Cloning of a swelling-induced chloride current related protein from rabbit heart.

Recently, pIcln has been reported to be a regulator of a swelling-induced chloride conductance. We have cloned a cDNA RCL-H1 from rabbit heart, of which primary structure is highly homologous to that of pIcln. Outwardly rectifying currents were recorded in oocytes expressing RCL-H1, which is consistent with the result of pIcln. RNA blot analysis revealed the widespread expression of RCL-H1 mRNA in rabbit tissues. RCL-H1 may play an important role in regulating cell volume and give a clue to revealing molecular structure of swelling-induced chloride channel(s).

Identification of thyroid hormone transporters in humans: different molecules are involved in a tissue-specific manner.

We have recently identified that rat organic anion transporters, polypeptide2 (oatp2) and oatp3, both of which transport thyroid hormones. However, in humans the molecular organization of the organic anion transporters has diverged, and the responsible molecule for thyroid hormone transport has not been clarified, except for human liver-specific transporter (LST-1) identified by us. In this study we isolated and characterized a novel human organic anion transporter, OATP-E from human brain. The isolated complementary DNA encodes a polypeptide of 722 amino acids with 12 transmembrane domains. A rat counterpart, oatp-E, was also identified. Homology analysis and the phylogenetic tree analysis revealed that OATP-E/oatp-E is a subfamily of the organic anion transporter. Human OATP-E transported 3,3',5-triiodo-L-thyronine (K(m), 0.9 microM), thyronine, and rT(3) in a Na(+)-independent manner. Although the clone was isolated from the brain, OATP-E messenger RNA was abundantly expressed in various peripheral tissues. The rat counterpart, oatp-E, also transported 3,3',5-triiodo-L-thyronine. In addition, in this study we revealed that human OATP, which is exclusively expressed in the brain, transported 3,3',5-triiodo-L-thyronine (K(m), 6.5 microM), T(4) (K(m), 8.0 microM), and rT(3). These data suggest that in humans, several different molecules are involved in transporting thyroid hormone: OATP in the brain, LST-1 in the liver, and OATP-E in peripheral tissues.

The voltage-dependent K+ channel (Kv1.5) cloned from rabbit heart and facilitation of inactivation of the delayed rectifier current by the rat beta subunit.

We have isolated a cDNA coding for a delayed rectifier K+ channel (RBKV1.5) from rabbit heart. The amino acid sequence of RBKV1.5 displays a homology to that of other K+ channels of Kv1.5 class. Overall amino acid identity between RBKV1.5 channel and Kv1.5 channel of other species is about 85%. RNA blot analysis revealed the expression of the primary transcript in various rabbit tissues, at the highest level in both the atrium and ventricle. When expressed in Xenopus oocytes, RBKV1.5 current showed a delayed rectifier type characteristics, which was converted to rapidly inactivating currents upon coexpression with a beta subunit.

Determination of KA values by controlled receptor expression in Xenopus oocytes.

1. In the present study we estimated the KA value of endothelin-1 (ET-1) for ETA-receptors by a new method in which the level of expression of ETA-receptors in Xenopus oocytes was altered in a controlled way. 2. Kvl.2 (a delayed rectifier type K channel) c RNA at the fixed concentration of 0.2 micro g micro l(-1) was mixed with ETA-receptor cRNA at various concentration ratios (10(-3)-3). Oocytes were examined 2-4 days after the injection of the cRNA mixtures. 3. In these oocytes, ET-1 suppressed the amplitude of Kvl.2 current in a dose-dependent manner in the range of 0.1-100 nM; the maximum inhibition produced by ET-1 was larger and the EC50 value for the inhibition by ET-1 was smaller as the mixture ratio was increased. Double-reciprocal plots of equiactive concentrations of ET-1 in 1/1- and 1/30-injected oocytes yielded a KA for ET-1 of 7.4 nM. The number of ETA-receptors in 1/30-injected oocytes was 13% of that in 1/1-injected oocytes, whereas the inhibition of the current in 1/30-injected oocytes was about 60% of that in 1/1-injected oocytes. This suggests the presence of spare receptors of ETA in the latter. 4. A saturation binding experiment estimated a KD value of 0.1 nM for ET-1 at ETA-receptors and the number of ETA-receptors in 1/30-injected oocytes was 23% of that in 1/1-injected ones. This value was not significantly different from that estimated by the above new method. However, there was a discrepancy between KA and KD, which could be due to factors unique to the expression system employed in the present study.

Inhibition of calmodulin function by CV-159, a novel dihydropyridine compound.

