Purification of swine carbonic anhydrase isoenzyme III and measurement of its levels in tissues and plasma.

The changes in the levels of carbonic anhydrase isozyme III (CA-III) in swine plasma and urine have not been previously determined or reported. CA-III is relatively specific to skeletal muscles, and should therefore be a useful diagnostic marker for muscle diseases. We isolated CA-III from swine muscle tissues and determined CA-III levels in the plasma and urine from both healthy and diseased pigs. The levels of CA-III in the tissues of female swine (age, 3 months) and plasma of young swine (age, 1-5 months) and adult female pigs (age, 2-3 years) were determined using the ELISA system for swine CA-III. The mean (± SD) levels of CA-III in the skeletal muscles were 3.8 ± 3.2 mg/g (wet tissue), and in the plasma, 230 ± 193 ng/ml at 1 month, 189 ± 208 ng/ml at 2 months, 141 ± 148 ng/ml at 3 months, 78 ± 142 ng/ml at 4 months and 53 ± 99 ng/ml at 5 months. The mean level of CA-III in the plasma samples from 2- to 3-year-old pigs was 18 ± 60 ng/ml. CA-III in the plasma samples was found to decrease from 1 month until 3 years of age (p < 0.01). We performed far-western blotting to clarify the cause of the observed decrease in CA-III in plasma. Our results demonstrated that CA-III is bound to the transferrin and albumin. In addition, we determined that the levels of CA-III in plasma and urine samples were higher in diseased swine compared with the healthy pigs.

Time-course effects of St John's wort on the pharmacokinetics of cyclosporine in dogs: interactions between herbal extracts and drugs.

To clarify the interaction between St John's wort (SJW) and cyclosporine (CsA) in dogs, the pharmacokinetics of CsA before and during the repeated administration of SJW were analyzed. In the SJW group, SJW (300 mg) was given orally to four dogs every 24 h for 14 days. A single dose of CsA (5 mg/kg) was given orally 7 days before and 7 and 14 days after the initiation of the repeated administration of SJW. In the Control group, a single dose of CsA (5 mg/kg) was given orally to four other dogs in accordance with that in the SJW group. Blood samples from both groups were collected, and whole-blood concentrations of CsA were determined using high-performance liquid chromatography with UV detection. The maximum whole-blood concentration and AUC(0-∞) of the SJW group were significantly lower and the CL(tot) /F and V(d) /F were significantly higher than those in the Control group 7 and 14 days after the initiation of repeated SJW. Thus, repeated administrations of SJW affect the pharmacokinetic profiles of CsA in dogs. Further studies are necessary to elucidate the mechanisms of interaction between SJW and CsA in dogs.

A pilot study investigating the effect of pimobendan on the cardiac rhythm and selected echocardiographic parameters of healthy cats.

INTRODUCTION: The effects of pimobendan on the heart rhythm in cats are unknown. The purpose of this pilot study was to evaluate the effect of pimobendan on the cardiac rhythm and selected echocardiographic parameters of cats. ANIMALS, MATERIALS, AND METHODS: Six clinically healthy cats received each of four medication protocols for 15 days, with a washout period of at least one month between each protocol. The protocols were, pimobendan 0.5 mg/kg twice daily (high dosage group), pimobendan 0.25 mg/kg twice daily (standard dosage group), pimobendan 0.125 mg/kg twice daily (low dosage group), and Biofermin R, one tablet twice daily (placebo group). Twenty-four-hour ambulatory electrocardiogram recordings, blood pressure measurements, and echocardiographic examinations were performed after two weeks of each medication protocol. Electrocardiographic, echocardiographic, and blood pressure parameters were compared between the four groups. RESULTS: The total number of escape/idioventricular/idiojunctional complexes in the high dosage group was significantly higher compared with the placebo, low dosage, and standard dosage groups (p < 0.001). The blood pressure; total number of heart beats per day; and mean, minimum, and maximum heart rates were not significantly different between the groups. The longitudinal strain rate and calculated cardiac output were significantly increased in the high and standard dosage groups. CONCLUSIONS: The administration of pimobendan, especially at high doses, was associated with increased numbers of escape/idioventricular/idiojunctional complexes in some cats and echocardiographic parameters. Further studies are warranted to investigate both the mechanism underlying the observed changes and what, if any, clinical implications these changes might have in cats with heart disease.

