Tomohiko Ishikawa

The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells

We reported that one of the isoquinolinesulfonamide derivatives, KN-62, is a potent and specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Tokumitsu, H., Chijiwa, T., Hagiwara, M., Mizutani, A., Terasawa, M. and Hidaka, H. (1990) J. Biol. Chem. 265, 4315-4320). We have now investigated the inhibitory property of a newly synthesized methoxybenzenesulfonamide, KN-93, on CaMKII activity in situ and in vitro. KN-93 elicited potent inhibitory effects on CaMKII phosphorylating activity with an inhibition constant of 0.37 microM but this compound had no significant effects on the catalytic activity of cAMP-dependent protein kinase, Ca2+/phospholipid dependent protein kinase, myosin light chain kinase and Ca(2+)-phosphodiesterase. KN-93 also inhibited the autophosphorylation of both the alpha- and beta-subunits of CaMKII. Kinetic analysis indicated that KN-93 inhibits CaMKII, in a competitive fashion against calmodulin. To evaluate the regulatory role of CaMKII on catecholamine metabolism, we examined the effect of KN-93 on dopamine (DA) levels in PC12h cells. The DA levels decreased in the presence of KN-93. Further, the tyrosine hydroxylase (TH) phosphorylation induced by KCl or acetylcholine was significantly suppressed by KN-93 in PC12h cells while events induced by forskolin or 8-Br-cAMP were not affected. These results suggest that KN-93 inhibits DA formation by modulating the reaction rate of TH to reduce the Ca(2+)-mediated phosphorylation levels of the TH molecule.

Effects of Cilostazol, a Selective cAMP Phosphodiesterase Inhibitor on the Contraction of Vascular Smooth Muscle

The effects of cilostazol (OPC-13013, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quin olinone) on cyclic nucleotide metabolism and Ca2+-induced contraction of intact and skinned rabbit arterial smooth muscles were investigated. The concentrations of cilostazol producing 50% inhibition of cyclic adenosine monophosphate phosphodiesterase and Ca2+-dependent cyclic nucleotide phosphodiesterase were 0.4 microM and above 100 microM, respectively. This compound has no significant effect on adenylate cyclase in concentrations of up to 100 microM. Addition of cilostazol increased significantly the cAMP content without significant effect on cyclic guanosine monophosphate level of rabbit thoracic aorta in the presence of forskolin. Moreover, the ED50 value of cilostazol in relaxation of rabbit mesenteric arterial strips was decreased selectively by addition of 0.01 microM forskolin, which alone at this concentration has no effect on vascular contraction. Cilostazol of up to 30 microM did not suppress the Ca2+-induced contraction of the chemically skinned rabbit mesenteric artery. Therefore, cilostazol may produce the relaxation of intact vascular smooth muscle by its inhibition of cyclic adenosine monophosphate hydrolysis.

Modification of cardiac sodium current by intracellular application of cAMP

We examined the effects of intracellular perfusion of cyclic adenosine monophosphate (cAMP) on the sodium current (INa) of guinea-pig ventricular myocytes, using the whole-cell clamp technique. INa was elicited by depolarizing voltage steps (−20 mV) from a variety of holding potentials (−120 to −50 mV), under conditions of 60 mM extracellular Na+ concentration ([Na+]0) and at the temperature of 24–26°C.Intracellular perfusion of cAMP decreased the INa elicited from the holding potentials less negative than −90 mV. In the presence of 1 mM cAMP, for example, the peak INa elicited from −80 mV decreased from 6.0±2.0 nA to 4.0±2.2 nA (mean±SD, P<0.02, n=7) within 3–6 min. In the presence of extracellular 3-isobutyl-1-methylxanthine (IBMX, 20 μM), much lower concentrations of cAMP (0.2 mM) yielded a comparable effect. On the other hand, intracellular perfusion of cAMP increased the INa elicited from very negative holding potentials (<−100 mV). For instance, the application of cAMP (1 mM) increased the INa elicited by step depolarizations from −120 mV (to −20 mV), from 9.9±2.1 nA to 11.0±3.1 nA (P<0.05, n=5).The former effect was attributed to a marked shift of the steady-state inactivation curve of INa to the negative direction; the voltage of half-inactivation shifted from −77.9±1.0 to −83.5±1.4 mV, or by −5.6 mV. The latter effect may be explained by increases in maximum available conductance of INa. Extracellular application of isoproterenol (1 μM) also decreased the INa evoked from a holding potential of −80 mV, whereas it increased the INa elicited from more negative potentials of −120 mV. These effects of isoproterenol were reversible and markedly attenuated in the presence of a specific inhibitor of cAMP-dependent protein kinase, H-89 {N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide}, an isoquinolinesulphonamide derivative, in the extracellular medium (2–10 μM) and a protein kinase inhibitor (Walsh inhibitor) in the pipette solution (40 μM). H-89 (10 and 30 μM) affected neither the adenylate cyclase activity prepared from rabbit ventricular muscles, nor the isoproterenol-mediated increases in the cAMP content in guinea-pig ventricular muscles. Our observations suggest that the increase in intracellular cAMP modulates the function of cardiac Na channels, preferentially by stimulating cAMP-dependent protein kinase, with subsequent phosphorylation of the channel protein.