1,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-dicarboxylic acid methyl 6-(5-phenyl-3-pyrazolyloxy)hexyl ester (CV-159), a new compound synthesized from dihydropyridine, was examined for its effect on calmodulin (CaM) function. The concentration of CV-159 producing 50% inhibition of Ca2+/CaM activated myosin light chain kinase (MLC kinase) was 6.2 microM. The apparent Ki value of CV-159 was 0.8 microM for MLC kinase. On the other hand, the concentration of CV-159 producing 50% inhibition of Ca2+/CaM activated cyclic nucleotide phosphodiesterase (Ca2+-PDE) was 0.55 microM. CaM antagonized competitively the CV-159-induced inhibition of activation of both MLC kinase and Ca2+-PDE. Interaction of CV-159 with CaM was also demonstrated by fluorescence studies using dansyl-CaM (5-dimethylaminonaphthalene-1-sulfonylated CaM). CV-159 produced a decrease in fluorescence intensity of dansyl-CaM, in a Ca2+-dependent fashion, and the concentration of this drug producing 50% inhibition of dansyl-CaM fluorescence was 1.2 microM. However, the concentration of nicardipine producing 50% inhibition of MLC kinase exceeded 100 microM. CaM did not antagonize the nicardipine-induced inhibition of Ca2+-PDE. These results suggest that the action of CV-159 is unique in that it inhibits both Ca2+-PDE and MLC kinase, through interaction with calmodulin. CV-159 seems to be a different class of drug from known dihydropyridine compounds.

Differential effects of flavonoids as inhibitors of tyrosine protein kinases and serine/threonine protein kinases.

The inhibitory potencies of bioflavonoids on various tyrosine protein kinases and serine/threonine protein kinases were investigated. The phosphotransferase activity of an oncogene product, pp130fps, and a growth factor receptor, insulin receptor, were inhibited by myricetin, a derivative of quercetin. However, tyrosine kinase activity in the particulate fraction from human platelets (PM-TPK) was resistant to myricetin. Apparent Ki values of myricetin for tyrosine protein kinases of pp130fps and insulin receptor were 1.8 and 2.6 microM, respectively. The Ki values for serine/threonine kinase activities of myosin light chain kinase (MLC-kinase), casein kinase I, casein kinase II, cAMP-dependent protein kinase, and protein kinase C were 1.7 microM, 9.0 microM, 0.6 microM, 27.5 microM, and 12.1 microM, respectively. Lineweaver-Burk plots revealed that myricetin competitively inhibits pp130fps tyrosine kinase, myosin light chain kinase, casein kinase I and II with ATP, but does not inhibit other protein kinases. Since myricetin is a hydroxylated derivative of quercetin, the inhibitory effects of a series of seven flavonoids with various numbers of hydroxy residues were examined. Structure activity studies exhibited that the inhibitory potencies of the flavonoids for tyrosine kinases of pp130fps and insulin receptor correlated with the number of hydroxy residues on the flavone rings (gamma = 0.974 and 0.926, respectively), whereas the hydroxylation influenced to a lesser extent the inhibitory potencies for serine/threonine protein kinase. The hydroxy residues at position 3' and 5' did not affect the activities of cAMP-dependent protein kinase, and protein kinase C, and the hydroxylation at position 5' is detrimental for the inhibition of MLC-kinase, and casein kinase I and II. Thus, flavonoids may be useful tools to elucidate the active site of tyrosine and serine/threonine protein kinases.

Receptor-mediated modulation of rat KV1.2 in Xenopus oocytes.

To investigate mechanisms for the receptor-mediated inhibition of a rat cardiac K+ channel clone (KV1.2), we coexpressed KV1.2 with a subtype of endothelin receptors (ETA) in Xenopus oocytes. Effects of endothelin ETA receptor stimulation were mimicked by application of PMA (4-beta-phorbol 12-myristate 13-acetate; 0.1 microM) or intracellular injection of CaCl2 (estimated concentration of 1 microM). These effects diminished in the presence of staurosporine (1 microM) or EGTA (estimated concentration of 5 mM). These results suggest that both activation of protein kinase C and an increase in intracellular Ca2+ contribute to the suppression.

Visualization of histamine H1 receptors in dog brain by positron emission tomography.

Histamine H1 receptors were visualized in the living dog brain using [11C]pyrilamine or [11C]doxepin by positron emission tomography (PET). The regional distribution of these carbon-11 labeled compounds in the brain corresponded well with that of the histamine H1 receptors separately determined by in vitro binding assay. The radioactivity in the brain was reduced by treatment with triprolidine (1 mg/kg), a histamine H1 antagonist. The results of our study indicate that it is feasible to visualize histamine H1 receptors in human brain using these 11C-labeled compounds and PET.

