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R A Janis

Publications and source records attributed to R A Janis.

At least 19 recordsLinked to original sources
PubMed1993

Effects of calcium channel antagonists on the phosphorylation of major protein kinase C substrates in the rat hippocampus.

K(+)-induced depolarization of rat hippocampal slices resulted in significant increases in the phosphorylation state of myristoylated, alanine-rich C kinase substrate (MARCKS; also known as 87K, pp80) and neuromodulin [also known as growth associated protein 43 (GAP43), B50, F1] as determined by back-phosphorylation using protein kinase C. The effect of organic and inorganic Ca2+ antagonists on the phosphorylation of these major protein kinase C substrates in the rat hippocampus was studied to determine whether Ca2+ influx through L- or N-type voltage-sensitive Ca2+ channels was required for the phosphorylation changes observed. The depolarization-induced changes appeared to be dependent on extracellular Ca2+, based on evidence indicating that the chelation of extracellular Ca2+ with ethylene glycol-bis (beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibited these changes. In addition, pretreatment of the slices with 500 microM Cd2+, but not 300 nM nimodipine, 10 microM omega-conotoxin GVIA or 10 microM MK-801, blocked the K(+)-induced change in phosphorylation. These results suggest that K(+)-induced changes in the phosphorylation of MARCKS and neuromodulin are mediated by Ca(2+)-dependent mechanisms other than, or in addition to, those sensitive to the organic Ca2+ channel antagonists employed.

Animals↗
PubMed1993

High affinity binding of a potassium channel agonist to intact rat insulinoma cells.

The specific binding of a novel tritiated K+ channel opener, [3H]BAY X 9228, has been characterized in a rat insulinoma (RINm5F) cell line. The KD was 2.1 nM and Bmax 50 fmol/mg total protein as determined by saturation analysis. The high affinity binding to intact cells was inhibited by pinacidil and by a series of BAY X 9228 analogs with an activity sequence correlating well with that for producing glyburide-reversible relaxation of partially depolarized rat aorta. This represents the first report of the specific binding of a K+ channel opener to cultured cells.

Animals↗
PubMed1993

Isolation, identification and synthesis of an endogenous arachidonic amide that inhibits calcium channel antagonist 1,4-dihydropyridine binding.

This study was part of a broad search for endogenous regulators of L-type calcium channels. The screening for active fractions was done by measuring inhibition [3H]1,4-dihydropyridine (DHP) binding to rat cardiac and cortex membranes. An inhibitory fraction, termed lyophilized brain hexane-extractable inhibitor (LBHI), was isolated from hexane extracts of lyophilized calf brain. The active substance was purified by a series of chromatographic steps. 13C nuclear magnetic resonance (NMR), 1H coherence spectroscopy (COSY) NMR and fast atom bombardment (FAB) mass spectroscopy suggested that LBHI was N-arachidonic acid-2-hydroxyethylamide. Synthesis of this substance and subsequent high performance liquid chromatography (HPLC) and NMR analysis confirmed this structure. Synthetic LBHI (SLBHI) inhibited [3H]DHP binding to rat cortex membranes with an IC50 value of congruent to 15 microM and a Hill coefficient of congruent to 2. Saturation analysis in the presence of SLBHI showed a change in KD (equilibrium dissociation constant), but not maximal binding capacity (Bmax). SLBHI produced an increased dissociation rate, which, along with the Hill slope of > 1, suggested a non-competitive interaction with the DHP binding site. The results suggest that arachidonic acid derivatives may be endogenous modifiers of the DHP calcium antagonist binding site.

Animals↗
PubMed1992

Effects of nisoldipine upon vasoconstrictor responses and binding of endothelin-1 in ischemic and reperfused rat hearts.