Low level hyperlipidemia impairs endothelium-dependent relaxation of porcine coronary arteries by two mechanisms. Functional change in endothelium and impairment of endothelium-dependent relaxation by two mediators

We evaluated the effect of a low level of hyperlipidemia and the effects of in vitro exposure to atherogenic lipoproteins (LDL, VLDL) on the vascular responsiveness of isolated porcine coronary arteries. Firstly we studied the change in vascular responsiveness induced by feeding a cholesterol-rich diet to pigs for 4 and 9 weeks (C4 and C9 pigs). The serum cholesterol level in pigs fed a cholesterol-rich diet reached 218.5 +/- 32.9 mg/dl compared with 85.5 +/- 8.4 mg/dl in the controls. Segments of the left descending coronary artery were examined. The contraction induced by KCl or prostaglandin F2 alpha was not altered significantly by hypercholesterolemia nor was the relaxation induced by the Ca2+ ionophore, A23187, or by nitroglycerin. Endothelium-dependent relaxation (EDR) evoked by high, but not low, concentrations of bradykinin was reduced in the C4 pigs as compared with those in normal animals. EDRs evoked by bradykinin, substance P, and serotonin were significantly reduced in C9 pigs. Histologically, as observed by light and electron microscopy, fatty changes or intimal thickenings were not seen in the coronary arteries of the C4 pigs. Minimal changes (intimal thickening and fragmentation of internal elastic lamina) were observed only in parts of arteries of the C9 pigs. Secondly, the direct effects of LDL and VLDL on vascular responsiveness were studied. Although preincubation with LDL inhibited the EDR caused by exposure to bradykinin and A23187 in the coronary arteries of normal and cholesterol-fed pigs, preincubation with LDL inhibited the arterial relaxation induced by exposure to substance P or serotonin in both the C4 and the C9 pigs, but not in the control animals. The degree of inhibition was especially marked in the C9 pigs. The inhibitory effect of VLDL on EDR was weaker than that of LDL. Indomethacin (5 microM) did not alter this inhibitory effect of lipoproteins. Neither LDL nor VLDL had any effect on the vascular relaxation induced by nitroglycerin. These results are consistent with the idea that endothelium-dependent arterial relaxation is attenuated even at the very early stage of cholesterol-induced atherosclerosis. Atherogenic lipoproteins may further impair the decreased EDR in the arteries of hyperlipidemic pigs by two factors: one released on stimulation with bradykinin and the calcium ionophore A23187, the other released on stimulation with substance P and serotonin.

Triphasic response of rat intracerebral arterioles to increasing concentrations of vasopressin In vitro