CT-based anatomical features of large airway and heart volume in dogs of different body size.

Differences in the prevalence and clinical signs of cardiopulmonary diseases in dogs of different body sizes have been reported. It was hypothesized that the anatomical features of the heart and large airways varies by body size in dogs and might influence clinical manifestations of cardiopulmonary disease. The purpose of this study was to compare various anatomical features of the thoracic organs (heart, trachea, etc.) in dogs according to body size using computed tomography (CT) images. Dogs without clinically significant heart and lung disease (n=226) that underwent CT were divided into three groups on the basis of bodyweight: small (<7kg), medium (7-20kg), and large (>20kg). The following parameters were calculated from CT images using OsiriX and compared among groups: relative heart volume (heart volume/thoracic volume), relative distance from mainstem bronchi to vertebra (distance from mainstem bronchi to vertebra/heart length), longitudinal/transverse diameter ratio of trachea, and angle of bronchus. Small dogs had larger hearts relative to their thorax, a shorter distance from the heart to the vertebra, and laterally-elongated oval-shaped tracheas, compared to medium and/or large dogs. These differences in anatomical features according to body size may potentially contribute to different clinical manifestations when the heart is enlarged.

Delta 9-tetrahydrocannabinol-induced catalepsy-like immobilization is mediated by decreased 5-HT neurotransmission in the nucleus accumbens due to the action of glutamate-containing neurons.

Delta(9)-tetrahydrocannabinol (THC) has been reported to induce catalepsy-like immobilization, but the mechanism underlying this effect remains unclear. In the present study, in order to fully understand the neural circuits involved, we determined the brain sites involved in the immobilization effect in rats. THC dose-dependently induced catalepsy-like immobilization. THC-induced catalepsy-like immobilization is mechanistically different from that induced by haloperidol (HPD), because unlike HPD-induced catalepsy, animals with THC-induced catalepsy became normal again following sound and air-puff stimuli. THC-induced catalepsy was reversed by SR141716, a selective cannabinoid CB(1) receptor antagonist. Moreover, THC-induced catalepsy was abolished by lesions in the nucleus accumbens (NAc) and central amygdala (ACE) regions. On the other hand, HPD-induced catalepsy was suppressed by lesions in the caudate putamen (CP), substantia nigra (SN), globus pallidus (GP), ACE and lateral hypothalamus (LH) regions. Bilateral microinjection of THC into the NAc region induced catalepsy-like immobilization. This THC-induced catalepsy was inhibited by serotonergic drugs such as 5-hydroxy-L-tryptophan (5-HTP), a 5-HT precursor, and 5-methoxy-N,N-dimethyltryptamine (5-MeODMT), a 5-HT receptor agonist, as well as by anti-glutamatergic drugs such as MK-801 and amantadine, an N-methyl-d-aspartate (NMDA) receptor antagonist. THC significantly decreased 5-HT and glutamate release in the NAc, as shown by in vivo microdialysis. SR141716 reversed and MK-801 inhibited this decrease in 5-HT and glutamate release. These findings suggest that the THC-induced catalepsy is mechanistically different from HPD-induced catalepsy and that the catalepsy-like immobilization induced by THC is mediated by decreased 5-HT neurotransmission in the nucleus accumbens due to the action of glutamate-containing neurons.

Effects of urine pH modification on pharmacokinetics of phenobarbital in healthy dogs.

