Effects of GTP analogues and activation of endogenous protein kinases on photoaffinity labeling with [3H](+)PN200-110 of crude membranes from rat heart and brain.

The effects of GTP analogues and conditions in which various endogenous protein kinases were activated on photoaffinity labeling with [3H](+)PN200-110 (PN) of crude membranes from rat cardiac muscle and whole brain were investigated. Photoaffinity labeling with 20 nM [3H](+)PN of these crude membranes was decreased by 100 microM GTP-gamma-S, but not by 100 microM GTP or 100 microM GDP-beta-S. Similar results were obtained on the effects of GTP and its analogues on the specific binding of 20 nM [3H](+)PN to these crude membranes under the same conditions. Activation of endogenous protein kinases in these crude membranes did not influence the photoaffinity labeling with [3H](+)PN. These results suggested the binding sites, or DPH-sensitive, or L-type, calcium channels in curde membranes from rat cardiac muscle and whole brain are directly or indirectly modulated by endogenous GTP-binding protein, but not by various endogenous protein kinases in these crude membranes.

Photoaffinity labeling with dihydropyridine derivatives of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain.

The characteristics of photoaffinity labeling with the calcium agonist [3H]Bay K 8644 (Bay) and the calcium antagonists [3H]nitrendipine (Nit) and (+)PN200-110 (PN) of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain were investigated. In all these crude membranes, [3H](+)PN (20 nM) was mainly photoincorporated into one protein band with a molecular weight of 30,000 - 41,000 Da. It was also incorporated into some other bands of all these crude membranes. The photoincorporation of [3H](+)PN into these crude membranes was inhibited by the presence of 20 microM unlabeled (+)PN. The photoincorporation of [3H](+)PN into these crude membranes depended on its dose and on the time of UV irradiation. No incorporation of [3H](+)PN was observed in the absence of UV irradiation. The incorporation was not affected by the presence of 1 mM CaCl2 and/or 0.15 M NaCl, but was significantly decreased by 20 microM (+)PN and slightly decreased by 20 microM (-)PN, 20 microM Bay, 1 mM diltiazem, or 1 mM verapamil. Namely, enantiomers of PN caused various extents of stereoselective inhibition of photoaffinity labeling by [3H](+)PN of specific protein bands in these crude membranes. [3H]Nit was photoincorporated into these crude membranes in the same way as [3H](+)PN, but [3H]Bay was not photoincorporated. However, 20 microM unlabeled Nit did not consistently inhibit photoaffinity labeling with [3H]Nit. These findings suggested that measurement of photoaffinity of crude membranes from rat skeletal, cardiac, and uterine muscles and whole brain with [3H](+)PN by UV irradiation is a useful method for investigating the characteristics of the voltage-dependent calcium channels that are affected by 1,4-dihydropyridine derivatives.

Characteristics of specific bindings of nitrendipine and PN200-110 to various crude membranes: induction of irreversible bindings by UV irradiation.

The characteristics of the specific bindings of [3H]nitrendipine (Nit) and [3H](+)PN200-110 (PN) to crude membranes from rat skeletal, cardiac, and uterine muscle and whole brain were investigated, with special interest in the effect of UV irradiation on these bindings. The specific bindings of [3H]Nit and [3H](+)PN to these crude membranes were saturable and reversible. The specific bindings of [3H]Nit to all these membranes except crude skeletal membranes was maximum in the presence of 0.15 M NaCl plus 1 mM CaCl2 and minimal in the absence of these ions, but the specific bindings of [3H](+)PN to these crude membranes was not affected significantly by these ions. A calcium agonist and antagonists inhibited the specific bindings of [3H]Nit and [3H](+)PN to these crude membranes, the order of their inhibitory effects on specific [3H]Nit bindings being roughly Nit greater than or equal to (+)PN greater than or equal to (-)PN much greater than Bay K 8644 (Bay) greater than verapamil (Ver) greater than diltiazem (Dil). In crude skeletal membranes only, PN caused significant stereospecific inhibition. The order of inhibitions of specific [3H](+)PN bindings to these crude membranes was generally (+)PN greater than Nit greater than or equal to (-)PN greater than Bay much greater than Ver greater than or equal to Dil. In all these crude membranes, UV irradiation completely prevented decrease in the amount of specific binding of [3H](+)PN binding on addition of excess unlabeled (+)PN. These findings suggested that [3H]Nit and [3H](+)PN bind to voltage-sensitive calcium channels in crude membranes from rat skeletal, cardiac, and uterine muscle and whole brain, and that UV irradiation changes the specific bindings of [3H]Nit and [3H](+)PN from reversible to irreversible bindings.

Mechanism of the lipid-lowering effect of ethyl all-cis-5,8,11,14,17-icosapentaenoate.

The effect of highly purified ethyl all-cis-5,8,11,14,17-icosapentaenoate (EPA-E) on cholesterol metabolism in rats was examined to clarify the mechanism of its hypolipidemic action. Pretreatment with EPA-E reduced the increase in plasma radioactivity after oral administration of [14C]cholesterol. The conversion of [14C]3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to [14C]mevalonic acid was significantly inhibited in liver microsomes obtained from rats treated with EPA-E. There was an increase in free cholesterol and a marked rise in the eicosapentaenoic acid (EPA) content of phospholipids in these microsomes. EPA-E restored the suppression of biliary secretion induced by feeding a casein-rich diet to bile duct-cannulated rats. Furthermore, when serum lipoprotein (d < 1.210) from rats given EPA-E was i.v. injected into normal rats, a more rapid elimination of cholesterol was observed as compared to that in rats injected with lipoprotein from EPA-E-untreated rats. This rapid clearance was found in the lipoprotein fractions of d < 1.006 and 1.006 < d < 1.063. These findings suggest that EPA-E has an inhibitory effect on intestinal cholesterol absorption and hepatic cholesterol biosynthesis, and an enhancing effect on hepatic biliary secretion. EPA-E would also seem to cause modification of serum lipoproteins, whereby their clearance from the serum is increased.