To determine how vasopressin affects the vascular tone of the smaller cerebral arterioles, we carried out an in vitro study of isolated and cannulated intracerebral arterioles of rats. We found that increasing concentrations of vasopressin induced a triphasic response of vasodilation (10−12–10−11 M), vasoconstriction (10−10–10−8 M), and vasodilation stabilizing to control diameter (10−7–10−6 M) and that the maximum constriction was twice the maximum dilation in these smaller arterioles [21.2 ± 13.1% (mean ± SD) decrease in diameter vs. 11.2 ± 5.7% increase]. Pretreatment of the arterioles with NG-monomethyl-l-arginine (10−4 M), a specific inhibitor of endothelium-derived relaxing factor, abolished the vasopressin-induced vasodilation and significantly increased the vasoconstriction. These results suggest that these arterioles were maintained in a dilated state by an endothelium-derived relaxing factor activated by vasopressin. Both vasodilation and vasoconstriction were found to be mediated through vasopressin V1 receptors in a study of arterioles pretreated with d(CH2)5Tyr(Me)arginine vasopressin (10−6 M), a vasopressin V1 receptor antagonist. These results support the hypothesis that vasopressin may constrict smaller cerebral arterioles while simultaneously dilating larger cerebral arteries. Our results also suggest that vasopressin may aggravate cerebral ischemia in pathological conditions, such as subarachnoid hemorrhage, when the arteriolar response to vasopressin shifts from vasodilation to vasoconstriction due to increased vasopressin levels in plasma and CSF and impaired endothelium-derived relaxation.

β-Migrating Very Low Density Lipoprotein Attenuates Endothelium-Dependent Relaxation in Rabbit Atherosclerotic Aortas

We studied the effects of beta-migrating very low density lipoprotein (beta-VLDL) on the vascular responses of isolated thoracic aortic preparations taken from normal and hypercholesterolemic rabbits. The endothelium-dependent relaxation induced by acetylcholine or adenosine triphosphate (ATP) was attenuated in the arteries from hypercholesterolemic rabbits that were fed a cholesterol-rich diet for 12 weeks. In these aortas, the lesional circumference of the atherosclerotic plaques (fatty streaks) was only 12.18 +/- 1.98%. The relaxation induced by the Ca2+ ionophore A23187 or nitroglycerin was not altered. Preincubation with beta-VLDL significantly inhibited the relaxation due to acetylcholine, ATP, or A23187, especially in the aortas of hypercholesterolemic rabbits. However, beta-VLDL did not alter the response to nitroglycerin. Preincubation with high density lipoprotein had no significant effect on vessel relaxation. These results indicated that endothelium-dependent relaxation was already inhibited in the early stages of atherosclerosis, and that the atherogenic lipoprotein, beta-VLDL, further inhibited endothelium-dependent relaxation in atherosclerotic aortas. It may be that beta-VLDL also plays a role in determining the level of vascular tonus in atherosclerosis.

Some Observations of the Caravaning Behavior in the Musk Shrew (Suncus Murinus)

Observations of caravaning were made on the domesticated musk shrew (Suncus murinus) with particular reference to its developmental aspects. In a rearing box (Experiment I), caravaning appeared from 10 to 22 days of age, peaking at 16 days. In the open-field test (Experiment II), it occurred over a range from 5 to 22 days, with its peak at around 13 days. Its formation patterns also changed with age of days; five patterns were identified in this situation. Another type of test (encounter test; Experiment III) showed a sensitive period similar to that obtained in Experiment II. Caravaning peaked at a period shifting from the mother-initiative stage to the young-initiative stage in the mother-offspring relation. Inanimated model released caravaning, as was expected (Experiment IV). It occurred for the same period, peaking at the same age of days, although on closer examination it was found that the smaller peak obtained in Experiment II disappeared. The findings of the present study suggest that caravaning plays an important role in the mother-offspring relation of this species. Developmental changes in caravaning seem to reflect the maturation process of sensory functions. Possibilities of releasers in caravan formation were discussed by referring to histological and electrophysiological investigations.

A new and potent calmodulin antagonist, HF-2035, which inhibits vascular relaxation induced by nitric oxide synthase