Although pH modification is one of the effective strategies for dissolving or preventing uroliths, little is known about its effects on the pharmacokinetics of phenobarbital in dogs. Five spayed, female Beagles were fed with a twice-daily diet that included potassium citrate and ammonium chloride for urine alkalinization and acidification, respectively. After a stabilizing period of 7 days, a single clinical dose of phenobarbital (3 mg/kg) was orally administered, and time-course changes in its serum and urine concentrations were determined by high-performance liquid chromatography. Total amounts of unchanged phenobarbital excreted into urine for 216 h were decreased by urine acidification and increased by urine alkalinization. The elimination half-life of serum phenobarbital in dogs with urine alkalinization was shortened and Cl(R) increased when compared with dogs with urine acidification. Other pharmacokinetic parameters, including C(max), T(max), AUC(0-216), Cl/F, and A(e0-216) were not changed by modification of the urine pH. These results suggest that the pH of urine is likely to be a determinant of the pharmacokinetics, especially urine excretion rate, of a clinical dose of oral phenobarbital. It is possible that the serum concentration of phenobarbital might be altered when a pH modifying-diet is administered for the purpose of dissolving or preventing uroliths.

Pharmacokinetics of zonisamide and drug interaction with phenobarbital in dogs.

The purposes of the present study were to elucidate the pharmacokinetics of zonisamide, determine the presence of a drug interaction with phenobarbital, and evaluate how long any interaction lasted after discontinuation of phenobarbital in dogs. Five dogs received zonisamide (5 mg/kg, p.o. and i.v.) before and during repeated oral administration of phenobarbital (5 mg/kg, bid, for 30-35 days). Zonisamide (5 mg/kg, p.o.) was also administered 8, 10, and 12 weeks after discontinuation of phenobarbital. Blood was sampled until 24 h after each zonisamide administration and serum concentrations of zonisamide were determined. Repeated phenobarbital decreased the maximum serum concentration, area under the serum concentration vs. time curve, apparent elimination half-life, and bioavailability of zonisamide. Total clearance increased. Time to maximum serum concentration and volume distribution were not changed. The maximum serum concentration and area under the serum concentration vs. time curve of zonisamide continued to be low until 10 weeks after the discontinuation of phenobarbital. They were restored to the same serum concentration as before phenobarbital administration 12 weeks after the discontinuation of phenobarbital. These data suggested that repeated administration of a clinical dose of phenobarbital enhanced the clearance of zonisamide and the enhanced clearance lasted at least 10 weeks after the discontinuation of phenobarbital. Caution may be necessary when zonisamide is given with phenobarbital and when antiepileptic therapy is changed from phenobarbital to zonisamide.

alpha(2)-adrenoceptor antagonist properties of OPC-28326, a novel selective peripheral vasodilator.