Ethyl all-cis-5,8,11,14,17-icosapentaenoate modifies the biochemical properties of rat very low-density lipoprotein.

The biochemical properties of serum very low-density lipoprotein (VLDL) were investigated in rats given highly purified all-cis-5,8,11,14,17-icosapentaenoate (EPA), an ethyl-ester derivative (EPA-E). The elution time (gel filtration) of VLDL from EPA-E-treated serum was increased significantly compared with that of the control. EPA-E-treated VLDL isolated by ultracentrifugation exhibited a marked decrease in triglyceride content with a relative increase in cholesterol. In treated VLDL, a significant increase in the ratio of apo E/apo C was observed. There was a remarkable increase in the content of EPA in all the fractions of phospholipids, cholesteryl esters and triglycerides after EPA-E treatment, resulting in n-3 polyunsaturated fatty acid-rich VLDL. EPA-E also reduced the incorporation of [14C]oleate into triglycerides in hepatic microsomes and the rate of hepatic triglyceride secretion. Moreover, lipoprotein lipase activity in heparin-injected plasma was increased in rats given EPA-E without there being an effect on hepatic triglyceride lipase activity. These findings indicate that EPA-E exerts an inhibitory effect on hepatic triglyceride synthesis/secretion and a stimulatory effect on triglyceride degradation, resulting in a reduction in particle size and an increase in the ratio of apo E/apo C. Triglyceride-poor and EPA-rich VLDL may rapidly be converted into the density of intermediate low-density lipoprotein and low-density lipoprotein and/or may be absorbed into the liver rapidly.

[Pharmacological and clinical studies on the long acting cefaclor (S6472)].

A study in which 375 mg of S6472 was orally given to 3 healthy adult volunteers before a meal, after a light meal, and after a usual meal in the cross-over method revealed the highest levels, both in serum and urine, in cases treated before a meal. In cases administered after a light or square meal, the serum level was less, but approximately 2 micrograms/ml over 6 hours after administration. No difference was seen in the AUC. The effective rate of S6472 when given at 750 approximately 1,500 mg/day was 74.6% in 62 patients with skin or soft tissue infectious diseases. Neither subjective or objective adverse reactions were seen in any case. Clinical laboratory testing revealed 1 case each of anemia and increased BUN, for which S6472 was not responsible.

Effects of synthetic omega-conotoxin on the contractile responses of segments of rat ileum, stomach fundus and uterus and guinea pig taenia coli.

The effect of synthetic omega-conotoxin (omega-CgTX) on the contractile responses of segments of rat ileum, stomach fundus and uterus and guinea pig taenia coli were investigated. Omega-CgTX (10(-9)-5 x 10(-6) M) did not inhibit the contractile responses of all smooth muscle segments to high KCl and/or ACh. However, unexpectedly, omega-CgTX (3 x 10(-7)-10(-5) M) alone caused dose-dependent contraction of segments of the stomach fundus and uterus. These contractile responses to omega-CgTX alone depended upon the presence and/or the influx of extracellular Ca2+; and they were inhibited by calcium antagonists such as diltiazem, nitrendipine and verapamil, with the exception that the segments of stomach fundus was not inhibited by verapamil. With the segments of uterus, but not those of other tissues, omega-CgTX (10(-7)-5 x 10(-6) M) significantly enhanced the contractile responses to various concentrations of ACh and high KCl. With rat ileum and guinea pig taenia coli segments, omega-CgTX (10(-9)-5 x 10(-6) M) did not induce a contractile response or have an enhancing effect. These findings suggest that omega-CgTX may have a calcium agonist-like effect on smooth muscles such as the stomach fundus and uterus of rats.

Hypolipidemic effect of ethyl all-cis-5,8,11,14,17-eicosapentaenoate (EPA-E) in rats.

We examined the effect of ethyl all-cis-5,8,11,14,17-eicosapentaenoate (EPA-E) with high purity on circulating lipids in rats under several experimental conditions. In normolipidemic rats, EPA-E decreased the lipids in a dose-dependent manner. Clofibrate (100 mg/kg/day) was more potent in lowering the lipids than EPA-E (1000 mg/kg/day). In high cholesterol diet-fed rats, EPA-E (300 mg/kg/day) decreased the total cholesterol. However, clofibrate (300 mg/kg/day) had little effect on the total cholesterol. In hypertriglycemic rats induced by corn oil, EPA-E (300 mg/kg/day) or clofibrate (100 mg/kg/day) reduced the rise of triglycerides. EPA-E (300 mg/kg/day), clinofibrate (100 mg/kg/day) or clofibrate (300 mg/kg/day) caused a significant reduction in the lipids induced by the injection of Triton WR-1339. Furthermore, EPA-E (300 mg/kg/day) or clinofibrate (100 mg/kg/day) decreased the elevation of lipids produced by feeding the rats a casein-rich diet. These results show that EPA-E possesses potent inhibitory activity on experimental hyperlipidemia induced either exogenously or endogenously.