Modification of the baroreceptor reflex by superfusion of the canine posterior hypothalamus with tetrodotoxin.

The role of the posterior hypothalamus in the baroreceptor reflex was investigated in dogs. The posterior hypothalamus was superfused bilaterally through push-pull cannulae with artificial cerebrospinal fluid. After bilateral superfusion with tetrodotoxin (10(-5) M) for 120 min, the increases in mean arterial pressure and heart rate induced by the electrical stimulation of the posterior hypothalamus were almost abolished, and the resting mean arterial pressure was significantly decreased. Under these circumstances the increases in mean arterial pressure and heart rate in response to bilateral carotid occlusion were significantly reduced. The decrease in mean arterial pressure induced by intravenous injection of sodium nitroprusside (5 micrograms/kg) was significantly augmented while the associated increase in heart rate was suppressed. These results suggest that the posterior hypothalamus plays an important role in maintaining the resting arterial pressure and in the baroreceptor reflex to increase the arterial pressure and heart rate.

Further evidence for postganglionic actions of prostaglandin F2 alpha and histamine in the submandibular gland of the dog.

To test the possibility that prostaglandin F2 alpha and histamine might produce salivary and vasodilator responses by stimulation of preganglionic parasympathetic nerve fibers in the submandibular gland the responses to the two substances of chronically decentralized glands were compared with those of acutely decentralized ones in anesthetized dogs. No significant differences in responses to either prostaglandin F2 alpha or histamine were noted between chronically decentralized glands and acutely decentralized ones. Thus, it was concluded that prostaglandin F2 alpha and histamine only stimulate parasympathetic postganglionic neurons within the gland and neither of the responses is related to non-cholinergic ganglionic transmission.

Characterization of neuronal and vascular histamine receptors mediating the salivary and vasodilator responses to histamine of the dog submandibular gland.

The submandibular gland in situ was perfused with blood through the glandular artery at constant pressure in anesthetized dogs, and all drugs were administered intra-arterially. During infusion of metiamide, histamine and 2-(2-pyridyl)ethylamine (PEA) produced salivary and vasodilator responses consisting of an early and a late component. The dose-response curves for respective components of the salivary and vasodilator responses to PEA were parallel with the corresponding curves for histamine and in producing these responses PEA was about 40 times less potent than histamine on a molar basis. During infusion of mepyramine, histamine and dimaprit produced only the early vasodilator response. The dose-vasodilator response curves to histamine and dimaprit were parallel, and dimaprit was about 750 times less potent than histamine on a molar basis. The present results support the conclusion obtained in a previous study that neuronal histamine receptors mediating the whole salivary and the late vasodilator response are exclusively of the H1-type and vascular histamine receptors mediating the early vasodilator response consist of both H1-and H2-type although the former is predominant.

Cloning and modulation by endothelin-1 of rat cardiac K channel.

We have cloned a delayed rectifier type K channel from rat heart (RH1). RH1 was identical to the rat brain K channel BK2 and differed from recently cloned rat cardiac K channel RAK by one amino acid residue. Endothelin receptors(ETRs)-mediated modulation of RH1 current (IRH1) was studied using Xenopus oocyte expression system. Activation of two different subtypes of ETRs by endothelin-1 equally suppressed the amplitude of IRH1. Stimulation of phosphatidylinositol turnover will probably be responsible for the suppression.

Amelioration of heart failure by MDL 17043 and MDL 19205, novel positive inotropic drugs, in dog heart-lung preparations.

The efficacies of MDL 17043 and MDL 19205 in ameliorating heart failure were assessed in dog heart-lung preparations in which cardiac function had been severely depressed by pentobarbital. Six preparations were used for each drug. Both drugs in doses of 1-30 mumol similarly improved cardiac function in a dose-dependent manner and at 30 mumol improved it beyond control values. AV conduction impaired by pentobarbital was restored by 30 mumol of the 2 drugs. In these doses, however, neither of the drugs produced a significant increase in heart rate or arrhythmias. These results indicate that the 2 drugs would be of use in the treatment of heart failure.

The mechanism underlying the vasodilator action of bunitrolol: contribution of alpha 1-adrenoceptor blocking action.