1. Antagonistic properties of OPC-28326 ([4-(N-methyl-2-phenylethylamino)-1-(3,5-dimethyl-4-propionyl-aminobenzoyl)] piperidine hydrochloride monohydrate), a selective peripheral vasodilator, were investigated by analysing the data from functional studies in various tissues from the rat and binding studies of the drug to alpha(2)-adrenoceptor subtypes. 2. Using a human recombinant receptor and rat kidney cortex, we found that OPC-28326 displays affinities to alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptors with K(i) values of 2040, 285, and 55 nM, respectively. The K(i) values of yohimbine for alpha(2A)-, alpha(2B)-, and alpha(2C)-adrenoceptors were 3.0, 2.0 and 11.0 nM, respectively. 3. B-HT 920, an alpha(2)-adrenoceptor agonist, produced a pressor response via peripheral postsynaptic alpha(2)-adrenoceptor stimulation (thought to be an alpha(2B)-subtype) in a reserpine-pretreated pithed rat preparation. OPC-28326 (3 - 30 mg kg(-1), i.v.) and yohimbine (0.3 - 3 mg kg(-1), i.v.) caused dose-dependent rightward shift in the pressor dose-response curve induced by B-HT 920. The apparent pA(2) values were 1.55 (0.87 - 2.75, 95% confidence interval) and 0.11 (0.06 - 0.21) mg kg(-1), respectively. The potency of OPC-28326 was about 14 times less than that of yohimbine. 4. Clonidine inhibited the tension developed by electrical stimulation, of the rat vas deferens, by its peripheral presynaptic alpha(2A/D)-adrenoceptor action. OPC-28326 (1 - 100 microM) and yohimbine (10 - 1000 nM) caused a rightward shift in the concentration-response curve of clonidine. The pA(2) values were 5.73 (5.54 - 5.91) and 7.92 (7.84 - 8.01), respectively, providing evidence for a potency of OPC-28326 of about 155 times less than that of yohimbine. 5. Mydriasis was induced by brimonidine via stimulation of central alpha(2A/D)-adrenoceptors in anaesthetized rats. Intravenous OPC-28326 had no effect on this action, even at a very high dose of 10 mg kg(-1) i.v., while yohimbine (0.1 - 0.3 mg kg(-1) i.v.) inhibited mydriasis in a dose-dependent manner, indicating that OPC-28326 was at least 100 times less potent than yohimbine in regard to the anti-mydriatic effect. 6. These data suggest that OPC-28326 preferentially exerts peripheral and postsynaptic antagonistic actions on the alpha(2B)- and alpha(2C)-adrenoceptor subtypes.

Aryl radical cyclizations: one-Pot syntheses of protoberberine and pavine alkaloids.

Treatment of 2-(2'-bromo-beta-phenethyl)isocarbostyrils 7 with AIBN-Bu(3)SnH in boiling benzene gave 8-oxoberbines 3 in good yields. A similar treatment of 2-(2'-bromo-beta-phenethyl)isoquinolinium bromides 6 and their nor- and homoanalogues (10,11) induced 6-, 5-, and 7-exo radical closures in a one-pot manner to give protoberberines 2, dibenzo[b,g]indolizidine 14a and, dibenzo[a, h]-1-azabicyclo[5.4.0]undecane 15a, respectively. A one-pot radical cyclization of 1-(2'-bromobenzyl)isoquinoline methiodide 18a gave a pavine alkaloid, (+/-)-algemonine (19a).

Involvement of ATP-sensitive K+ channels in the sustained coronary vasodilator response to adenosine in dogs.

Coronary vasodilator mechanisms of adenosine were investigated in isolated canine papillary muscle preparations cross-circulated through the anterior septal artery with support dogs. Infusions of adenosine (1-3000 nmol/min) for 3 min into this artery caused a transient increase (the initial phase) in blood flow followed by a sustained increase (the sustained phase), and single-bolus intra-arterial injections of adenosine (0.3-1000 nmol) produced only a transient increase in blood flow. The force of contraction of the papillary muscle was virtually unaffected. The increase in blood flow in the sustained phase was antagonized by glibenclamide (6 mumol/kg i.v.), a blocker of ATP-sensitive K+ channels, administered to support dogs whereas increases in both phases were antagonized by 8-phenyltheophylline (12 mumol/kg i.v.), a non-selective adenosine receptor antagonist, administered in a similar way. The transient increase in blood flow caused by single-bolus injections of adenosine was reduced in duration rather than in peak value by glibenclamide. Thus, it appears that opening of ATP-sensitive K+ channels gradually becomes involved in the vasodilator mechanisms for adenosine A2 receptor-mediated, sustained vasodilation.

Venodilator effects of pranidipine, a 1,4-dihydropyridine Ca2+ channel antagonist, in rats: comparison with nifedipine and amlodipine.