Changes in the vascular response of isolated, perfused rat hearts to endothelin-1 (ET-1) and binding of [125I]ET-1 to cardiac membranes were examined following ischemia (30 min, zero flow) and reperfusion (15 min). Infusion of ET-1 (0, 2.5, 5, 7.5, and 10 x 10(-10) M) increased the control heart perfusion pressure (61, 73, 88, 102, and 117 mm Hg, respectively). Ischemic and reperfused hearts were more sensitive to ET-1 infusion (p less than 0.05 at all concentrations). Nisoldipine (NIS, 1 nM) prevented the rise in sensitivity to ET-1 following ischemia and reperfusion. Two binding sites for [125I]ET-1 were identified in cardiac membranes. High-affinity (Kd = 0.04 nM, Bmax = 0.46 pmol/mg of protein) and low-affinity (Kd = 13.8 nM, Bmax = 5.4 pmol/mg of protein) sites were unchanged by ischemia and reperfusion, and NIS did not change binding constants in control or ischemic and reperfused hearts. Increased ET-1 sensitivity after ischemia may be due to other factors. Endothelium-dependent vasodilation and endothelium-independent vasodilation were significantly reduced following 30 min of ischemia. Inhibition of dilator responses may account for increased ET-1 responses following transient ischemia.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1991

Characterization of binding of the ATP-sensitive potassium channel ligand, [3H]glyburide, to neuronal and muscle preparations.

Binding of the hypoglycemic sulfonylurea, [3H]glyburide, to crude membrane fractions from brain, heart and smooth (intestinal) muscle was saturable, linear with protein concentration and reversible. Saturation analysis revealed high affinity sites (KH values, 7 x 10(-11) M, 5 x 10(-11) M and 6 x 10(-11) M), with Bmax-H values 209, 36 and 23 fmol/mg protein in the brain, heart and smooth muscle, respectively. High affinity [3H]glyburide binding was pharmacologically specific, insensitive to a variety of receptor-active ligands, but sensitive to a series of sulfonylureas, and good, essentially 1:1, correlations were obtained between binding affinities and literature-derived pharmacologic activities. The K+ channel activators, cromakalim, nicorandil, pinacidil and minoxidil were not effective as inhibitors of [3H]glyburide binding. However, diazoxide was a modestly effective inhibitor. Putative low affinity sites (KL values, 3 x 10(-7) M, 1 x 10(-7) M and 2 x 10(-9) M) with Bmax-L values 4956, 336 and 53 fmol/mg protein in brain, heart and smooth muscle, respectively, were identified. Their significance remains to be established. Except for ATP gamma S, the ability of nucleotide triphosphates to inhibit high affinity [3H] glyburide binding was dependent on the presence of Mg++. ADP, in the presence of Mg++, inhibited binding with an IC50 value of 6.3 x 10(-4) M. Nucleotide monophosphates did not inhibit [3H] glyburide binding in the presence or absence of Mg++, whereas in the presence of Mg++, nucleotide triphosphates were equally potent inhibitors of binding. The rank order potency for nucleotide diphosphate inhibition of binding, in the presence of Mg++, is ADP greater than GDP greater than IDP = UDP. In the absence of Mg++, [3H]glyburide binding shows a biphasic response to ADP, and the inhibition of binding by ADP was prevented by ATP. It is suggested that this biphasic response is the result of a second nucleotide binding site.

Adenosine Triphosphate↗
PubMed1989

Recent development in calcium channel antagonists.

The introduction of the Ca2+ channel antagonists, including the clinically available verapamil, nifedipine and diltiazem, into cardiovascular medicine served to initiate much work directed to the elucidation of their mechanisms of action at voltage-dependent Ca2+ channels. The Ca2+ channel ligands (both activator and antagonist) interact with the channel in a state-dependent manner associating preferentially with open states (activators) or open and inactivated states (antagonists). Both frequency- and voltage-dependent interactions occur with activator and antagonist drugs. These effects underlie the antiarrhythmic activity of verapamil and the vascular smooth muscle selectivity of 1,4-dihydropyridines. Selectivity of action occurs from a combination of factors including the relative Ca2+ demands of the system, the stimulus mode and state dependence of interaction, the agonist/antagonist character of the channel ligand and the category of Ca2+ channel involved.