The mechanism of the vasodilator action of bunitrolol was investigated in pentobarbital-anesthetized dogs. When injected intraarterially, bunitrolol increased blood flow through the femoral arterial bed more effectively than that through the vascular bed of the left anterior descending coronary artery (LAD). The former is rich in alpha-adrenoceptors and tonically controlled by the sympathetic nerves, whereas the latter is not. Intraarterial prazosin increased femoral flow but not LAD flow, whereas intraarterial nitrendipine increased equieffectively both femoral and LAD flows. In the saphenous arterial bed of dogs that also underwent spinal anesthesia and received atropine and nadolol, intravenous bunitrolol suppressed more effectively vasoconstrictor responses to saphenous nerve stimulation than those to intraarterial norepinephrine. These effects of bunitrolol were similar to those of prazosin and dissimilar to those of yohimbine. In similarly treated dogs, bunitrolol suppressed more effectively increases in mean systemic arterial pressure in response to methoxamine than those to B-HT 920. From these results, it was concluded that an alpha 1-adrenoceptor blocking action is mainly involved in the acute vasodilator effect of bunitrolol. This action may also contribute to the decrease in total peripheral resistance seen in hypertensive patients treated chronically with bunitrolol.

Activation of purified calcium channels by stoichiometric protein phosphorylation.

Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of 45Ca2+ uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of 45Ca2+ uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd2+, Ni2+, and Mg2+. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels.

Improvement by denopamine (TA-064) of pentobarbital-induced cardiac failure in the dog heart-lung preparation.

The efficacy of denopamine, an orally active beta 1-adrenoceptor agonist, in improving cardiac failure was assessed in dog heart-lung preparations. Cardiac functions depressed by pentobarbital (118 +/- 28 mg; mean value +/- SD) such that cardiac output and maximum rate of rise of left ventricular pressure (LV dP/dt max) had been reduced by about 35% and 26% of the respective controls were improved by denopamine (10-300 micrograms) in a dose-dependent manner. With 100 micrograms denopamine, almost complete restoration of cardiac performance was attained, associated with a slight increase in heart rate. No arrhythmias were induced by these doses of denopamine. The results warrant clinical trials of denopamine in the treatment of cardiac failure.

Selectivity and potency of agonists for the three subtypes of cloned human beta-adrenoceptors expressed in Chinese hamster ovary cells.

The selectivities, potencies and efficacies of beta3-adrenoceptor (beta3-AR) agonists on human three beta-AR subtypes expressed in Chinese hamster ovary (CHO) cells were investigated using radioligand binding assay and cyclic AMP (cAMP) accumulation assay. The three beta-AR subtypes showed the nature of G protein-coupled receptors with the constitutive activity. BRL37344, CL-316,243 and a newly synthesized beta3-AR agonist N-5984, 6-[2-(R)-[[2-(R)-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-2,3-dihydro-1,4-benzodioxine-2-(R)-carboxylic acid, were compared for the potency and selectivity for the beta3-AR. In the radioligand binding assay, the affinity of N-5984 for beta3-ARs was 14, 70 and 220 times more potent than those of BRL37344, isoproterenol and CL-316,243, respectively. N-5984 had higher selectivity than BRL37344 for human beta3-ARs compared with either for beta1-ARs or beta2-ARs. N-5984 showed higher potency and intrinsic activity of cAMP production than BRL37344 in CHO cells expressing the beta3-ARs. CL-316,243 had almost no activity of cAMP production in CHO cells expressing any subtype of beta-ARs. These results indicate that N-5984 is the most potent and selective agonist for human beta3-ARs than any other agonists tested.

Differential sensitivity of Kv1.4, Kv1.2, and their tandem channel to acidic pH: involvement of a histidine residue in high sensitivity to acidic pH.

We studied the effects of acidic pH on the currents through voltage-gated K+ channels, Kv1.2, Kv1.4, and their tandem construct (Kv1.4-Kv1.2). Kv1.4 currents were inhibited considerably under acidic pH in a voltage-independent manner, whereas Kv1.2 currents were less inhibited in an apparently voltage-dependent manner. The apparent voltage-dependent block of Kv1.2 currents was mostly ascribed to the shift of activation voltage, which is probably due to surface charge effects of H+ ions. Mutagenesis analysis identified the histidine residue at 508 (H508) in the S5-H5 linker as a molecular determinant of pH sensitivity of Kv1.4. Currents through the tandem channel showed intermediate characteristics between the two parent channels in both sensitivity and voltage dependence of pH effects. Our results suggest that 1) the H508 plays a critical role in determining pH sensitivity of Kv1.4; and 2) the two parent channels, Kv1.2 and Kv1.4, are involved in determining pH sensitivity and apparent voltage dependence of the tandem channel.