying experimental conditions.     

Disopyramide elimination in the isolated perfused rat liver

The elimination kinetics of disopyramide, [14C]disopyramide and [2H]disopyramide have been studied in the isolated perfused rat liver. Disappearance of disopyramide from perfusate was dose- and time-dependent over the dose range 0.3-7.5 mg. Although the mechanism underlying these observations is unclear, the data are consistent with the presence of enzyme saturation and product inhibition. Biliary secretion of conjugated metabolites appeared to be the rate-limiting step in the perfusate clearance of total radioactivity. At doses of 0.3 and 7.5 mg the kinetics of [2H]disopyramide showed a small isotope effect probably of negligible importance.     

Disopyramide elimination in the isolated perfused rat liver

1. The elimination kinetics of disopyramide, [¹⁴C]disopyramideand [²H]disopyramide have been studied in the isolated perfused rat liver.

2. Disappearance of disopyramide from perfusate was dose- and time-dependent over the dose range 0-3-7-5 mg. Although the mechanism underlying these observations is unclear, the data are consistent with the presence of enzyme saturation and product inhibition.

3. Biliary secretion of conjugated metabolites appeared to be the rate-limiting step in the perfusate clearance of total radioactivity.

4. At doses of 0-3 and 7-5 mg the kinetics of [²H]disopyramide showed a small isotope effect probably of negligible importance.     

Cadmium toxicity in the isolated perfused rat liver

Isolated perfused livers from male and female Sprague-Dawley rats were exposed to cadmium chloride (50 and 200 microM). Acute hepatotoxicity was investigated by measuring cadmium-induced changes in bile flow, urea synthesis and alanine aminotransferase (ALT) leakage. Cadmium-induced lipid peroxidation was estimated by formation of conjugated dieners and thiobarbituric acid (TBA) reactants. Cadmium, at both concentrations, caused a rapid decrease in bile flow (within 40 min) and complete cholestasis within 70 min exposure in livers perfused from both male and female rats. Cadmium exposure (50 and 200 microM) also resulted in the leakage of ALT into the perfusate within 60 min. In contrast, exposure of isolated rat hepatocytes to as high as 500 microM cadmium did not result in enzyme leakage until 180 min exposure. Sex differences in cadmium-induced cholestasis and ALT leakage were not observed at these concentrations. Malondialdehyde was not detected in the perfusate nor were conjugated dienes detected in liver tissue following 90 min cadmium exposure. These data demonstrate that the isolated perfused rat liver (IPRL) is a sensitive system in which to study chemically induced hepatotoxicity. Cadmium rapidly causes functional alterations and cellular damage in perfused livers from both male and female rats. Cadmium-induced liver injury was apparently not related to lipid peroxidation.     

Benzene metabolism by the isolated perfused lung

Benzene is an occupational hazard and environmental toxicant whose toxic effects are dependent on its metabolism by cytochrome P-450. Most physiologically based pharmacokinetic models assume that benzene is metabolized only in the liver. They may not be completely accurate in predicting metabolism, especially following inhalation exposure, if metabolism by the lung is important. In the current study, the metabolizing capability of the lung was examined in an in vivo simulation using the isolated perfused lung. Lungs from the rabbit, rat, and mouse were used to mimic benzene metabolism following exposure via the pulmonary vasculature. With the isolated perfused mouse lung, three concentrations (55 microM, 120 microM, and 200 microM) were used to evaluate concentration dependence. To evaluate the ability of the lung to metabolize inhaled benzene, the isolated perfused mouse lung was exposed to benzene (approximately 175 ppm) via the trachea. Benzene was metabolized in all species, with phenol being the major metabolite. Phenylsulfate was also detected in perfusate from rabbits and mice but at much lower levels. Benzene metabolism was concentration dependent in mice. The ability of the lung to metabolize benzene during inhalation exposure was demonstrated in the isolated perfused mouse lung. These results demonstrate that the lung can metabolize benzene in an in vivo simulation when exposed via the pulmonary vasculature or via inhalation.     

Induction of paracetamol metabolism in the isolated perfused kidney

1. At a perfusate concn. of 3±5-4±0 mM, 59/Pm9 nmol of paracetamol/h per?g wet wt. were oxidized by isolated rat kidney.

2. Approx. half the paracetamol undergoing oxidation was converted to a mercapturic acid metabolite and the remainder was covalently bound to kidney protein.

3. Addition of GSH to the perfusate decreased the level of covalent binding. Depletion of cellular GSH, by prior administration of diethyl maleate, significantly decreased formation of the mercapturic acid metabolite.

4. The metabolic pathways of glucuronylation, sulphation and mercapturic acid formation were induced either by 3-methylcholanthrene pretreatment or by prolonged feeding of aspirin or paracetamol; covalent binding of paracetamol to kidney protein was not increased.