Animals↗
PubMed1988

Isolation and characterization of a fraction from brain that inhibits 1,4-[3H]dihydropyridine binding and L-type calcium channel current.

Bovine brain was subjected to acid extraction and several purification steps. A fraction from brain that eluted from C18 reverse-phase columns at 30-35% acetonitrile inhibited [3H]nitrendipine binding to cardiac membranes. Further purification of this fraction on a sizing column in the presence of 40% acetonitrile yielded a low molecular mass fraction (less than 1 kDa) that produced a time- and voltage-dependent inhibition of L-type (but not T-type) Ca2+-channel current in GH3 cells. The results suggest that this fraction contains an endogenous substance that binds directly to slowly-inactivating Ca2+ channels and thereby inhibits current flow.

Animals↗
PubMed1987

Comparative aspects and temperature dependence of [3H]1,4-dihydropyridine Ca2+ channel antagonist and activator binding to neuronal and muscle membranes.

Binding of [3H]nitrendipine, [3H]nimodipine, and (+)[3H]PN 200-110 to microsomal preparations of guinea pig smooth and cardiac muscle and brain synaptosomes revealed high affinity interaction with KD values in the sequence, (+)PN 200-110 greater than nitrendipine greater than nimodipine. Bmax values for a particular tissue were independent of the 1,4-dihydropyridine employed in radioligand binding at 25 degrees C. The temperature dependence of [3H]nitrendipine binding in cardiac and smooth muscle microsomal preparations and brain synaptosomes was measured from 0 degrees to 37 degrees C and for skeletal muscle preparations from 0 degrees to 30 degrees C. Bmax values increased with temperature for cardiac membranes, but did not vary in other tissues. van't Hoff plots were nonlinear in all tissues, enthalpy and entropy changes becoming increasingly negative with increasing temperature. Competition binding of the activator-antagonist enantiomeric 1,4-dihydropyridine pairs of Bay k 8644 and PN 202-791 for [3H]nitrendipine in smooth muscle did not reveal significant thermodynamic differences between activator and antagonist molecules.

Animals↗
PubMed1987

[3H]BAY K 8644, a 1,4-dihydropyridine Ca++ channel activator: characteristics of binding to high and low affinity sites in cardiac membranes.

The binding of [3H]BAY K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)- pyridine-5-carboxylate] to high and low affinity sites in rabbit ventricular membranes was characterized. Binding affinities were 0.66 and 138 nM at 15 degrees C and 9.1 and 72 nM at 37 degrees C, for the high and low affinity sites, respectively, and binding site densities were 0.3 and 14 pmol/mg at 15 degrees C and 0.41 and 1.4 pmol/mg at 37 degrees C, for the respective sites. The modification of high affinity [3H]BAY K 8644 binding by verapamil, diltiazem, tiapamil, Ca++ and EDTA appeared to be the same as that for nitrendipine binding, consistent with the hypothesis that the high affinity binding site for [3H]BAY K 8644 on isolated membranes is the same as the 1,4-dihydropyridine antagonist binding site. The binding of [3H]BAY K 8644 to a low affinity binding site was modified by temperature, Ca++ and diltiazem, but the lack of stereoselectivity, lack of denaturation by heat and the large number of sites indicated that most of the low affinity binding sites were not associated with Ca++ channels. It is concluded that the high affinity binding site for BAY K 8644 is associated with Ca++ channels, and is modified by at least some of the factors that modify the binding site for Ca++ channel antagonists, whereas many or all of the low affinity binding sites detected are not related to Ca++ channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1986

Photoaffinity labelling of a 33-35,000 dalton protein in cardiac, skeletal and smooth muscle membranes using a new 125I-labelled 1,4-dihydropyridine calcium channel antagonist.