The effects of pranidipine, a dihydropyridine Ca2+ channel antagonist, on mean circulatory filling pressure, an index of body venous tone, were compared with those of other dihydropyridines (nifedipine and amlodipine) and nitroglycerin in anaesthetized hexamethonium- and norepinephrine-treated rats. In this study, the compounds were used at doses having a equi-hypotensive effect. Intravenous bolus injection of pranidipine (10 and 30 microg/kg) significantly decreased mean circulatory filling pressure in a dose-dependent manner, as did nitroglycerin (30 and 100 microg/kg). Nifedipine (30 and 100 microg/kg), however, did not affect mean circulatory filling pressure. Amlodipine (1000 and 3000 microg/kg) decreased mean circulatory filling pressure only at the higher dose. These results suggest that pranidipine has a greater venodilator effect than nifedipine and amlodipine.

Differential properties of the optical-isomers of pranidipine, a 1,4-dihydropyridine calcium channel modulator.

Pranidipine is an optically-active 1,4-dihydropyridine (DHP) voltage-dependent L-type calcium channel inhibitor. Certain enantiomeric pairs display opposite effects, i.e., inhibition and activation of the calcium channel while others exhibit the same qualitative actions. We investigated pranidipine, a new DHP, using a paradigm of vascular smooth muscle reactivity. In isolated rat aorta, depolarized with 80 mM KCl, both isomers of pranidipine caused a right-ward shift of the concentration-contraction curves for extracellular Ca2+. The apparent pA2, values of the S-isomer and R-isomer were 10.03 and 8.36, respectively, providing evidence that the calcium channel blocking action of the S-isomer was 50 times more potent than that of the R-isomer. Antihypertensive actions of these two isomers studied in pentobarbital-anaesthetized spontaneously hypertensive rats, revealed that the S-isomer, at doses of 3-30 microg/kg i.v. decreased blood pressure in a dose-dependent manner, while the R-isomer had no effect on blood pressure at those doses. We conclude that the pair of enantiomers of pranidipine qualitatively display the same Ca2+ channel blocking action and that neither isomer exhibits Bay K 8644-like activation. Pranidipine may be useful in studies on the architecture of the DHP receptor 'pocket'.

Pharmacokinetics of nicorandil in dogs with mild mitral regurgitation.

The aim of this study was to examine the pharmacokinetics of nicorandil, a hybrid of an adenosine triphosphate-sensitive potassium channel opener and a nitrate, and to estimate its clinical doses in dogs with mild mitral valve regurgitation (MR). Nicorandil (0.1, 0.3, and 1.0 mg/kg) was administered orally to normal dogs and those with experimentally-induced MR, and its plasma concentrations were analyzed using high-performance liquid chromatography. Plasma concentrations increased dose-dependently after the administration of nicorandil, and were not different between normal dogs and those with MR. Similar to the effective plasma values obtained in cardiac disease in humans, the findings of this pharmacokinetic study may indicate that a dose of 0.3-1.0 mg/kg has the same effectiveness in dogs with cardiac dysfunction.

Long-lasting enhancement of CYP activity after discontinuation of repeated administration of phenobarbital in dogs.

We investigated how long in vivo hepatic cytochrome P450 (CYP) activity is enhanced even after discontinuation of repeated oral administration of phenobarbital (PB) in dogs using antipyrine clearance, which reflects hepatic CYP activity. A single antipyrine (5 mg/kg) was administered intravenously before and 34 days after the repeated oral administration of PB (5 mg/kg, bid) and 2, 4, 6, and 8 weeks after the discontinuation of PB in 5 dogs. Antipyrine clearance was increased by the repeated administration of PB, and remained increased 2 and 4, but not 6 and 8 weeks after the discontinuation of PB. The result suggests that hepatic CYP activity was enhanced by the repeated administration of PB, and this enhancement may last for at least 4 weeks even after its discontinuation.

ATP-sensitive K+ channels are gradually recruited in the vasodepressor response to adenosine in spinally-anesthetized dogs.