HF-2035, 2-[N-(2-aminoethyl)-N-(2,4,5-trichlorobenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, was synthesized and its effects on calmodulin-dependent enzymes were investigated. HF-2035 inhibited calmodulin kinase I, calmodulin kinase II and myosin light-chain kinase with IC50 values of 1.3 microM, 1.6 microM and 68 microM, respectively. HF-2035 also inhibited the activity of recombinant rat neuronal nitric oxide synthase, one of the calmodulin-dependent enzymes, with a Ki of 0.78 microM. Partially purified nitric oxide synthase of rat brain was also inhibited by HF-2035 with an IC50 of 3.2 microM. Kinetic analysis indicated that this inhibitory effect of HF-2035 was competitive with respect to calmodulin. We examined the effects of HF-2035 on constitutive nitric oxide synthase in a bioassay using vascular strips of rabbit carotid artery with and without endothelium. HF-2035 inhibited acetylcholine- and calcium ionophore, A23187 (6S-[6 alpha (2S*,3S*),8 beta (R*),9 beta, 11 alpha]-5- (methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)- ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazol ecarboxylic acid)-induced relaxation of endothelium-intact strips with an ED50 of 1.5 +/- 0.5 microM and 2.8 +/- 1 microM, respectively. This compound, however, did not inhibit N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A), an exogenous nitric oxide donor, -induced relaxation of endothelium-denuded strips. W-7 (N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide) inhibited acetylcholine-induced relaxation with an ED50 of 46 +/- 7 microM, which was 30-fold less potent than HF-2035. HF-2035 was unable to inhibit the activity of the inducible form of nitric oxide synthase in isolated thoracic aorta of rat treated with Escherichia coli lipopolysaccharide. These findings suggest that HF-2035 is a new and potent calmodulin antagonist, and may be used as a mother compound to develop more selective inhibitors of constitutive nitric oxide synthase.

Developmental Changes in the Caravaning Behaviour of the House Musk Shrew (Suncus Murinus)

Development of caravaning behaviour in the house musk shrew (Suncus murinus) was described in detail and discussed in relation to sensory and motor development. With age of pups, caravaning changed in its formation pattern (I-V) as well as its quantitative aspects such as frequency of occurrence, duration and distance. The shift in formation pattern from II to III was simultaneous with the onset of vision (Experiment I). In darkness, frequency of caravaning significantly decreased at ages later than 11 days. This was due to the selective disappearance of Patterns III and V (Experiment II). These findings suggest the role of vision in caravan formation at the later stage of the period of occurrence. After the onset of vision, caravaning is released mostly by visual motion of the object, whereas caravaning at the earlier stage is released nonvisually. Caravaning and the pups locomotor activity were suppressed and delayed until the age of weaning in the enclosure set up to simulate the natural habitat. The period of occurrence was reduced by disappearance of some formation patterns observable in the laboratory open-field and the onset of caravaning exactly synchronized with that of spontaneous and independent locomotion. Development of caravaning in the outdoor enclosure closely resembled the development of Pattern V in the open-field (Experiment III). The adaptive significance of caravaning can be fully understood only by comparing behaviours which occur in different situations. Such an approach could bridge the gap between observations in the laboratory and those in the natural habitat. In Experiment I, caravaning was compared between domesticated laboratory animals and animals at the first two generations of nonsib-mating from wild ones. The age-dependent tendency was more distinct in the latter group. This suggests that mother-offspring relations changed in the process of domestication.

Chelation of copper reduces inhibition by oxidized lipoproteins of endothelium-dependent relaxation in porcine coronary arteries

SummaryWe examined the effect of dialyzing oxidized low-density lipoprotein (oLDL) against Krebs-Ringer solution, in the absence (yielding d-oLDL) or presence (yielding EDTA-oLDL) of ethylenediamine tetraacetic acid (EDTA), to investigate the mechanism that underlies the inhibition of endothelium-dependent relaxation (EDR) by o-LDL. Oxidation of LDL by exposure to Cu2+ resulted in the formation of a thiobarbituric acid-reacting substance (TBARS) and lipid hydroperoxide (LPO). At a concentration of 5mg/dl, d-oLDL markedly attenuated EDR in the porcine coronary artery. Analysis of doLDL by gel filtration revealed that TBARS was distributed in both the lipoprotein and the aqueous phases, whereas LPO was present only in the lipoprotein particles. Lysophosphatidylcholine (LPC), which has been suggested to be responsible for the impairment of EDR by oLDL, was present not only in the lipoprotein but also in the aqueous phase. However, EDR inhibitory activity was observed only in the oLDL particles, not in the aqueous phase. Almost all Cu2+ associated with the oLDL particles was removed by dialysis of oLDL against Krebs-Ringer solution containing EDTA. EDTA-oLDL or native LDL, at concentrations as high as 75mg/dl, exerted only a moderate inhibitory action on EDR, Both TBARS and LPO in EDTA-oLDL were distributed only in the lipoprotein particles, not in the aqueous phase. These results demonstrate that the impairment of EDR by oLDL is related both to LPO and to transition metal ions such as Cu2+ associated with the lipoprotein particles, not to the amount of the TBARS or negative charge, and that factors other than LPC may affect EDR.