The binding sites for Ca2+ channel antagonists were probed using Bay P 8857 [2-iodoethyl isopropyl 1,4-dihydropyridine-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarbox ylate] that has been radiolabelled with 125I. This drug was shown to bind with high affinity to cardiac, smooth, and skeletal muscle membranes, with a KD approximately equal to 0.3 nM. A protein of molecular weight 33-35,000 daltons was specifically and irreversibly radiolabelled after irradiation of cardiac, skeletal and aortic smooth muscle membranes, incubated with the [125I]-Bay P 8857. The peptide labelled by 1,4-dihydropyridine binding therefore appears similar in size for cardiac, skeletal, and smooth muscle. This data suggests that of the three peptide subunits which reportedly comprise the skeletal and cardiac muscle 1,4-dihydropyridine receptor complex, the 33-35,000 dalton peptide contains the dihydropyridine binding site.

Affinity Labels↗
PubMed1984

Interaction of phenoxybenzamine with muscarinic receptors and calcium channels.

Phenoxybenzamine (POB, 10(-6) - 10(-4) M) inhibited the responses of guinea pig ileal longitudinal smooth muscle to both muscarinic agonists and K+-depolarization but was more effective against the agonist-induced responses. POB inhibited binding of both the muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) and the Ca2+ channel antagonist [3H]nitrendipine and was, paralleling its effects on mechanical responses, more effective against [3H]QNB binding. POB reduced specific [3H]QNB binding by a reduction in Bmax with no change in KD, but inhibited [3H]nitrendipine binding by reducing KD with no effect on Bmax. It is suggested that the activity of POB against Ca2+ channels may underlie the ability of POB, and other 2-halogenoethylamines, to inhibit a wide variety of apparently discrete pharmacological events.

Animals↗
PubMed1984

Comparison of high affinity binding of calcium channel blocking drugs to vascular smooth muscle and cardiac sarcolemmal membranes.

The binding of the 1,4-dihydropyridine calcium channel blocker [3H]nitrendipine to canine cardiac sarcolemmal and bovine aortic membranes was found to be rapid, specific, saturable, and reversible. Dissociation constants (Kd) determined by Scatchard analysis were 0.14 and 0.16 nM and the maximal numbers of binding sites (Bmax) were 0.96 +/- 0.2 and 0.08 +/- 0.01 pmole/mg protein for cardiac and aortic membranes respectively. Values of Kd calculated from kinetic data were approximately 0.10 nM for both membrane preparations. Competition assays with the enantiomers of a nisoldipine derivative indicated that [3H]nitrendipine binds stereoselectively. The order of potency of several nifedipine analogs for inhibition of binding of [3H]nitrendipine to cardiac and aortic membranes paralleled their relative potencies for inhibition of contraction in smooth muscle. It is concluded that the high affinity binding sites for nitrendipine in bovine aortic smooth muscle membranes are similar to those of canine ventricular sarcolemma.

Animals↗
PubMed1984

Specific binding of a calcium channel activator, [3H]BAY k 8644, to membranes from cardiac muscle and brain.

BAY k 8644 is a member of a new class of drugs that directly activates Ca2+ channels. This 1,4-dihydropyridine was found to bind to both high and low affinity sites on rabbit ventricular microsomes and guinea pig brain synaptosomes. The dissociation constant obtained from Scatchard analysis with [3H]BAY k 8644 was 2 to 3 nM for the high affinity binding site, and the estimated maximal number of binding sites was 0.8 and 0.4 pmol/mg protein for heart and brain membranes, respectively, at 15 degrees C. Competition between nitrendipine and [3H]BAY k 8644 indicated a common high affinity binding site for Ca2+ channel activators and antagonists. The results suggest that the 1,4-dihydropyridine Ca2+ channel antagonists do not act as simple channel plugs.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1984

Stimulation of Na+,K+-ATPase of isolated smooth muscle membranes by the Ca2+ channel inhibitors, nimodipine and nitrendipine.

Nimodipine (0.015 to 1.5 microM) increased Na+, K+-ATPase activity by 70-120% in isolated smooth muscle membranes. At 0.015 microM, nitrendipine, but not nifedipine, verapamil or diltiazem, also activated this enzyme. Nimodipine stimulated this Na+, K+ATPase three times more than nitrendipine at 15 nM. Marked stimulation of Na+,K+-ATPase by nimodipine was seen in membranes from rat and guinea pig aorta and rat vas deferens, but not in membranes from guinea pig heart or brain. Although it is not known whether these results are applicable to intact cells, the results are consistent with the hypothesis that vasodilation produced by nimodipine and nitrendipine may be due not only to inhibition of Ca2+ entry but also to the stimulation of the Na+ pump.