Vasodepressor mechanisms of adenosine were investigated in spinally-anesthetized dogs. An i.v.-infusion of adenosine (0.1-10 mumol/kg/min) caused a slowly developing and sustained decrease in blood pressure (BP). This vasodepression was antagonized by glibenclamide, a blocker of ATP-sensitive K+ (KATP) channels. On the other hand, a transient decrease in BP caused by a single bolus i.v.-injection of adenosine was not antagonized by glibenclamide in our previous study. These results suggested that the opening of KATP channels is gradually recruited in the vasodepressor mechanisms for adenosine-induced sustained vasodepression.

Cardiovascular profile of Ro 40-5967, a new nondihydropyridine calcium antagonist, delineated in isolated, blood-perfused dog hearts.

Coronary and cardiac effects of Ro 40-5967 were compared in isolated, blood-perfused dog heart preparations. Intraarterial Ro 40-5967 increased blood flow in all preparations. In sinoatrial preparations, Ro 40-5967 decreased sinus rate and produced atrial stand-still when administered at high doses. In paced atrioventricular (AV) node preparations, Ro 40-5967 injected into the posterior septal artery (which perfuses the AV node) increased AV conduction time and at high doses produced second- or third-degree AV block. In the same preparations, Ro 40-5967 had little effect on AV conduction time, even at higher doses, when injected into the anterior septal artery (which perfuses the His-Purkinje ventricular system). In paced papillary muscle preparations, Ro 40-5967 reduced the force of contraction only at high doses. In spontaneously beating papillary muscle preparations, Ro 40-5967 decreased the force of contraction only at high doses and had no effect on the rate of automaticity even at higher doses. The dose that doubled blood flow was about one-fifth of the dose that produced a 15% decrease in sinus rate and also one-fifth of the dose that produced a 15% increase in AV conduction time. The dose that reduced the force of contraction by half was > 10 times the dose that doubled blood flow. The results indicate that the cardiovascular profile of Ro 40-5967 differs from those of verapamil and diltiazem but instead resembles that of dihydropyridine derivatives, which are classified as vasoselective calcium antagonists.

A possibility that the ATP-sensitive potassium channel in coronary artery has a high-affinity internal binding site for tetraalkylammonium.

The functionally responsible sites for the blocking action of tetraalkylammonium ions (TAAs) in ATP-sensitive K+ (KATP) channels opened by levcromakalim were estimated in canine coronary artery. Tetraethylammonium (TEA) and tetrabutylammonium (TBA) inhibited the levcromakalim-induced relaxation in a noncompetitive manner. Analyses of the noncompetitive antagonism revealed that the binding constant of TBA was about 900 times lower than that of TEA, although the reported affinity of TBA for the internal binding site in various K+ channels was only 10 times higher than that of TEA. TBA is much more lipid-soluble and permeable through membranes than TEA. Thus, TBA blocks KATP channels by binding to a possible high-affinity internal site for TAAs, whereas TEA seems to bind to the external site.

Vasodilator mechanisms of action of amrinone do not involve the opening of either ATP-sensitive or Ca(2+)-activated K+ channels.

Amrinone is more coronary vasodilatory than positive inotropic compared with other newer phosphodiesterase inhibitors. In order to explore possible involvements of ATP-sensitive K+ (KATP) and large-conductance Ca(2+)-activated K+ (KCa) channels in the vasodilator effect of amrinone, we investigated whether amrinone-induced vasodilation would be antagonized by glibenclamide, a blocker of KATP channels, or by charybdotoxin or tetraethylammonium, blockers of KCa channels. In isolated, blood-perfused canine papillary muscle preparations, the amrinone-induced increase in blood flow was not affected by glibenclamide given to support dogs. In canine large coronary arteries contracted with high (25 mM) KCl, amrinone-induced relaxation was not affected by glibenclamide, charybdotoxin and tetraethylammonium. These results suggest that the vasodilator mechanisms of amrinone do not involve the opening of either KATP or KCa channels.