Animals↗
PubMed1984

BAY K 8644, a 1,4-dihydropyridine Ca2+ channel activator: dissociation of binding and functional effects in brain synaptosomes.

K+-stimulated 45Ca2+ uptake into rat brain and guinea pig cerebral cortex synaptosomes was measured at 10 s and 90 s at K+ concentrations of 5-75 mM. Net increases in 45Ca2+ uptake were observed in rat and guinea pig brain synaptosomes. 45Ca2+ uptake under resting or depolarizing conditions was not increased by the 1,4-dihydropyridine BAY K 8644, which has been shown to activate Ca2+ channels in smooth and cardiac muscle. High-affinity [3H]nitrendipine binding in guinea pig synaptosomes (KD = 1.2 X 10(-10) M, Bmax = 0.56 pmol mg-1 protein) was competitively displaced with high affinity (IC50 2.3 X 10(-9) M) by BAY K 8644. Thus high-affinity Ca2+ channel antagonist and activator binding sites exist in synaptosome preparations, but their relationship to functional Ca2+ channels is not clear.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1984

Some recent pharmacological findings with nitrendipine.

The available evidence indicates that nitrendipine and other dihydropyridines with a similar pharmacological action exert their therapeutic effects by inhibiting Ca2+ channels. In our recent experiments, nitrendipine was shown to block K+-stimulated 45Ca2+ uptake and K+-induced contractions of isolated rabbit aortic rings. Its IC50 were 4.7 and 8.9 nM for inhibition of Ca2+ uptake and of contractions, respectively. There was no statistically significant difference between the two values. At higher concentrations, nitrendipine also reduced norepinephrine-induced 45Ca2+ uptake and norepinephrine-induced contractions of rabbit aortic strips. The norepinephrine-induced contractions were only slightly (21%) reduced by nitrendipine at 10 microM. Nitrendipine at 10 nM and higher concentrations inhibited K+- or angiotensin-II-(AII) induced release of aldosterone from isolated bovine adrenal glomerulosa cells. The drug was more potent and more effective in inhibiting K+- than AII-induced aldosterone release. Dantrolene, 25 microM, enhanced the inhibitory activity of nitrendipine on AII-stimulated aldosterone release. Acute renal failure produced by either glycerol or gentamicin in rats was antagonized by nitrendipine at oral doses of 15-25 mg/kg/day. Our studies confirmed previously reported observations that the usefulness of nitrendipine in the treatment of hypertension may be determined not only by its vasodilator action. We demonstrated that nitrendipine has a direct inhibitory effect on the release of aldosterone from adrenal glomerular cells. In addition to a previously described diuretic action, nitrendipine was shown to have renal cytoprotective activity.

Acute Kidney Injury↗
PubMed1984

The 1,4-dihydropyridine receptor: a regulatory component of the Ca2+ channel.

Evidence that nitrendipine (2,6-dimethyl-3-carbomethoxy-5-carbomethoxy-4-(3-nitrophenyl)1,4- dihydropyridine) and other 1,4-dihydropyridines interact at a specific site to antagonize Ca2+ channel function is reviewed. This evidence derives from electrophysiologic and pharmacologic studies and is confirmed by [3H]radioligand binding to excitable tissues. The availability of 1,4-dihydropyridine activator ligands including BAY K 8644 (2,6-dimethyl-3-carbomethyl-5-nitro-4-(2-trifluoromethyl)1,4- dihydropyridine) suggests that this site is intimately concerned with Ca2+ channel regulation. The actions of the 1,4-dihydropyridine ligands are discussed with particular reference to their tissue selectivity and the functional relationships of binding sites to Ca2+ channels.

Animals↗